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Second Sphere Effects on Oxygen Reduction and Peroxide

Activation by Mononuclear Iron Porphyrins and Related Systems
Sarmistha Bhunia,† Arnab Ghatak,† and Abhishek Dey*

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ABSTRACT: Activation and reduction of O2 and H2O2 by synthetic and biosynthetic iron
porphyrin models have proved to be a versatile platform for evaluating second-sphere effects
Downloaded via CARNEGIE MELLON UNIV on May 31, 2023 at 14:50:20 (UTC).

deemed important in naturally occurring heme active sites. Advances in synthetic techniques
have made it possible to install different functional groups around the porphyrin ligand,
recreating artificial analogues of the proximal and distal sites encountered in the heme
proteins. Using judicious choices of these substituents, several of the elegant second-sphere
effects that are proposed to be important in the reactivity of key heme proteins have been
evaluated under controlled environments, adding fundamental insight into the roles played by these weak interactions in nature. This
review presents a detailed description of these efforts and how these have not only demystified these second-sphere effects but also
how the knowledge obtained resulted in functional mimics of these heme enzymes.

CONTENTS Acknowledgments 12417

Abbreviations 12417
1. Introduction 12370 References 12417
1.1. Heme Proteins in Nature 12371
1.2. Definition of Second Sphere 12372
2. Role of Second Sphere in Synthetic Models of
CcO 12372 1. INTRODUCTION
2.1. Role of the Distal Cu Center 12372 Reduction of O2 to H2O during respiration provides the energy
2.2. Role of the Distal Phenol Group 12384 needed to sustain all aerobic life forms� from unicellular
3. Mononuclear Metallo-porphyrins as ORR Cata- bacteria to higher mammals.1,2 The solar energy stored in O2
lysts 12386 during photosynthesis is utilized to generate ATP, the energy
3.1. Role of Hydrophobicity and Steric Effect in carrier molecule, in the mitochondria. Thus, apart from being a
the Distal Pocket 12387 process fundamentally important to life on earth, the process
3.2. Role of Distal Water-Mediated H-Bonding 12389 has direct implication to the chemical storage of energy.
3.3. Role of Distal Redox Active Center 12391 Conventionally, O2 has served as oxidant in most abundant
3.4. Role of Distal H-Bonding Interactions 12395 combustion processes that release energy.3−5 The release of
3.5. Role of Electrostatic Interactions 12403 CO2 and other unamicable gases in the atmosphere associated
4. Biosynthetic Models of CcO 12405 with combustion of carbon-based fuels has led to an emprise
4.1. Role of Distal Cu 12405 for cleaner energy sources where sustainable and noncarbona-
4.2. Role of Tyrosine Residue 12406 ceous fuels like H2, which can be oxidized at the anode and
4.3. Electrocatalytic ORR by Biosynthetic Models utilized for O2 reduction at the cathode in a fuel-cell to
of CcO 12408 generate electricity, is being hailed as a potent replace-
4.4. Role of Distal Arginine Residue in Amylin ment.6−11 Consequently, the understanding of this fundamen-
Peptide 12410 tally important 4e−/4H+ reduction of O2 to H2O has now
5. Role of Second-Sphere Distal Residues in generated considerable interest in the research community as it
Peroxide Activation 12411 may aid in access to clean and sustainable energy.12−15
6. Conclusion 12416
Author Information 12416
Special Issue: Catalysis beyond the First Coordination
Corresponding Author 12416
Sphere
Authors 12416
Author Contributions 12417 Received: December 9, 2021
Notes 12417 Published: April 11, 2022
Biographies 12417

© 2022 American Chemical Society https://doi.org/10.1021/acs.chemrev.1c01021

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Figure 1. Active site structures of (A) myoglobin (pdb id: 1mbo),39 (B) cytochrome P450 (pdb id: 2a1m),40 (C) horse radish peroxidase (pdb id:
1hh5),41 cytochrome c oxidase (pdb id: 2y69),42 Sir (pdb id: 3 mm5),43 and Nir (pdb id: 3d1i).48
To begin conceiving or designing a catalyst for O2 reduction
a logical place to start is the active sites of metallo-enzymes
that are known to do it in nature.16 These would be either the
heme-based cytochrome c oxidase or copper-based multi-
copper oxidases.17−21 There has been a substantial volume of
work in the development of O2 reduction catalysts in the past
several decades, which include different cathode materials,
molecular catalysts, often adsorbed on cathodes, and enzymatic
catalysts.15,22−26 Many of these, at least the molecular ones,
were often prescribed following the design of the active sites of
these natural metalloenzymes. Such efforts have been reviewed
extensively very recently.10,27−31 In this paper, the intension is
to review the recent developments in understanding the effect
of weaker, often noncovalent, interactions that are known to
play a major role in controlling the reactivity of heme proteins
and could be integrated in the design of artificial catalysts to
enhance their reactivity.
1.1. Heme Proteins in Nature
One of the astounding features of the heme-based proteins is
the ability to catalyze diverse sets of chemical reactions with
apparently small modifications of the active site�such
diversity is perhaps only matched by iron−sulfur proteins.32−38
The heme active sites vary in the nature of the heme cofactor,
in the ligation to the protein, and in the environment around
the heme active sites.39−43 While the different porphyrin
macrocycles and axial ligand to the iron are ostentatious
changes in the active site and may be expected to affect the
chemical reactivity of the active site, the environment around
the heme is a major enabler of the reactivity of these active
sites and is the inspiration behind the topic of this review.44−47
In the water-soluble O2 binding/storage proteins like
myoglobin, the distal environment, the active site in the
vicinity of the exchangeable axial ligand, contains hydrophobic
residues and a histidine residue (Figure 1A) which decelerate
the hydrolysis of the FeIII−O2− adduct by both limiting access
of water and hydrogen bonding to the bent Fe−O2 unit.49−51
In the active site of cytochrome P450 superfamily of enzymes,
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while the axial thiolate ligand is key to the monooxygenase
activity, the threonine residue in the distal site (Figure 1B)
transfers the protons needed to cleave the O−O bond to
generate the reactive oxidant such that the mutation of this
residue results in loss of monooxygenase activity.38,52 The
distal site of horseradish peroxidases (HRP) has an arginine
residue and a histidine residue (Figure 1C) which are
responsible for the efficient binding of the oxidant H2O2 and
the substrates and heterolytic cleavage of the O−O bond to
generate the highly oxidizing compound I species. Mutation of
either of the two species is deleterious to the function of
peroxidases.47,53−55 The distal site of cytochrome c oxidase
(CcO) contains a CuB (Figure 1D) which is held by three
histidine residues, one of which is post-translationally cross-
linked with a tyrosine residue. The CuB and tyrosine residues
are indispensable for the 4e−/4H+ reduction of O2 catalyzed by
this active site. Additionally, the coupling between the O−O
bond cleavage and proton transfer from the tyrosine residue is
likely responsible for creating vectorial proton transfer during
O2 reduction which is responsible for generating the proton
gradient across the mitochondrial membrane to be later
utilized for the synthesis of ATP from ADP and phosphate
during oxidative phosphorylation.56−59
The organization of the protein residues around the
metalloporphyrin active site in sulfite reductases is very
impressive to say the least. This siroheme active site catalyzes
the 6e−/6H+ reduction of SO2/SO32− to H2S and plays a major
role in the geochemical sulfur cycle.60 The roles of the distal
lysine and arginine residues (Figure 1E) include binding and
orientation of the substrate and transfer of protons to the
catalytic site.61 In addition to the distal residues there is a Fe4S4
cubane bridged to the cluster which provides electrons to the
active site, which may achieve the transformation in three 2e−
steps.61−65 The same siroheme active site is found in
assimilatory nitrite reductases which reduce NO2− to
NH4+.66 The cytochrome cd1 nitrite reductase, on the other
hand, does not possess the Fe4S4 cluster and uses heme d1
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Figure 2. Active site of sulfite reductase (SiR) used to demonstrate the second sphere interactions: hydrogen bonding (gray lines), electrostatic
potential (columbic charge surfaces; generated using Chimera), and a second Fe4S4 cluster.
cofactor and can reduce NO2− to NO and not further.67 The
active site of Cd1 NiR contains arginine and histidine residues
(Figure 1F) which are believed to serve the same purpose as
the ones present in the active site of SiRs.68−71
1.2. Definition of Second Sphere
A metallo-protein active site design can thus be divided into
two distinct components, the first being the immediate co-
ordination sphere of the metal. In the case of heme proteins
this will include the porphyrin ring that forms the organic part
of the heme cofactor and the axial ligand used to bind the
heme cofactor to the proteins.72,73 There are several different
varieties of the porphyrin ring known to exist in different heme
active sites.74,75 Similarly, axial ligands reported to date for
heme include histidine (imidazole headgroup), cysteine
(thiolate headgroup), tyrosine (phenolate headgroup), me-
thionine (thioether headgroup), and lysine (primary amine
headgroup).76−78 Variations in the primary co-ordination
sphere affect the electronic structure of the heme active site
which leads to very discernible changes in spectroscopic
features of heme proteins and plays a dominant role in
stabilizing, and thereby determining, the nature of the key
reactive intermediate in these proteins.79−83
The second component to the active-site design is the
noncoordinated amino acids which may or may not affect the
electronic structure of the heme active site in its resting state
but manifest themselves in the reactivity. The distal site of the
SiR protein, for example, has several amino acids like arginine
and lysine which are likely to be protonated under
physiological conditions as their head groups guanidine and
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lysine have pKa > 9.60,84,85 Some of these directly hydrogen
bond with the substrate (shown with gray lines, Figure 2),
while some, that are further away, create a positive electrostatic
field (blue surfaces, Figure 2) in the vicinity of the substrate;
both of these effects will aid binding, orientation, and
reduction of the bound substrate.62 At the same time, the
Fe4S4 cubane, albeit bridged, stores electrons and shuttles it to
the heme cofactor during catalysis.85−87 These components of
the metallo-enzyme active sites can broadly be classified as the
second sphere, and their effect on the reactivity of the active
site can be summarized as second-sphere effects on catalysis.88
Second-sphere effects comprise effects induced by amino
acids such as hydrophobicity, hydrogen bonding, and electro-
statics as well as the effect of introducing another redox active
metal in the vicinity of the iron porphyrin active site. Over the
past decade or so, bioinspired modeling of heme active sites
has advanced to include these second-sphere effects on
spectroscopy and reactivity of artificial analogues. A
comprehensive review of such efforts toward the understanding
and development of ORR is presented in this paper.
2. ROLE OF SECOND SPHERE IN SYNTHETIC MODELS
OF CCO
2.1. Role of the Distal Cu Center
The role of a distal Cu ion in the reduction of O2 by iron
porphyrins has been extensively investigated by several groups
with the aim of generating functional mimics of naturally
occurring enzyme CcO.29,35 CcO, the terminal enzyme of the
human respiratory chain, catalyzes the 4e−/4H+ reduction of
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Figure 3. Synthetic CcO model complexes (A−E) reported by Collman and co-workers.109−111
dioxygen to water in mitochondria.89,90 A proton gradient
along the mitochondrial membrane resulting from this process
is used to synthesize ATP.4,5,91 This process of oxidative
phosphorylation in mitochondria stores the energy obtained by
O2 reduction by NADH/NADPH released from different
metabolic pathways in a eukaryotic cell.92−94 The exact
mechanism of the binuclear hemea3-CuB enzyme active site
that performs the O2 reduction without any leakage of partially
reduced oxygen species (PROS) like peroxide and superoxide,
which cause oxidative stress, has been a fundamentally
important question.95,96 Apart from biochemists investigating
the membrane bound protein itself, synthetic inorganic
chemists have forwarded some noteworthy artificial mimics
that allowed investigating the inorganic active site outside of
the protein environment.28,97,98
The initial attempts focused on mimicking the O2 reduction
activity electrochemically under heterogeneous conditions
where the synthetic mimics were adsorbed on electrodes
(generally graphitic) such that the electrons were directly
provided from the electrode and protons and oxygen could be
obtained from aqueous solutions where these electrodes were
dipped in.99,100 Rotating disc electrochemistry (RDE) and
rotating ring disc electrochemistry (RRDE) are two convenient
methods that have been extensively used to estimate the rate
and selectivity for the 4e−/4H+ oxygen reduction proc-
ess.101,102 In RDE, the plot of 1/icat (icat is the catalytic
current) and 1/ω1/2 (ω is the angular rotation velocity of the
electrode) at a potential in the region where the catalytic
current is mass transfer limited (i.e., current independent of
potential) yields a straight line where the Y-intercept yields the
rate of O 2 reduction (kcat ) at infinite ω where the
concentration of the substrate at the electrode surface is the
same as the concentration in bulk solution, while the slope
yields the number of electrons supplied to the substrate (n).
Ideally, the n should be 4 for a 4e+/4H+ ORR and the highest
value of kcat can be the diffusion rate of O2 or H+ in solution at
a given pH.103−105 In RRDE, the working electrode of RDE is
encircled by a Pt ring secondary electrode that can be
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independently addressed and either a constant or sweeping
potential can be applied.106−108
Collman and co-workers synthesized some earlier model
complexes of CcO such as a cobalt(II) porphyrin which
consists of a copper triazacyclononane macrocycle fastened to
the distal side along with a linker bearing an imidazole
headgroup at the proximal side (Figure 3A). This model could
bind O2 irreversibly in 1:1 ratio to form a stable adduct. This
oxygen-derived adduct is a bridging peroxide which was
characterized by electrospray ionization mass (ESI-MS)
spectra and IR spectra [ν(O−O) at 804 cm−1, Δ16/18O2 =
46 cm−1], and this adduct regenerated the deoxygenated form
when titrated with 4 equiv of a reducing agent like cobaltocene,
i.e., it catalyzes the 4e−/4H+ reduction. On the contrary, the
Cu free form of the complex 3A showed reversible O2 binding
but not O2 reduction. RRDE data of complex 3A at
physiological pH showed 4e−/4H+ O2 reduction to H2O.
The selectivity is proposed to be derived from the presence of
Cu in the distal structure which aids the O−O bond
cleavage.28,109
The electrochemical behavior of two well-defined synthetic
FeII−CuI model complexes of CcO (Figure 3B,C) were
explored. These two complexes differ only in the nature of the
distal ligand structure to which the Cu ion binds. Compound
3B has a 1,4,7-triazacyclononane (TACN) cap which can bind
the Cu ion with three nitrogen atoms, while complex 3C has a
N,N′,N”-tribenzyltris(aminomethyl)amine (TBTren) cap con-
taining four nitrogen atoms. When both of the complexes were
deposited over the edge-plane graphite (EPG) electrode and
RRDE experiments were performed, 3B was found to catalyze
2e− reduction of O2 to generate H2O2, while 3C catalyzed 4e−
O2 reduction at pH 7. The difference in selectivity for O2
reduction was ascribed to the different CuII/I potential induced
by the TACN cap in 3B and TBTren cap in 3C. The 3C
complex with TBTren cap in the distal structure was proposed
to stabilize the side-on FeIII(O22−)-CuII peroxo intermediate,
and as a result, the conversion to H2O2 is endergonic and only
H2O formation is exergonic, which favors the 4e− O2
reduction. On the contrary, for the 3B complex, both H2O2
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Figure 4. Synthetic CcO model complexes (A−F) studied by Boitrel and co-workers.112−115
formation and H2O formation become exergonic. Thus, the
selectivity of oxygen reduction depends on the rate of
hydrolysis of the peroxo intermediate, which is less stable for
3B due to TACN cap in the distal structure, and hence, it
hydrolyses and releases H2O2, thereby catalyzing the 2e− O2
reduction. Notably, this was one of the earliest reports where
PROS formation during electrochemical O2 reduction was
attributed to hydrolysis of peroxide intermediates. The model
complex 3C, which selectively reduces O 2 to H 2 O,
demonstrates the role of CuII/I potential in controlling the
stability of the peroxide intermediate and in turn controlling
the fate of O2 reduction.10,110
Collman and co-workers, in their continued pursuit,
synthesized even closer structural analogues of Fea3/CuB active
site of CcO where the Cu atom is coordinated by three
imidazole ligands.111 Both compound 3D and 3E contain three
distal imidazole ligands tethered to the porphyrin core via
acetamide linkages, but they differ in the nature of the
covalently attached axial base. Molecular modeling suggested
that due to the presence of acetamide linkages in both the
complexes the Cu···Fe distance was in the ∼4.5−5 Å range but
does not coordinate with the iron atom. RRDE experiments,
with these catalysts adsorbed on EPG electrode, revealed that
both these complexes showed 4e− reduction of oxygen across a
pH range of 3.5−8.5. The complex 3E exhibited electro-
catalytic reduction of O2 at very low overpotential (ηORR)
compared to other reported analogues. At pH 7 buffer
solution, addition of anions like chloride, bromide, and
carboxylate did not affect the O2 reduction, suggesting that it
is the water-derived ligands (e.g., aquo-hydroxo or hydroxo
ligands) which determine the redox potential of the Fe/Cu pair
during the catalytic cycle.28,111 In these series, the selectivity
for 4e−/4H+ reduction of O2 was rationalized mostly through
the stability and O−O activation of FeIII−(O22−)−CuII
intermediates, although its presence was never established
directly.
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Around the same time, Boitrel and co-workers synthesized a
series of biomimetic models of CcO in order to exact the role
of the distal Cu center and Cu ion binding environment in the
electrocatalytic O2 reduction. The quinolinyl picket porphyr-
ins, containing external nitrogen base (4A) and tailed nitrogen
base (4B) were specifically designed (1) to evaluate their
efficiency of electrocatalytic O2 reduction to water, (2)
introduce structural similarity with the natural enzyme by
incorporating nitrogen atoms that is part of aromatic system to
stabilize Cu and off-centring of two metal atoms which is often
neglected, and (3) prevention of any intermolecular reaction
on the distal side of the porphyrin between the copper atoms
of two such constructs (Figure 4). Both of the Fe−Cu-
containing porphyrins were found to bind oxygen irreversibly,
while the Fe-only one (not shown) could bind O2 reversibly.
Surprisingly, when these compounds were physiadsorbed over
the EPG electrode and RRDE experiments were performed at
pH 6.86, they found that the Fe only model complexes are
better 4e− catalysts than the Fe−Cu analogues (4A and 4B),
which catalyze both the 2e− and 4e− reductions. Boitrel and
co-workers explained this deviation considering two possible
scenarios: (1) the copper center does not interfere with the O2
molecule bound to the iron during the reduction, it only
interferes during O2 binding and (2) the labile copper ion of
the tris(N-base) complex is no longer coordinated when the
catalyst adsorbed onto the graphite electrode is in contact with
the aqueous solution.10,112
The same group had almost similar observations with their
porphyrins capped with a tris(2-aminoethyl)amine (tren)
motif as functional models for CcO. The Fe−Cu porphyrin
4C was obtained by grafting the tren moiety on a
tris(chloroacetamide) picket porphyrin, leading to a ligand in
which the tren is held by three arms of the porphyrin and is
more off-centered and closer to the porphyrin than in model
4D (where all four of the porphyrin arms hold the tren ligand).
The Cu free forms of 4C and 4D were found to be more
selective toward 4e−/4H+ O2 reduction as well when the
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Figure 5. CcO active site structure (A). Synthetic CcO model complexes (B−D) studied by Collman and co-workers.125,126
RRDE experiment was performed in a pH 6.6 buffer solution
over EPG electrode. These observations could be related to the
fact that the μ-peroxo intermediate between iron and copper
atoms (FeIII(O22−)−CuII) might not be a prerequisite for O2
reduction, whereas the involvement of a protonated end-on
ferric peroxo unit was likely to be more compatible with the
reactivity. These results seem to be in contradiction with that
of Collman’s results where the nature of the FeIII(O22−)−CuII
species was deemed crucial. In these cases, the ferric
hydroperoxide complexes might have been stabilized through
H-bonding from the secondary amino groups present in the
distal structure, which likely remain protonated at pH 6.6 in
aqueous solution.113
In subsequent work, they showed that the substituents on
the secondary amino groups significantly influence the
electrocatalytic O2 reduction activity over the electrode
surface. The alkylated secondary amine in the distal structure
of 4C with −CO2Et and benzyl groups, respectively (4E and
4F), showed higher ηORR in reducing O2 compared to 4C. In
particular, the alkylation by benzyl group increased the ηORR by
0.5 V. In addition, the Fe-only forms of 4E and 4F showed
partial O2 reduction reaction, while 4C reduces O2 selectively
via the 4e− pathway only. The authors concluded that the
secondary amines in the distal structures were much more
efficient than tertiary amines in catalyzing the selective 4e−/
4H+ reduction of O2. The results from Boitrel’s work suggested
that a mononuclear iron porphyrin adsorbed on an electrode is
an intrinsically efficient catalyst for the reduction provided the
rate of flow of electrons and protons are very rapid, which is
exactly the case over EPG electrode in aqueous neutral pH
buffer solution.114,115 This conclusion was vindicated two
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decades later by the Dey’s group and is discussed in the later
sections of the manuscript.
In native CcO enzyme, the electrons required for O2
reduction arrive via cytochrome C (cyt C) at very slow rates
(∼4 s−1). While the fully reduced CcO does not require
electron transfer (ET) from Cyt C as the ET from reduced
CuA and heme b are rapid (104−105 s−1), Wickstrom and
others had postulated that during turnover the mixed valent
state where the heme−Cu site is reduced, and not the CuA and
heme b, is more likely to be catalytically active.1,57,116,117 This
was supported by the isolation of a oxoferryl-tyr• radical
intermediate PM (FeIV�O, CuII OH/H2O, and Tyr•) where
out of the 4e− needed for O2 reduction three are obtained from
the heme−Cu site and one is obtained from the Tyr244
residue.56 The X-ray crystallographic structure of the CcO
active site is shown in Figure 5A for clarification (it includes a
binuclear site composed of hemea3 with a proximal histidine
along with a histidine bound copper (CuB) in the distal site).
One of these three ligating histidine residues is covalently
cross-linked to a nearby tyrosine residue which acts as a source
of electron and proton in the reduction of O2 and aids vectorial
proton translocation.57,118,119 To address this, Collman et al.
synthesized a CcO model that mimic both CuB and Tyr244 at
the distal site. To mimic the slow ET rate of Cyt C, self-
assembled monolayer (SAM) on Au electrodes were
used.120−122 The electron tunnelling rates across SAM
exponentially decays with its length and ET rates across
SAM can be attenuated between 105 and 10 s−1 by controlling
the chain length.123,124 Immobilization of CcO models using
CuI-catalyzed azide−alkyne cycloaddition (click chemistry)
atop SAM allowed control of the electron flux, which enabled
understanding the exact role of redox active centers under
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varying the rates of electron delivery. In the new structural and Scheme 1. (A) Schematic Representation of Two Different
functional CcO model complex synthesized, a proximal SAM (Slow SAM S1 and Fast SAM S2) Modified Au
imidazole, a distal copper bound to a tris-imidazole ligand, a Electrode Used by Collman and Co-workers for Variation in
phenol group covalently attached to one of the copper-ligating Electron Flux, Where the Model Complex Was Covalently
imidazoles and an alkyne functional group for immobilization Attached. (B) PROS Bar Diagram of the Three CcO Model
were assembled in what can only be described as one of the Complexes over Two Different SAM-Modified Au
most elaborate, albeit arduous, feats of synthetic inorganic Electrode125
chemistry in the known literature (Figure 5B).125 Distal copper
was installed over the heme plane in such a way that the Fe···
Cu distance remained 5 Å, which is very close to the natural
mammalian CcO enzyme, and the covalently attached phenol
group which mimics the tyrosine residue of CcO, was
introduced close to the O2 binding site of Fe porphyrin. The
proximal imidazole ligand was covalently introduced as a trans
axial ligand to Fe, mimicking His376 of the natural enzyme and
the purpose of introducing terminal alkyne was to selectively
attach these systems over azide terminated SAM solution
through azide−alkyne cycloaddition. The control model 5C
lacks the external phenol which is masked with a −OMe group
and their electrochemical behavior was also compared with the
iron only model 22A (discussed in section 3.2) which lacks
both Cu and Tyr244.
The role of the phenol groups introduced in O2 reduction
was evaluated by comparing the selectivity for O2 reduction
using RRDE to detect any PROS released under slow ET
conditions. Model complexes were covalently attached onto
mixed SAM combining of 1-azidohexadecanethiol and
hexadecanethiol (slow SAM S1) where the ET from electrode
is rate limiting having rate constant kET = 6.0 ± 0.1 s−1 and, for
comparison, a fast SAM S2, comprising azidophenylene-
ethynylenebenzylthiol and octanethiol, was also used which
has an ET rate constant (kET) as high as 104 s−1 (Scheme
1A).123,127 Although different SAMs do not change the
potential at which ORR takes place (0.3 V vs NHE in both
SAMs), the kET affects the kinetics of the catalysis, i.e., while in
slow SAM S1 ET is rate limiting and in fast SAM S2 O2
diffusion to the electrode is rate limiting. When RRDE was
performed over an S2-modified Au electrode, it was found that active site. As a result, more than 96% selectivity toward 4e−/
the Fe-only model 22A showed 89 ± 2% 4e−/4H+− ORR 4H+ O2 reduction was recorded over a slow SAM S1-modified
selectivity. While incorporation of Cu in 5C increased the electrode (Scheme 1B). Thus, these model complexes help to
selectivity to 97 ± 1%, incorporation of both Cu and phenol propose that both CuB and Tyr244 are required in the distal
(5B) did not alter the selectivity further (Scheme 1B). The site as electron donors for selective four-electron reduction of
situation on fast SAM S2 was similar to that of fully reduced O2 under rate-limiting ET conditions.125 The role of the
CcO in a single turnover experiment, and the involvement of phenol group alone could not be evaluated in these
distal redox-active tyrosine was not crucial as long as electrons investigations. Different groups have later investigated this in
could be readily delivered from the electrode which surrogates details, and these results are discussed later (sections 2.2 and
at heme b and CuA. Alternatively, the Slow SAM S1 might have 3.3).
better mimicked the physiological conditions, where CcO is Apart from the electrocatalytic investigations, stoichiometric
not normally at its fully reduced state during turnover due to reaction with O2 in organic solvents at low temperature was
very slow electron delivery from Cytochrome C. Over slow monitored using different spectroscopic techniques. The
SAM S1 modified Au electrode, complex 22A lacking both Cu heme/Cu complex 5D, which has a built-in histidine-tyrosine
and phenol produced ∼29% PROS in RRDE experiments and cross-link, reacted with O2 resulting in the formation of a heme
resulted in rapid degradation during O2 reduction. The superoxide species which was generally stable in organic
analogue which includes Cu only in absence of phenol, 5C solutions at low temperatures and could be characterized using
was more stable than 22A but still showed marked degree of resonance Raman (rR) and 1H nuclear magnetic resonance
PROS leakage. Complex 5C could store up to three electrons (1H NMR) spectroscopy. When the phenol group was
at the active site (two from iron and one from copper) and, included, the reaction with O2 proceeded via the heme−
thus, one additional electron is required from the electrode for superoxide intermediate to eventually result in a species which
the 4e− reduction of oxygen. Since the ET rate from the had electron paramagnetic resonance (EPR) spectroscopic
electrode to the catalyst was slow in SAM S1, PROS was being features like that of PM in CcO. It is important to note that this
generated before the electron reaches the active site. In case of series of heme/Cu complexes did not present a strong case of a
5B, an additional redox active phenol is present along with Cu FeIII−(O22−)−CuII intermediate in contrast to previous reports
in the distal site and four electrons could be supplied from the from this group. Rather, a direct 3e− reduction of the bound
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Figure 6. Structural representation of the TMPA−Cu complex and the porphyrin (A) used by Karlin and co-workers for stoichiometric reaction
with O2. The formation of the corresponding peroxo and oxo complexes are also shown in (B) and (C), respectively.131

Figure 7. (A) Tethered Cu−TMPA with Fe−TPP complex (A) and its constitutional isomer (C) used by Karlin and co-workers. The
corresponding peroxo complex after reaction of (A) with O2 is shown in (B).132

Fe−O2 species was proposed which is similar to the earlier
conclusions from Boitrel and co-workers.128 Recently, Solo-
mon et al. determined the geometric and electronic structural
contribution in heme Cu oxidase (HCO) active site for the
O−O bond cleavage and how this is coupled to proton
pumping by spectroscopically defining the PM intermediate.112
In the previous literature reports although evidence for the
Tyr• radical in PM intermediate had been extensively discussed,
the presence of both CuII and Tyr• in HCOs was still elusive,
and this was the motivation for this particular work.129,130
12377
In their attempts to mimic the O2 reduction process in CcO,
Karlin and co-workers utilized Cu-TMPA complex (where
TMPA = tris(2-pyridylmethyl)amine) and FeII tetrakis (2,6-
difluorophenyl)porphyrin FeII(F8TPP) (Figure 6A).131 When
O2 was made to react with the above two in a 1:1 ratio in
acetonitrile at −40 °C, heterobinuclear FeIII−(O22−)−CuII
peroxo complex (Figure 6B) was formed. The peroxo complex
was not stable and underwent thermal degradation to produce
the μ-oxo (FeIII−O−CuII) complex subsequently (6C). The
formation of peroxo species was established using rR
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Figure 8. Oxygen-bound species (A−C) formed by the reaction of tridentate Cu ligand LMe2N with 6A and O2 as studied by Karlin and co-
workers.133
spectroscopy, where the stretching frequency, ν(O−O), was
observed at 808 cm−1 (ν16/18O2 = 46 cm−1). Solution magnetic
moment measurement and 1H NMR indicated that 6B has an
S = 2 ground state having spin only magnetic moment of 5.1
μB at −40 °C, which arose because of antiferromagnetic
coupling between the (S = 5/2) FeIII and (S = 1/2) CuII
centers. Formation of a peroxo complex was followed using
stopped-flow kinetics in the temperature range of −94 to −75
°C in acetone solvent. The data revealed that prior to the
formation of 6B (λmax = 556 nm) from 6A, a transient heme−
superoxide species was generated having λmax= 537 nm, and
this transformation was a first-order process with ΔH⧧ = 45 ±
1 kJ mol−1, ΔS⧧ = −19 ± 6 J mol−1K−1, and k = 0.007 s−1 at
−90 °C. Formation of this transient ferric superoxo species
instead of the corresponding Cu(II)−superoxo species
indicated that heme was preferred for O2 binding. The peroxo
complex (t1/2= 1015 ± 20 s, at room temperature) finally
transformed to the μ-oxo complex 6C by releasing 0.5 equiv of
oxygen.35,131
The same group synthesized a binuclear complex 6L−FeCu
(7A) in which a Cu−TMPA moiety was tethered to a
(TPP)Fe II−porphyrin derivative and reported their O 2
reactivity instead of separately reacting TMPA, FeII(F8TPP),
and O2 in a 1:1:1 ratio.132 When 7A reacted with O2 in 1:1
ratio in acetonitrile solvent, the peroxo adduct (7B) (FeIII−
O22−−CuII) was formed which was characterized by spectro-
scopic data and MALDI-TOF-MS, this time with much higher
stability having t1/2 = 60 min at room temperature (Figure 7).
The rR spectra revealed that ν(O−O) of 7B was at 787 cm−1
(ν16/18O2 = 43 cm−1). The 1H, 2H NMR and EPR spectra
revealed that 7B was a paramagnetic high spin (Stotal = 2)
peroxo complex which was formed by the antiferromagnetic
coupling between high spin Fe (S = 5/2) and Cu (S = 1/2).
Upon reaction of 7A in a 1:1 ratio with O2, a short-lived
intermediate was formed (λmax = 538 nm) apart from 7B (λmax
= 561 nm) which Karlin and co-workers postulated as a
superoxo species [Fe−(O2)···CuI]+ and/or a bis-O2 adduct
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[Fe−(O2)···CuII−(O2−)]+. These two species were formed
independently within 1 ms, and the ratio of these two products
depended on the reaction temperature and O2 concentration.
At low temperature, intermediate having λmax = 538 nm was
favored, and it was converted to the peroxo complex 7B with a
first-order kinetics at −94 to −60 °C where ΔH⧧ = 37.4 ± 0.4
kJ mol−1 and ΔS⧧ = −28.7 ± 2.3 J mol−1K−1. Surprisingly,
while performing the same O2 reactivity with the constitutional
isomer of 7A, i.e., 7C, it was found that the resulting peroxo
complex (not shown) was diamagnetic as compared to the
paramagnetic 7B. Also, spectrophotometric titration revealed
that stoichiometry of the reaction is 7C/O2 = 2:1 and the
ν(O−O) of 809 cm−1 was higher compared to 7B. The
contrasting nature of these two peroxo complexes might have
been due to the ligand architecture. Nevertheless, this study
showed that if a Cu unit can be tethered and appended
properly in the distal site of an iron porphyrin moiety, a μ-
peroxo species might be formed upon the reaction of the fully
reduced complex with O229,35,132
Tridentate ligands for the Cu instead of tetradentate TMPA
ligands such as PY2, where PY2 = N,N-bis[2- (2-pyridyl)-
ethyl]amine, when mixed in 1:1 ratio with FeII(F8TPP), 6A, in
the presence of dioxygen, resulted in a heme superoxo species
[(S)(F8TPP)Fe-(O2)] (8A, S = acetone or THF) which
subsequently reacted with the Cu to produce the bridging μ-
peroxo species (Figure 8). However, unlike the tetradentate
Cu chelate, in this case the peroxo species was unstable even at
low temperature and transformed to an isolable and character-
izable μ-oxo species [(F8TPP)FeIII−(O2−)−CuII(MePY2)]+
immediately. When a more electron-donating Cu binding
LMe2N was used, a short-lived heme superoxo species
[(S)(F8TPP)Fe-(O2)] (8A) was formed further, reacting
with the CuI to form the bridging peroxo species [(F8TPP)-
FeIII−(O22−)−CuII(LMe2N)]+(8B) having slightly more stability
at low temperature. The 2H and 1H NMR spectroscopic data
indicated that this species was also an antiferromagnetic
coupled S = 2 system, arising from high spin FeIII and CuII
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Figure 9. (A) PImHFeII complex with its O2 reactivity in absence of Cu (B) and in the presence of Cu (C−E) reported by Karlin and co-workers.134
centers. However, rR spectra showed two distinct peroxo
species (arising from different isomer or conformer) with
ν(O−O) vibrations at 767 and 752 cm−1 (ν16/18O2 = 41 and
45 cm−1), respectively, and these values were significantly
lower than those observed for tetradentate systems. A μ-η2:η2
side-on bound heme−peroxo−copper structure with a bent
Cu−O2−Fe core was proposed in contrast to the (μ-1,2) end-
on peroxo structure for the tetradentate Cu ligands. The
compound 8B converted thermally to the μ-oxo complex
[(F8TPP)FeIII−O2−−CuII(LMe2N)]+, 8C, as seen for other
related systems.35,133
In a recent study, Karlin et al. reported a CcO model system
where a histamine moiety is appended to the periphery of a
fluorinated tetrapheylporphyrin to produce a new binuclear
tridentate ligand system PImH (Figure 9).134 They investigated
the dioxygen reactivity of the PImHFeIIcomplex (9A) both in
the presence and absence of copper ion. Reaction of 9A with
O2 in THF at −80 °C produced the PImHFe−O2 (9B) species,
which was characterized by UV−vis absorption spectroscopy.
Complex 9B was EPR silent and showed upfield-shifted pyrrole
resonance (δ = 9.12 ppm) in 2H NMR, indicating a six-
coordinated low spin (S = 0) species where the tethered
imidazoyl arm serves as the sixth coordination. The rR spectra
of 9B showed the characteristic ν(O−O) at 1171
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cm −1 ((ν 16/18 O 2 = 61 cm −1 ) and ν(Fe−O) at 575
cm−1(ν16/18O2 = 24 cm−1) and the reaction was reversible;
i.e., warming 9B at room temperature gave back 9A. They
obtained the fully reduced heterobinuclear heme copper
complex [(PImH)FeIICuI]+(9C) at low temperature by addition
of 1 equiv of [CuI(CH3CN)4](B(C6F5)4] to the reduced
complex 9A. When dry O2 gas in THF was passed through 9C
at low temperature, a new species [(PImH)FeIII−(O22−)−
CuII]+(9D) was formed, which was characterized using
absorption spectral features at 420, 545, and 565 nm.
Surprisingly, for this high-spin peroxo complex 9D, ν(O−O)
stretching was found at 799 cm−1 (ν16/18O2 = 48 cm−1), which
was higher compared to other tridentate ligands with CuII ion,
studied by Karlin et al. where side-on μ−η2:η2−O22− binding
to both FeIII and CuII had been postulated.133,135,136 The
vibrations [ν(Fe−O) = 524 cm−1; ν(O−O) = 799 cm−1] of
9D were closer to the previously reported HS−TMPA
complexes. Based on the X-ray absorption fine structure
(EXAFS) data and density functional theory (DFT)
calculations, it was found that PImH tridentate chelate induced
considerable geometric distortion in the CuII coordination
sphere, which forced the peroxo to binding to the Cu ion in an
essentially η1 fashion instead of η2 fashion. So, the formulation
of 9D peroxo species was proposed to be [(PImH)FeIII−
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Figure 10. Parent TMPA heme−peroxo−copper complex (A) along with eight other low-spin heme−peroxo−copper complexes bearing
derivatized TMPA-based copper chelates (B−I) indicating their roles as reported by Karlin and co-workers.142

(μ−η2:η1−O22−)−CuII]+. The species 9D was also EPR silent S
= 2 resulting from the antiferromagnetic coupling between FeIII
(S = 5/2) and CuII (S = 1/2) centers.134 On the contrary, a
low-spin analogue of [(PImH)FeIII−(O22−)−CuII]+(9E) was
obtained when an axial base DCHIm in THF was introduced
at −80 °C. In absorption spectroscopy, incorporation of
DCHIm changed the Soret and Q-band to 425 and 538 nm
from 9D, and relatively intense low energy features were
observed in the 800−900 nm region originating from the
peroxo to metal LMCT band indicating an LS μ-1,2 peroxo
either lowering the energy barrier and/or selectively enhancing
heterolytic (over homolytic) O−O bond cleavage. Previously,
reaction of biomimetic heme−peroxo−copper complexes with
exogeneous substrates like phenol or 4-nitrophenol was
investigated to understand the role of H-bonding in “O−O”
bond cleavage.137,138 Mimicking H-bonding with exogeneous
substrates in synthetic systems is complicated by the loss of
entropy in positioning a H-bond participant with the reactive
unit which is absent in the enzyme systems.137−139 Therefore,
_
Karlin et al. prepared 16 novel synthetic heme−(μ-O22 )− d

species. In addition, the 2H NMR spectra confirmed the spin
change from 9D to 9E with the change in chemical shift value
from 93.0 to 10.2 ppm. The S = 0 EPR silent ground state of
the 9E species resulted from antiferromagnetic coupling of the
LS FeIII (S = 1/2) and CuII centers. rR spectra indicated that
ν(Fe−O) dramatically increased by 86−610 cm−1 due to the
formation of LS peroxo species 9E.
H-bonding plays a crucial role in the heme−Cu oxidase
chemistry, and it is likely that the reductive cleavage of the
“O−O” bond in the active site is often controlled by it by
12380
Cu(XTMPA) complexes using the parent TMPA ligand
(mentioned earlier) where they incorporated intramolecular
H-bonding moieties to stabilize and spectroscopically detect
the proposed Ip intermediate where a putative metal-bound
hydroperoxide species in the active site is H-bonded with
tyrosine residue through a water molecule to facilitate the
necessary proton coupled electron transfer (PCET) from
tyrosine residue29,140,141 and to understand the steric and
electronic effects of H-bonding on O2-reduction. A series of
TMPA-based Cu ligands which differ in the substituents in the
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6
pyridyl position like CH3TMPA, (CH3)2TMPA, OCH3TMPA, peroxo−Cu core. Therefore, optimal H-bonding interactions
PV
TMPA, A 2 TMPA, N H 2 TMPA, ( N H 2 ) 2 TMPA, and were required to the peroxo core for activating the O−O bond,
NH2,CH3
TMPA (Figure 10) were paired with tetrakis(2,6- which was exactly served by the variant with two amine groups
difluorophenyl)porphyrinate (F8) heme for generation of low- (10I) revealing a key orientational requirement of H-bonding
spin heme−peroxo−copper synthetic model complexes for O−O bond cleavage.
[(DCHIm)(F8)FeIII−(O22−)−CuII(TMPA)]+, (DCHIm = Naruta and co-workers prepared a binuclear complex,
1,5-dicyclohexylimidazole) and were characterized using [(TMP)Fe I I -(5MeTPA)Cu I ]BPh 4 (TMP-5MeTPA =
absorption and rR spectroscopy.142 It was found that 10,15,20-tris(2,4,6-trimethylphenyl)-5-(2′-bis((5′′-methyl-2′′-
derivatives of the parent ligands which contain one or two pyridylmethyl)aminomethyl)pyridine-5′-
amine ligands preferentially formed strong H-bonding with the carboxyamidophenyl)porphyrin (11A) which undertook re-
Cu bound oxygen atom of the heme−(μ-O22−)−Cu(XTMPA) action with O2 in CH3CN at −30 °C (Figure 11).143
complex (10G, 10H, and 10I), which was evidenced by the Absorption spectra showed that bands at 428 and 534 nm
blue shift of the peroxo to Fe charge-transfer band in the 800− were replaced by new bands at 420, 557, and 612 nm
900 nm region compared to the parent LS−(DCHIm)− indicating the formation of a peroxo-bridged FeIII−O22−−CuII
(F8)FeIII−(O22−)−CuII(XTMPA) complex (10A) and the species (11B). This μ-peroxo complex could be crystallized
control samples LS−CH3TMPA (10E), LS−(CH3)2TMPA from its CH3CN solution and yielded dark purple crystals on
(10F). This was due to the fact that the H-bonding interaction prolonged standing at −30 °C, without decomposition. This
lowers the energy of peroxo π* donor orbitals more compared was the first reported crystal structure for a heme−Cu μ-
to Fe acceptor orbitals. Remarkably, these set of complexes peroxo complex, whose single-crystal X-ray analysis showed
allowed the detection of the ν(Cu−O) which was typically that the FeIII−O22−−CuII moiety has a μ−η2:η1 coordination;
unidentified in similar LS CuII−(μ-O22−)−FeIII complexes. both the oxygen atoms (O(2) and O(3)) of the peroxo ligand
The rR data indicated that with the increase in the steric factor bind to Fe(1), while only O(2) binds to Cu(1). The observed
(presence of ligand methyl substituents) the stretching O−O bond length was found to be 1.460 Å, with a Fe(1)−
frequency of the Fe−O and Cu−O bonds gradually decreased O(2)−Cu(1) bond angle of 166. In additon, the crystal data
(Table 1). However, in the case of H-bonding amine showed that the iron atom lies 0.595 Å above the porphyrin
plane. Solution EXAFS data and rR spectroscopy were
Table 1. Spectroscopic Properties of LS Heme−Peroxo consistent with the solid-state structure, the latter exhibiting
Complex with TMPA-Derived Cu-chelate142 an isotope-sensitive ν(O−O) at 790 (16O2)/746 (18O2) cm−1.
Raman 16O2 (cm−1) The Mossbauer spectrum of 11B at 77K (zero field) showed a
sharp quadrupole doublet with parameters (ΔEQ = 1.17 mm
(DCHIm)
F8FeIII−O22−− ν ν ν s−1, δ = 0.56 mm s−1) that are typical for an HS FeIII
CuII(L) UV−vis (nm) (O−O) (Fe−O) (Cu−O) ν2, ν4 compound and suggested that this was a peroxo bound
10A 426, 535, 850, 812 623 535 1572, iron(III) complexes. The ν(O−O) and high spin FeIII ground
950, 1020 1364 state of the heme iron observed in 11B adds credence to the
(br)
assignment of 10B discussed earlier based on its reported
10G 426, 536, 790, 775 625 533 1570,
900 (br) 1364 ν(O−O) and high spin FeIII ground state.143
10I 425, 538, 785, 760 614 506 1571, Apart from investigating O2 reactivity in homogeneous
897 (br) 1367 solutions, Karlin and co-workers also studied the electro-
10H 425, 533, 789, 765 610 510 1571, catalytic behavior of the Fe−Cu complexes that were
897 (br) 1367
synthesized with tetradentate Cu ligands (TMPA).109 Electro-
10E 422, 534, 855, 789 612 525 1570,
980 (br) 1363 catalysis was carried out over the EPG electrode in 0.1 M
10F 420, 538, 845, 776 593 501 1570, CF3COOH by physiadsorbing three different complexes:
940 (br) 1363 mononuclear porphyrin complex 6A along with the tethered
10B 429, 536, 840 806 573 489 1571, binuclear Fe−Cu complex, 6L−FeCu (7A), and the Cu free
(br) 1365 form of 7A, i.e., 6L−Fe with the Cu free tether. RDE and
10C 426, 535, 823 802 580 484 1571, subsequent K−L analysis showed that both 7A and its Cu free
(br) 1366
variant catalyzed oxygen reduction by 4e− to produce water,
10D 424, 539, 853, 805 575 483 1569, while 6A, the simple F8TPP−FeII-reduced oxygen primarily in
946 (br) 1365 the 2e− pathway produced H2O2. RRDE data quantitatively
showed that efficiency of 4e− reduction of 7A was 74%, while
substituents in 10H and 10I, there seemed to be no significant its Cu free analogue had 59% efficiency and was only 20% for
effect in the Fe−O and Cu−O bonds and only the O−O bond 6A. These data supported the prior results of Boitrel and co-
became significantly weak (Table 1). This weakening of the workers showing that the Cu ion might not be necessary for
O−O bond by the amine mediated H-bonding could help in the 4e− ORR; rather, it was the stability of the superoxide/
the heterolytic cleavage facilitating selective protonation of the peroxide intermediates formed during O2 reduction that
oxygen atom bound to Cu in these CuII−(μ-O22−)−FeIII mattered. In the Cu free form of 7A, the pyridyl groups of
complexes, which was also supported by DFT calculations. the empty distal tether were likely protonated in acidic 0.1 M
When amide functionalities (10B, 10C) were used instead of CF3COOH solution which stabilizes the negatively charged
their amine counter-parts, it distorted the local geometry to heme-superoxide species [(S)(F8TPP)Fe-(O2)], and hence, it
such an extent that H-bonding to the peroxo core could only showed higher 4e−/4H+ ORR selectivity. The role of Cu in 7A
impart a weak electronic effect despite the fact that amide was to do the same by stabilizing the heme superoxide leading
groups could provide much stronger H-bonds to the heme− to the formation of 7B and the corresponding μ-oxo species.144
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Figure 11. (A) CcO model complex studied by Naruta and co-workers. The side-on peroxo complex (B) formed by the reaction of (A) with O2
has a single-crystal XRD structure.143

Scheme 2. Proposed Mechanism of Homogeneous O2 Reduction by 6L−FeCu and Its Cu Free Form, 6L−Fe in Organic
solution (A) at rt and (B) at −60 °C27,a

a
Reproduced with permission from ref 27. Copyright 2011 PNAS.

The authors further established the role of distal Cu in ORR
mechanism by demonstrating selective and efficient (turnovers
1000) 4e− reduction of O2 to water using 7A and its Cu free
form as catalyst, where decamethylferrocene (Fc*) was used as
electron source and trifluoroacetic acid (TFA) as proton
donor. A detailed kinetic investigation revealed that at room
temperature (25 °C) O2 binding to heme (FeII) was the rate-
determining step for both 7A and its Cu-free form as was
evident from accumulation of the reduced heme−FeII species
with soret at 422 nm (Scheme 2A). The spectral changes
observed at −60 °C, were quite different from that at room
temperature. At −60 °C, the species observed under steady
state was assigned to be a FeIII−OOH species for both the
complexes. This (FeIII−OOH, CuII) species was characterized
using UV−vis absorption (415 and 538 nm), ESI-MS, and
EPR spectroscopy and could also be independently generated
by adding excess TFA to the EPR silent (FeIII−(O22−)−CuII)
12382
complex at −80 °C. The change in Soret band as a function of
the reaction temperature represented a change in the identity
of the steady state intermediate. The 422 nm band of the
reduced heme (FeII) in the steady state at room temperature
shifted to the 415 nm band representing the FeIII−OOH
species at low temperature. The O−O bond cleavage of this
newly observed FeIII−OOH species was assigned to be the
rate-determining step at −60 °C (Scheme 2B). At room
temperature, the rate of O2 binding to 6L−FeCu as measured
by the turnover frequency (TOF = 41 s−1) was almost twice
compared to 6L−Fe (TOF = 24 s−1), whereas the rate of O−O
bond cleavage of the FeIII−OOH species at −60 °C was same
for both 7A and its Cu free form. Therefore, Cu in the distal
site was proposed to enhance the rate of O2 binding during the
catalytic cycle, but it did not seem to contribute to the O−O
bond cleavage process for these model systems during
homogeneous catalytic ORR.27
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The mechanistic details of ORR by CcO model complexes
were mostly gained under single turnover conditions in organic
solvents and in the presence of a strong acid. The mechanistic
details in aqueous solvent (more relevant to the working
conditions of the natural enzyme) remained unexplored due to
a lack of direct spectroscopic evidence for any intermediate
involved. Dey et al. developed a setup where dynamic
electrochemistry is combined with a spectroscopic technique,
surface-enhanced resonance Raman (SERRS), which allows
direct in situ observation of intermediates under steady-state
conditions, and from that mechanistic insight of ORR
catalyzed by iron porphyrin can be obtained in aqueous
medium.145 In this technique, all those species whose rate of
formation is greater than the rate of decay get accumulated
over the electrode during ORR. The synthetic CcO model
complexes of Karlin and co-workers, 6L−FeCu (7A), its cu free
form (12A), and the imidazole adduct of 6L−FeCu (12B),
were used by Dey et al. for electrocatalytic ORR under
physiological conditions to understand the role of Cu and
imidazole and to detect the intermediates formed during ORR
(Figure 12).80
Figure 12. Synthetic CcO model complexes of Karlin et al. used for
electrocatalytic ORR in aqueous medium.80
RDE data of these three complexes and their subsequent K-
L analysis revealed that the selectivity and efficiency for
complete 4e− reduction of O2 over the EPG electrode
exhibited by 6L−Fe(12A) and binuclear 6L−FeCu (7A) were
higher (n = 4) than its imidiazole adduct (12B) (n = 3.4). The
second-order rate constants of ORR (kcat) were obtained as
(3.65 ± 1.1) × 106 M−1 s−1, (4.49 ± 0.9) × 106 M−1 s−1, and
(1.28 ± 0.11) × 106 M−1 s−1 for complexes 12A, 7A, and 12B,
respectively. It should be noted that the kcat value of 6L−FeCu
(7A) is 1 order of magnitude greater than synthetic heme−
copper complexes reported by Collman previously.28 Addi-
tionally, RRDE experiments provided quantitative detection of
PROS during ORR as well as its dependence with the variation
of ET rates from the electrodes. In order to control the ET rate
to the catalyst, self-assembled monolayers (SAM) of thiol-
modified Au electrodes were used as working electrode (as
previously demonstrated by Collman and Chidsey) by Dey and
co-workers, where the modification in the chain length of the
thiols can vary the ET rate from 105 s−1 to 10 s−1.123,127 RRDE
data of 7A and 12B over the EPG electrode, C8−SAM
(equivalent to fast SAM S2) modified Au electrode, and C16−
SAM (equivalent to slow SAM S1, used by Collman) modified
Au electrode suggested that the presence of an additional
redox center like Cu reduced the amount of PROS generation
as compared to the Fe-only analogue, and the effect was more
prominent under slow electron flux as has been reported by
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Collman.125 These complexes showed the general trend of
increasing the amount of PROS generation with a decrease in
ET rate as observed for other CcO models. However, the 6L−
Fe(Im)Cu complex showed higher 4e−/4H+ selectivity (4 ±
1% PROS only) under slow electron flux (C16−SAM)
compared to Fe−Cu (13.5 ± 1%) and Fe-only (23 ± 3%)
complexes which showed almost 94% selectivity for the 4e−
reduction of O2 to H2O under fast ET conditions over the
EPG electrode. Complex 12B followed the opposite trend, and
the amount of PROS was 25% over EPG, i.e., showed only
75% selectivity for the complete 4e−/4H+ reduction of O2 to
water. SERRS-RDE data of all the three complexes over the
C8−SAM-modified Ag electrode at cathodic ORR potential
revealed that both 6L−FeCu (7A) and 6L−Fe(Im)Cu (12B)
showed a LS−FeIII component as the major species under
steady-state conditions with ν4 and ν2 values (oxidation and
spin state marker band) at 1366 and 1567 cm−1 and at 1368
and 1569 cm−1 (red traces in Figure 13A,B along with their
Lorentzian fitted data in the right side of the spectra),
respectively. Apart from the LS−FeIII species, two minor
components HS−FeII denoted by 1349 cm−1 (ν4) and 1542
cm−1 (ν2) for 7A (Figure 13A, Lorentzian fit, green trace) and
1345 cm−1 (n4) and 1544 cm−1 (n2) for 12B (Figure 13B,
Lorentzian fit, green trace) and HS−FeIII denoted by 1359
cm−1(ν4) and 1555 cm−1 (ν2) for 7A (Figure 13A, Lorentzian
fit, purple trace) and 1357 cm−1 (ν4) and 1559 cm−1 (ν2) for
12B (Figure 13B, Lorentzian fit, purple trace) were also found
under the steady state. In contrast, complex 12A, the Fe-only
form, showed an HS−FeIII species as the major component
with ν4 and ν2 values at 1361 and 1556 cm−1 (Figure 13C, red
trace in the Lorentzian fit), while the minor components were
HS−FeII (green trace in 13C), LS−FeIII (sky blue trace in
13C), and FeIV�O (purple trace in 13C). SERRS-RDE data in
the low-frequency region indicated the formation of a bridging
peroxide intermediate [no H/D shift on ν(O−O)] during O2-
reduction by both complexes 7A and 12B under steady-state
reaction conditions, suggesting that O−O bond heterolysis was
likely to be the rate-determining step (rds) at the mass transfer
limited region. The O−O vibrational frequencies were at 819
cm−1 (759 cm−1 in 18O2 saturated pH 7 buffer) for the 6L−
FeCu complex and at 847 cm−1 (786 cm−1 in 18O2 saturated
pH 7 buffer) for the 6L−Fe(Im)Cu complex, indicating the
formation of side-on and end-on bridging peroxo intermedi-
ates, respectively (Figure 14A,B). Alternatively, in the case of
6
L−Fe, no such bridging peroxide was formed in the absence
of a Cu center. The peroxo adduct in the organic solution was
a HS FeIII−O22−−CuII compound having a ν(O−O) value 30
cm−1 less compared to the one in aqueous medium. The
binding mode of the bridging side-on peroxo to the Fe and Cu
centers was likely six coordinated LS μ−η1:η2 (η2 at Cu and η1
at Fe) in aqueous medium. These data suggested that side-on
bridging peroxide intermediates could indeed be involved in
fast and selective O2 reduction in these synthetic complexes.
Accumulation of end-on bridging peroxide during the ORR
mechanism by 12B indicated that at first O2 bound to the Cu
center, which went through 1e−/1H+ O2 reduction to produce
higher PROS under fast ET conditions (Scheme 3B). The
plausible mechanism of O2 reduction by complexes 7A and
12B is shown in Scheme 3A,B.80 These results clearly indicated
the proposals from Boitrel, Karlin, and Naruta on the likely
importance and involvement of a μ-peroxo species in O2
reduction by heme/Cu systems.
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Figure 13. SERRS-RDE data of (A) 6L−FeCu (complex 7A), (B) imidazole adduct of 6L−FeCu (complex 12B), and (C) iron only complex (12A)
in the high-frequency region.28

Figure 14. SERRS-RDE data in the low-frequency region under steady-state conditions in the presence of air and 18O2-saturated pH 7 buffer for
(A) 6L−FeCu (complex 7A) and (B) the imidazole adduct of a 6L−FeCu (complex 12B) on C8SH-modified Ag surfaces.28

2.2. Role of the Distal Phenol Group
In order to thoroughly investigate the role of the redox-active
tyrosine residue (Tyr244) in CcO for the selective and efficient
4e−/4H+ reduction of O2 to water, Karlin and Solomon used a
combination of experimental and theoretical methods on the
heme−peroxo−copper model complexes in the presence of
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externally added phenolic substrates.137,138,146 The reactivity of
two different low-spin bridging peroxo complexes, LS-
4DCHIm (15A) and LS-3DCHIm, toward O2 were compared
using different proton sources and Fc* as a reductant in an
organic solvent.138 In 15A, the weakly acidic 4-NO2−phenol
H-bonded with the Cu-bound Operoxo atom and with the
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Scheme 3. Proposed Mechanism of O2 Reduction in
Aqueous Medium by the Complex 7A (A)28 and by the
Complex 12B (B),28 Respectivelya
a
Reproduced from ref 28. Copyright 2015 American Chemical
Society.
addition of 2 equiv of Fc*, and the O−O bond was cleaved
yielding Fe(III) and Cu(II) as the final products. The kinetics
suggested that a transient ferryl species [(DCHIm)F8FeIV�O]
Figure 15. Overall proposed mechanism for the reaction of LS-4DCHIm (15A) with 4-NO2−phenol and Fc*.138
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was formed by a rate-limiting ET process to [LS-4DCMIm-
(ArOH)] which was then followed by another 1e−/1H+
reduction step at a rate 3 times faster than the earlier rate
(Figure 15). The activation of the O−O bond in the (15A +
PhOH) adduct, [LS-4DCHIm(ArOH)], was supported by the
rR data where ν(O−O) decreased from ∼870 to 827 cm−1
(>40 cm−1) on H-bonding and the corresponding ν(Fe−O)
increased slightly (∼10 cm−1) from ∼590 to 598 cm−1; i.e., the
O−O bond gets weakened and Fe−O bond strengthens. The
H-bonding interaction was also indicated in the DFT-
optimized structure of [LS-4DCHIm(ArOH)], and on the
basis of a combination of the experimental and theoretical
results, it was concluded that the observed shift (∼10 cm−1) of
the ν(Fe−O) mode was due to H-bonding of the PhOH with
the distal O atom of the FeIII−O2−CuII species as reported
previously for the same in heme metalloenzymes.147,148
Deuterated PhOD indicated a kinetic isotope effect (KIE) of
1.6 in the PhOH association step and 1.9 in the rds that is
quite lower than what is expected for phenolic H-atom transfer
(HAT), but it lies in the range of a PCET reaction149,150
confirming the involvement of phenolic O−H/O−D bond in
both H-bond association and rds. The analogous heme−
peroxo−copper complex having three monodentate DCHIm
groups around the copper, LS-3DCHIm, did not react with the
weakly acidic phenol due to steric hindrance as phenol would
not have access to the relatively compressed peroxo core for
the H-bonded adduct formation unlike 15A, which mainly
contributes to the difference in reactivity between these
complexes. In contrast, a relatively stronger acid like [DMF-
H+](OTf−) resulted in M−O bond cleavage via protonation to
the proximal oxygen atom with respect to the iron-releasing
H2O2 while reacting with both of these complexes. Thus, the
O−O bond homolysis was affected by the hydrogen-bonding
network of the properly oriented phenol group close to the
peroxo, which facilitated the PCET reaction (here, from 4-
NO2−phenol and Fc*, and in CcO the cross-linked Tyr
residue) in a fashion similar to the generally proposed CcO
enzymatic mechanism.57
In a related work, Solomon and Karlin groups established
the mechanism of O−O bond cleavage by the weakly acidic
redox-active phenol in a reaction with biomimetic low-spin
heme−peroxo−-copper complex, {[(DCHIm)(F8)−FeIII]−
(O22−)−[CuII(AN)]}+ (16A, AN = bis[(3-(dimethylamino)-
propyl)amine).137 This involved an H-atom transfer from
external electron-rich phenol (4-OMe-phenol) to the distal
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Figure 16. Two possible reaction mechanisms for phenol-induced O−O cleavage of 16A.137
oxygen atom of the bridging peroxide leading to the formation
of FeIV�O, CuII−OH, and phenoxyl radicals analogous to the
reaction mechanism of HCOs. Two different possible
mechanisms were considered: one involved the proton-
initiated pathway (PI), where the proton transfer from the
phenol occur before the barrier, and the other involved H-
bond initiated pathway (HB) where the O−O bond hemolysis
could be the predominant reaction with the phenol remaining
H-bonded to the Cu-bound Operoxo atom in the TS and PT
primarily occur after the barrier (Figure 16). In both
mechanisms, ET from phenol occurred after the PT (and
after the barrier) and the barrier of O−O bond cleavage was
only influenced by the interaction with the phenolic proton.
Furthermore, a KIE value of 1.7 was obtained, and the barrier
for this process (ΔG⧧expt) was estimated to be ∼15 kcal/mol
(at −70 °C), which is very similar to the theoretically
predicted value of HB mechanism exhibiting smaller secondary
KIE (<2 for the HB mechanism and >5 for the PI mechanism
independent of the functional used), therefore suggesting the
reaction of 16A with 4-OMePhOH proceeded via the H-bond-
assisted O−O homolysis mechanism.137 To understand the
relevance of such reaction (16A + PhOH), a computational
model of CcO active site was considered in which a cross-
linked tyrosine residue is widely known to participate in the
O−O cleavage step. It was determined that the active-site
tyrosine residue likely acts as the proton donor generating
tyrosinate which again provides an electron to favor the O−O
bond cleavage. It was proposed that in CcO the water
molecules present around the active site could interact with the
peroxide by means of PT or H-bonding interactions lowering
the barrier for O−O bond cleavage in a manner analogous to
the phenol in this model complex. The effect of PhOH in O2
reduction was also investigated later in detail in mononuclear
Iron porphyrins where phenol was covalently attached to the
porphyrin ring and is discussed in the next section.148
3. MONONUCLEAR METALLO-PORPHYRINS AS ORR
CATALYSTS
In order to design efficient molecular ORR catalysts as
alternatives of elegant but synthetically challenging binuclear
CcO mimics, which are known to retain high selectivity and
faster kinetics, the understanding of the oxygen reduction
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mechanism and the factors controlling the overall rate and
selectivity are crucial. In the case of complexes dissolved in
solutions, the intermediates involved in a reaction can be
investigated by trapping them at low temperatures and
interrogating them with spectroscopic methods. However,
mechanistic investigation of reaction catalyzed by surface-
adsorbed thin layer (often monolayers) of catalysts is
challenging as conventional spectroscopic techniques are not
amenable to such systems and the amount of sample is too low
to generate good signal. For this purpose, Dey et al. developed
SERRS-RDE technique which probes a system under steady
state while performing electrocatalytic ORR in aqueous
medium (Figure 17).145,153 To start with, simple mononuclear
Figure 17. Mononuclear iron porphyrins FeTPP(A) and FeEs4(B),
studied by Dey and co-workers in SERRS-RDE.145
iron porphyrin, FeTPP (17A), which does not have any distal
residue, was immobilized over a C8−SAM-modified Ag
electrode to carry out the mechanistic investigation along
with another mononuclear iron porphyrin, FeEs4 (17B), which
had a distal H-bonding cavity supported via triazole
substituents on the meso-phenyl rings of the TPP framework.
The high-frequency region of the SERRS-RDE data indicated a
high population of FeIII-LS species during the ORR (Figure
18A, red trace) compared to its data at 0 V (Figure 18A, blue
trace). The Lorentzian fit of the ν4 region obtained during
catalytic ORR showed a strong peak at 1369 cm−1 at an
applied potential of −0.50 V (Figure 18B), and the difference
spectra (reduced at −0.50 V - oxidized resting state) showed
that the resting FeIII−HS species (1360 cm−1) (Figure 18C,
yellow trace) was almost fully converted to FeIII−LS species at
steady state along with very small population of FeIV�O
species denoted by a vibration at 1371 cm−1 (Figure 18C,
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Figure 18. (A) Overlay of oxidized (blue at 0 V) and aerobically reduced (red at −0.5 V) SERRS data in the high-frequency region for FeEs4. (B)
Lorentzian ν4 fit of the aerobically reduced spectra (at −0.5 V) of FeEs4. (C) Difference spectra of the same (−0.5 V) to resting oxidized state along
with Lorentzian ν4 fit. All potentials are expressed with respect to Ag/AgCl (saturated KCl) reference electrode.145
brown trace). To obtain metal ligand stretches, labeling
experiments with 18O2 saturated pH 7 as well as pD 7
phosphate buffer solution was done. It was found in the low-
frequency region of the data for both 17A and 17B, a 830 cm−1
vibration gained intensity as the potential was gradually
lowered to cathodic potential where ORR took place (Figure
19A,C for 17B, data not shown for 17A). This 830 cm−1 band
was shifted to 782 cm−1 in 18O2 buffer (Figure 19B,D), which
was denoted as ν(O−O) of a LS FeIII−OOH species based on
previous literature reports.147,154 The corresponding ν(Fe−O)
of FeIII−OOH species were obtained at 631 cm−1 (ν16/18O2 =
37 cm−1) (see the difference spectrum at different potentials
from 16O2 to 18O2 in Figure 19E) and at 634 cm−1 (ν16/18O2 =
48 cm−1) (not shown) for 17B and 17A, respectively. The
ν(Fe−O) of high valent FeIV�O species were observed at 780
cm−1 (Figure 19A,C) (ν16/18O2 = 28 cm−1) and at 778 cm−1
for 17B and 17A, respectively. This spectro-electrochemical
data represented the first report of intermediates formed
during ORR by iron porphyrins on electrodes and revealed
that the accumulation of LS FeIII−OOH species dominated
under steady state, allowing more hydrolysis and larger PROS
production for porphyrins having no distal residues (FeTPP)
or porphyrins having hydrogen-bonding triazole residues
(FeEs4).145,155 Note that the accumulation of FeIV�O species
under steady state at cathodic potential is surprising as the
driving force for its reduction >1 V. An FeIV�O species was
also observed during O2 reduction by heme/Cu complexes
when Fc* was used as a reductant. This suggests that the
reduction of FeIV�O to FeIII−OH (a PCET step) is slow,
likely due to a large reorganization energy. The presence of
FeIV�O was evidenced later by using it to oxidize an external
substrate.
Different second-sphere interactions were investigated using
mononuclear metallo-porphyrins. These platforms are gen-
erally synthetically amenable, allowing the integration of
different types of second-sphere residues in the molecule
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which were then used to investigate O2 and H2O2 activation
and subsequent reactivity and are discussed below.
3.1. Role of Hydrophobicity and Steric Effect in the Distal
Pocket
Collman and co-workers first demonstrated reversible oxygen-
ation of myoglobin (Mb) and hemoglobin (Hb) by
introducing sterically hindered hydrophobic matrix over
porphyrin in the 1970s. They synthesized “Picket fence”
porphyrin (20A) which prevented the irreversible bimolecular
condensation of iron(II) porphyrins to form μ-oxo-bridged
dimer upon reacting with O2 (Figure 20).156 This was the first
instance where Fe−O2 adduct was trapped and characterized
to be a diamagnetic species. It was shown that oxygen binds
the Fe in an angular end-on fashion. This model complex, 20A,
has higher O2 binding affinity close to that of Hb and Mb. The
hydrophobic distal pocket in picket fence makes the redox-
active metal center accessible for the nonpolar diatomic oxygen
molecule.
H-bonding interaction in a hydrophobic environment is the
key to dioxygen activation in the natural enzyme Cyt P450, as
has been reflected in synthetic model systems by Naruta and
co-workers.157 The authors synthesized a unique ligand system
which contains a bulky binaphthyl-bridged porphyrin ligand
around the central Fe-atom which creates a hydrophobic
environment and pendant hydroxyl groups from the binaphthyl
moieties ensure secondary interaction.157 This system was
called single coronet porphyrin (20B), and using this, they
examined reactions with both dioxygen and carbon monoxide
by the help of flash photolysis. Surprisingly, the single coronet
had greater affinity toward oxygen than CO, whereas normal
heme centers have ∼20000 times more affinity toward CO.
This remarkable property is likely due to the hydrogen
bonding between the terminal oxygen atom of the dioxygen
molecule and the hydroxy groups attached to the binaphthyl
bridges in 20B. Ligand-iron bond strength and H-bonding was
investigated using IR and rR spectroscopy. Factors like polarity
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Figure 19. SERRS-RDE data of FeEs4 obtained at various potentials
under 16O2-containing (A) and 18O2-containing (B) pH 7 buffers. The
spectra obtained from the difference of various potentials from 0 V for
16
O2- and 18O2-containing buffers are shown in (C) and (D),
respectively. (E) Difference spectra of 18O2 from 16O2 obtained at
respective potentials. (F) Shifts obtained, along with their fits.145
could also be responsible for this discrimination between O2
and CO binding in enzymes as indicated by Naruta and co-
workers. The role of the hydroxyl group was further verified by
replacing hydroxy groups of the single coronet binaphthyl
bridge with methoxy group where CO affinity of the porphyrin
was found to be strong relative to O2 as expected.
For the last several decades, Co porphyrins have been largely
investigated as ORR electrocatalysts as these complexes usually
function at low ηORR in comparison with their analogous iron
Figure 20. Synthetic model complex FePf (A) and single coronet system (B) synthesized by Collman and Naruta et al., respectively.156,157
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porphyrins.99,158−161 The main drawback is that these Co-
containing catalysts generally tend to favor the two-electron
reduction of O2 to H2O2 except for the bimolecular
path.160,162−165 Therefore, several attempts have been made
successfully to date for the design of efficient and selective
mononuclear Co catalysts introducing hydrogen bonding,166
proton and electron transfer,167,168 electrostatic interactions,169
as these effects are found to be crucial for iron porphyrins to
exhibit 4e−/4H+ ORR.
In a recent work, Cao and co-workers reported electro-
chemical ORR with the four atropisomers of a cobalt−
porphyrin complex, each containing a bulky ο-amide
substituent on the meso-phenyl groups (Figure 21).170 They
Figure 21. Molecular structures of the four atropisomers of Co
porphyrins: αααα (A), αααβ (B), αβαβ (C), and ααββ (D).170
demonstrated a new approach for control over product
selectivity, i.e., 2e−/2H+ vs 4e−/4H+ ORR, by tuning the
steric effect of the different atropisomers. These complexes
were physiadsorbed over carbon black or CNTs and examined
for electrocatalytic O2 reduction in 0.1 M KOH solution under
aerobic condition. All the four atropisomers exhibited similar
ORR activity with the E1/2 values of around 0.73 V vs NHE but
diverse selectivity due to their dissimilar steric effect around
the redox-active Co center. The selectivity was determined
from RRDE and K−L analysis. The αβαβ isomer, 21C showed
high selectivity for 2e− ORR (n = 2.10) in the absence of any
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bimolecular path because of the large steric encumbrance on
both sides of the porphyrin ring. Without such a pronounced
steric effect in ααββ (21D) and αααβ isomers (21B), poor
selectivity was obtained for either 2e− or 4e− ORR with n =
2.89−3.10, which is commonly observed for most reputed Co
porphyrins. In contrast, the αααα isomer (21A) catalyzed the
4e−/4H+ ORR with high selectivity (n = 3.75) by improving
O2 binding inside the pocket as observed in UV and EPR data
even in the absence of any axial ligand to block the open side
of the porphyrin ring. Moreover, there is strong spectroscopic
evidence including IR and NEXAFS (K-edge near-edge X-ray
absorption fine structure) for the formation of stable Fe−O2
adduct in the αααα isomer, 21A. In FTIR, the changes in
ν(N−H) and ν(C�O) of amide functional group indicated
potential H-bonding interactions between the Fe−O2 adduct
and amide groups in its distal pocket resulting in stronger O2
binding affinity in 21A than the other three isomers. Therefore,
it was concluded that the hydrophobicity as well as steric effect
in the distal site employed by bulky tert-butyl groups covalently
attached to the porphyrin ring by the amide functionality can
strongly impact the higher O2 binding affinity to the metal
center (both Fe and Co) and result in stable Fe−O2 adduct
formation, which could enhance selectivity of ORR. Very
recently, Cao et al. further demonstrated that similar cobalt
porphyrins can be grafted over metal organic frameworks
(MOF) through ligand exchange. This porphyrin−MOF
hybrid synthetic assembly was shown to be a very reactive
and robust electrocatalyst for ORR having low overpotential
compared to simple Co porphyrins. This hybrid set up was
found to be more selective toward 4e−/4H+ ORR owing to
further reduction of H2O2 by active MOF surfaces, which
enabled it to be used successfully in Zn−air batteries.171
3.2. Role of Distal Water-Mediated H-Bonding
Investigation using biomimetic metallo-porphyrins with distal
superstructure has aided in a better understanding of the role
of distal structure in O2 binding. Collman et al. showed the
role of water present in the distal pocket of Fe-only CcO
model complexes during catalytic O2 reduction.126 Heteroge-
neous electrochemical analysis revealed that the PROS
production during O2 reduction sometimes depends on the
presence or absence of water cluster in the distal site of the
catalyst. Using the azide SAM-coated Au−Pt interdigitated
array electrodes (IDAs), this study demonstrated how the
factors that affect the rate of oxygen binding changed the
selectivity of ORR. Among the three complexes investigated
(Figure 22), 22B and 22C bound O2 quite rapidly with rates as
high as 107−108 M−1 s−1. The PROS produced for 22C was
much higher compared to 22B. On the contrary, the tris-
imidazole complex 22A bound O2 rather slowly, but it
produced almost half as much PROS compared to that of
the picket fence porphyrin, 22B. Collman et al. showed from
titration experiments that six water molecules were present
inside the triazole cavity of 22A among which two to three
water molecules were displaced during O2 binding. The
remaining water molecules form a stable H-bonding network
with the Fe−O2 species which was confirmed by FTIR
spectroscopy. Free superoxide is known to disproportionate at
high concentration in water, but at low concentrations they
could be spectroscopically identified as superoxide di-, tetra-,
or hexahydrate water clusters. Ab initio calculations also
supported this observation and indicated that the concen-
tration of superoxide-bound porphyrin was low for 22A and
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Figure 22. Synthetic model complexes (A−C) studied by Collman
and co-workers.126
the H-bonded array present in its superstructure (through H-
bonding with the nitrogen centers in the imidazole pendants)
increased the basicity of the water, which is required for the
stability of superoxide. This H-bonded array of the water
cluster was absent for picket fence 22B. For the complex 22C
where there is no distal residue, solvent access promoted the
hydrolysis of Fe−O2 instead of stabilizing it like 22A.
Therefore, PROS increased in these complexes from 22A to
22B to finally 22C, lowering the selectivity of 4e−/4H+
ORR.126 The H-bonded water cluster present in 22A which
increases the basicity of water as well as kinetic stability of the
superoxide or Fe−O2 species may also be operational in the
gas binding pocket of natural enzyme CcO.
Dey et al. synthesized a family of porphyrins with triazole-
containing distal superstructures having electron-withdrawing
and electron-donating groups. The tetra-triazole distal pocket
entrapped H2O molecules as evidenced by X-ray and FTIR
analysis. They showed the effect of H-bonding in both FeIII−
hydroxide and FeIII−superoxide complexes using electro-
chemical and spectroscopic data.172,173 In the low-frequency
region of rR spectra, an isotope-sensitive band in the region of
540−580 cm−1 was identified as the Fe−OH stretching
frequency for these set of complexes, which was found to be
574 cm−1 (544 cm−1 in deuterium) for FeFc4 (26A).When
electron-donating substituents were introduced in the triazole
ring as in the case of Fe(tBu)4 (23B) and Fe(PhOMe)4 (23C),
the Fe−OH stretching frequency of the hydroxide complex
decreased to 571 and 568 cm−1, respectively. In the case of
porphyrins having electron-withdrawing substituents in the
triazole ring like FeEs4 (17B) and Fe(PhCN)4 (23A), the
ν(Fe−O) was observed at somewhat higher values at 576
cm−1. For example, solution FTIR quantification indicated that
two water molecules were encapsulated inside the triazole
cavity in the distal structure which could form H-bonding with
the triazole N-atoms allowing the external ligand to bind Fe in
the sterically hindered side through H-bonding with the water
molecules (Figure 23, right). When electron-withdrawing
substituents were introduced in the triazole ring, the electron
density of the ring decreased resulting in weaker H-bonding
interactions with water molecules, which in turn increased the
H-bonding of the trapped water molecules with the distal
bound OH group; hence, the O−H bond strength decreased
and the Fe−O bond strength increased. This was further
verified with FePf (20A), where no H-bonding is present and
therefore the Fe−OH stretching was very weak and the
corresponding Fe−OH vibration was observed at 556 cm−1. As
the H-bonding to the bound axial hydroxide decreased, the π
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Figure 23. Synthetic model complexes (A−C) studied by Dey and co-workers.172

bonding between the dyz orbital of Fe and the 2p orbital of OH

ligand increased with the increase in the Fe−O bond strength,
which imparted more covalency in the Fe−O bond. As a result,
the hydroxide complex of FeEs4 with a stronger Fe−O bond
had a lower pKa value of 3.8, while for Fe(PhOMe)4 the pKa
value increased to 5.5 among the complexes investigated by
Dey at al. They also demonstrated the same effect of water-
mediated H-bonding in the Fe−O bond strength of the ferric
superoxide complexes of these porphyrins, measured in the rR
spectra.172−174 Here too. the ν(Fe−O) of the Fe−O2 species
shifted from 585 cm−1 in FeFc4 to 577 cm−1 in Fe(PhOMe)4
and the effect was mediated by competitive hydrogen bonding Figure 24. Representative structure for [Fe{porphyrin(pyH)4}(O2)·
between the Fe−O2 unit and the distal triazole for the H2O (H2O)2]4+ species with a water cluster as studied by Mayer et al.175
trapped in the cavity.
Mayer et al. reported soluble iron meso-tetra(pyridyl)- Table 2. Electrochemical ORR Data of 25A at Different
porphyrins as efficient electrocatalysts for ORR in acidic pH176
aqueous solution with the goal of affecting proton addition to
pH kcat (M−1 s−1) × 105 PROS (%)
the O2-derived ligand in iron center.175 They synthesized
FeIII−meso-tetra(2-pyridyl)porphyrin (29A), with the pyridyl 4 (59.8 ± 0.7) (2.52 ± 0.3)
nitrogens pointing inward, albeit far away from the iron center, 4.56 (19.9 ± 0.5) (1.82 ± 0.15)
4.67 (65.9 ± 1.2) (0.39 ± 0.4)
and compared its electrocatalytic behavior with 4-pyridyl
5 (27.4 ± 0.4) (2.09 ± 0.25)
isomer (29B) with the pyridines pointing outward. Higher
6 (6.09 ± 0.09) (3.86 ± 0.2)
selectivity toward 4H+/4e− ORR for the 2-pyridyl derivative
7 (1.85 ± 0.02) (5.01 ± 0.15)
compared to the 4-pyridyl isomer was proposed to originate
8 (29.0 ± 0.6) (3.03 ± 0.1)
mainly due to the differences in the proton delivery mechanism
9 (123 ± 11.0) (1.43 ± 0.2)
to the O2-derived intermediates formed during the catalytic
10 (121 ± 10.0) (0.03 ± 0.1)
cycle likely via the protonated pyridines in acidic aqueous
11 (169 ± 15.0) (0.03 ± 0.05)
medium. Computational models suggested that indeed the
protonated pyridine nitrogens were quite far from the [Fe−
O2] complex (pyH+···O = 3.80 Å). In the iron(II) complex increase in second order ORR rate (kcat) compared to neutral
[Fe{porphyrin(pyH)4}]4+, the pKa for the pyridinium groups pH, while almost 40-fold increase in kcat values was observed at
was computed to be about 4.4. Nonetheless, when more than low pH (Table 2). A plot of log kcat vs pH indicated a pKa
one H2O molecule was included between pyridinium ions and value of (5.6 ± 1) at low pH where rate enhancement occurred
bound O2, pyridinium ions seemed to organize them into a which matched with the pKa of phenanthroline (∼5) in water.
cluster. From DFT calculations, this water cluster mediated Therefore, at low pH the phenanthroline moieties were
system was supposed to help in proton delivery through H- protonated and behaved like protonated histidine at the active
bonding to the FeIII−OOH derivative (Figure 24). This was site of HRP which exerts a “pull effect” to the O−O bond of
likely the reason behind the higher 4H+/4e− ORR selectivity of the FeIII−OOH intermediate during ORR, increasing the ORR
these electrocatalysts, especially the 2-pyridyl isomer (29A). rate (Figure 25C). On the other hand, at alkaline pH the
Recently, a synthetic complex Fe−bisPhen (25A), where the putative (H2O)−FeIII−OOH intermediate was likely to be
proton relay along with water mediated H-bonding from distal deprotonated to become a trans OH− bound species. The pKa
structure to the Fe center of porphyrins facilitating ORR value of this change was estimated by them to be 7.9 ± 1. The
activity, was reported by Dey and co-workers.176 Taking OH− has a stronger push effect compared to neutral H2O
inspiration from the two distal residues (Arg, His) of HRP, molecule, which was proposed to enhance O−O heterolysis
complex 25A was synthesized where two 1,10-phenanthroline rates. Apart from the push effect from trans OH− ligand,
moieties above the porphyrin plane were envisaged. This was pyridine groups of phenanthroline directly H-bonds to the
shown to be a very effective and selective 4H+/4e− ORR axial ligands of metal center and through water-mediated H-
electrocatalyst when physiadsorbed over EPG electrode in bonding in aqueous medium. This simultaneous pull effect
aqueous medium throughout a pH range of 4−11. RRDE data from the distal site and push effect from the trans axial OH−
revealed that catalyst maintained >95% selectivity for water ligand might increase the polarity of O−O bond in FeIII−OOH
formation throughout a wide pH range (Table 2). RDE and species, which could explain the 100-fold ORR rate increase at
the subsequent K−L analysis in phosphate buffer solution alkaline pH. The “push−pull” mechanism resulted in a bell-
indicated that at high pH, 25A showed almost 100-fold shaped pH dependence against log kcat which is generally
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Figure 25. Single-crystal X-ray structure of Fe-bisPhen (A), plot of log (kcat) vs pH (B), and the proposed schematic representation of “O−O”
bond activation via “push” and “pull” effects at different pH by Fe−bisPhen (C) as reported by Dey and co-workers.176

typical of enzymatic active sites (Figure 25B). The direct porphyrin, namely, α4-FeFc4 (26A) (Figure 26).173 They
delivery of proton through water-mediated H-bonding or showed that α4-FeFc4 could reduce O2 under homogeneous
directly through phenanthroline nitrogen atoms to the distal
oxygen atom of FeIII−OOH species likely helped to increase
the 4H+/4e− ORR selectivity (Figure 25C).176

Table 3. Cobalt Porphyrins as ORR Catalysts under

Heterogeneous Conditions
catalyst medium Ecat/2 (mV) vs NHE PROS (%) ref
28A 0.5 M H2SO4a 455 29 165
28B 440 67 165
28C 365 51 165
28D 444 65 165
28E 400 61 165
Figure 26. Pictorial representation of the triazole moiety in the
28F 425 52 165 second sphere of porphyrin-based catalysts (A) α4-FeFc4 and (B) α4-
32A 1 M H2SO4b 440 73 163 FeFc1Es3.185
32B 495 30 163
32C 510 55 163
conditions in an organic solvent in the presence of acid and
32D 450 39−47 166
further could catalyze oxygen reduction with equal efficiency in
32E 480 30−35 166
aqueous solution under heterogeneous conditions over a pH
32F 430 5−13 166
a
range of 1−9. In heterogeneous aqueous medium when the ET
Catalysts physiadsorbed over MWCNT films dispersed on GC rate was very fast, i.e., over the EPG electrode, 26A reduced O2
electrode. bCatalysts physiadsorbed over EPG electrode. with >95% 4H+/4e− selectivity. Under moderate SAM (C8−
SAM with ET rate 103 s−1) and slow SAM (C16−SAM with ET
rate 10 s−1), α4-FeFc4 produced 2 ± 1 and 10 ± 1% PROS,
3.3. Role of Distal Redox Active Center respectively, during ORR to H2O (Table 4).123 They also
As discussed in the earlier sections, several metallo-enzymes in decreased the number of Fc groups in the distal structure of
nature which catalyze fundamental reactions like O2 activation, the porphyrin and synthesized α4-FeFc1Es3 (26B) and α4-
reduction to H2O (CcO and multicopper oxidases), O2 FeEs4 (17B) to evaluate this possibility. It was found that when
reduction, and organic substrate oxidation (Cyt. P450) or the number of Fc groups was reduced from 4 to 1 in the distal
nitrogen reduction (nitrogenases) require multiple protons and site, FeFc1Es3 produced 16 ± 1% and 42 ± 2% PROS,
electrons.17,114,177,178 The multiple electrons required for these respectively, over C8 and C16−SAM, while in the absence of all
reactions are generally not supplied by the substrate. Rather, the Fc groups, FeEs4 followed the 2H+/2e− pathway of ORR in
these are derived from ET sites; e.g., in CcO the electrons are the case of slow C16−SAM. Thus, under slow ET rates from
obtained from the redox-active centers present in distal site of the electrode, the hydrolysis of Fe−O2 or FeIII−OOH was
the heme.38,89,179−184 Using a click chemistry approach, the faster than the electron transfer rate from the electrode. When
Dey group incorporated four ferrocene (Fc) groups over the the Fc groups were present, the oxidation of Fc to Fc+ (E0 of
H-bonding triazole residues in the distal structure of Fc+/Fc0 couple in aqueous medium is 0.38 V vs Ag/AgCl)
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Table 4. Iron Porphyrins Used as ORR Catalysts under Heterogeneous Conditions

PROS analysis (%)
catalyst medium Ecat/2 (mV) vs Ag/AgCl kcat(×107) (M−1s−1) EPG C8-SAM C16-SAM Imz-SAM ref
22A pH 7 −30 0.51 × 10−2 10 >20 122
5C pH 7 −30 0.12 × 10−1 7.7 11 122
5B pH 7 5 122
12A pH 7 −50 0.36 6±1 9 ± 0.5 23 ± 3 78
7A pH 7 −80 0.45 6.3 ± 0.1 7 ± 0.1 13.5 ± 1 78
12B pH 7 −50 0.13 25 ± 2 10 ± 1 4±1 78
20A pH 7 10 ± 1 rapid degradation 170
26A pH 7 2 ± 1 10 170
17B pH 7 −145 0.50 × 10−2 15.6 ± 0.2 28.0 ± 4 100 10.0 ± 2.0 170
33B pH 7 −155 0.73 2.30 ± 0.20 6.15 ± 0.3 10.45 ± 0.9 193
33C pH 7 −103 1.80 1.25 ± 0.05 5.20 ± 0.3 4.90 ± 0.5 193
33D pH 7 −105 1.35 2.00 ± 0.10 5.10 ± 0.3 4.00 ± 0.5 2.9 ± 0.1 193
17A pH 7 −320 10 29 12.0 ± 1 193
33E pH 7 −196 0.42 2.54 ± 0.10 8.85 ± 0.1 18.0 ± 0.2 4.95 ± 0.4 194
33E-(N-Me)Imd pH 7 0.18 1.43 ± 0.2 3.10 ± 0.5 7.00 ± 0.1 2.90 ± 0.1 194
33E-(Me) pH 7 2.40 ± 0.3 5.43 ± 0.5 7.90 ± 0.1 2.64 ± 0.1 194
33E-(Ph) pH 7 1.80 ± 0.1 4.73 ± 0.5 9.20 ± 0.4 1.32 ± 0.1 194
31A 1 M H2SO4 60 1.4 22 ± 2 163
25A pH 7 −100 1.85 × 10−2 5.01 ± 0.2 172
27A pH 7 −160 0.19 3.3 ± 0.2 5.5 ± 0.6 3.3 ± 0.4 149
27B pH 7 −220 0.71 4.0 ± 0.4 3.2 ± 0.3 3.6 ± 0.4 149
27C pH 7 −240 5.31 1.8 ± 0.5 4.6 ± 0.2 10.2 ± 0.5 149

Scheme 4. (a) Proposed ORR Mechanistic Cycle in Heterogeneous Medium by α4-FeFc4 in the pH Range 1−9 Indicated by
Pathway A, at pH = 0, when the Triazole Rings Get Protonated by Pathway B Is Observed, while Pathway C Denotes an
Alternative 2e−/1H+ Pathway via Superoxide Protonation. (b) Proposed ORR Mechanistic Cycle in Homogeneous Organic
Medium by α4-FeFc4, in the Presence of 2-3 equiv of Triflic Acid Following Pathway A and Following Pathway B in the
Presence of Excess Acid, Where Triazoles Are Protonated185

during ORR maintained a rapid electron flow to the
intermediate Fe−O2 species formed during ORR, which
decreased their hydrolysis rate, promoting 4H+/4e− selectivity.
A plot of EPORR (peak potential of ORR catalytic current) vs
pH in aqueous medium showed a 28 mV slope for per unit pH
unit in the case of α4-FeFc4-catalyzed 4H+/4e− reduction of
O2, which indicated a 2e−/1H+ PCET step involvement in
ORR cycle. The proposed reaction pathway in heterogeneous
aqueous medium both in the pH 1−9 range and at pH = 0 is
shown in Scheme 4a. The triazole residues in the distal
environment having pKa ∼ 1.5 acted as a local buffer in the
vicinity in the O2 reducing iron porphyrin center, promoting
4H+/4e− selectivity, while at pH <1, triazoles were protonated,
promoting the hydrolysis pathway of decreasing selectivity of
4H+/4e− ORR.
12392
On the contrary, in spite of the presence of four redox-active
Fc groups in nonpolar organic solvent, in the absence of acid
source, 26A formed a Fe−O2 intermediate that led to the 1e−
reduction of O2 to O2− upon hydrolysis, which in turn
disproportionated to yield 48 ± 3% H2O2, detected by xylenol
orange assay of the resultant solution. This difference in
reactivity between aqueous and organic medium was due to
the shift in reduction potential of Fc+/Fc0 redox couple in
nonpolar organic medium to 0.73 V from 0.38 V in aqueous
medium. Note that in aqueous medium there is enhanced
solvation of the oxidized Fc+ ion, which enables the oxidation
of Fc centers at lower potential, allowing it to participate in a
PCET process during ORR, which was not possible in organic
medium. Alternatively, addition of 2−3 equiv of triflic acid to
the organic medium ensured the 4H+/4e− ORR to H2O by
26A. Three out of four Fc were oxidized to Fc+ (monitored by
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absorption spectra) in the process, and the other electron
needed to reduce O2 to H2O was derived from the iron in the
porphyrin ring. Thus, protonation of the ferric superoxide
species could cause oxidation of the Fc moieties resulting in
the 4e−/4H+ reduction of O2. The fact that the authors were
not able to trap any FeIII−OOH species or high-valent FeIV�
O species even at −80 °C suggested that the decay of FeIII−
OOH via oxidation of Fc was very fast. Only the final product
of ORR cycle FeIII−OH was observed in the EPR spectra at
−80 °C as indicated by a rhombic signal at g = 6 after addition
of 3 equiv of triflic acid to the Fe−O2 species. Thus, in organic
medium the Fe−O2 adduct underwent a proton transfer
followed by electro transfer (PTET) (Scheme 4b). However,
as shown by Dey et al., an excess equivalent of acid caused
hydrolysis of iron superoxide and increased the PROS
(Scheme 4). The proton delivery pathway to the Fe-bound
O2 species during the course of ORR by these porphyrins was
made possible by the fact that O2 bound to the site where
triazole moieties were present and not to the open side. This
was supported in the rR spectra at −80 °C of the Fe−O2
adduct of 26A showing a diamagnetic low-spin, six-coordinated
(CH3OH as trans ligand) Fe−O2 adduct with ν(Fe−O) at 585
cm−1 which shifted to 561 cm−1 in 18O2. If oxygen bound to
the open side, a S = 0, μ-oxo dimer having different
spectroscopic features would have formed.173,174,185
Recently, a covalently attached redox-active hydroxyquinone
group was introduced in the second sphere of iron porphyrin
(FeQH2, 27A) by Dey et al. to evaluate the reactivity of heme
ferric superoxide species toward H atom donors151 which may
also help to understand the mechanism of action of heme
dioxygenases like indoleamine 2,3 dioxygenase (IDO) and
tryptophan 2,3 dioxygenases (TDO) (Figure 27).186−188 The
Figure 27. Synthetic iron porphyrin complexes having hydroquinone
(FeQH2, A), phenol (FePh, B), and 2,5-dimethoxyphenyl rings
(FeQMe2, C) studied by Dey and co-workers.151
dioxygen adduct of reduced FeQH2 at first underwent an
intramolecular hydrogen atom transfer (HAT) reaction (kH/kD
= 4) from the attached hydroxyquinone to form FeIII−OOH,
and the hydroxyquinone was oxidized to form semiquinone
(SQ). The FeIII−OOH species was characterized by the
formation of 574 and 617 nm band in the UV absorption
spectra along with isotope sensitive ν(Fe−O) and ν(O−O) at
531 cm−1 (ν16/18O = 21) and at 783 cm−1 (ν16/18O = 35),
respectively, in the rR spectra. On the other hand, the
formation of SQ species was confirmed by the SQ vibrations at
834 and 816 cm−1 in the rR spectra, which shifted to 830 and
811 cm−1, respectively, on deuteration of the quinolinic group.
The LS FeIII−OOH[SQ] intermediate species, which resulted
in the simultaneous growth in the g = 2.38, 2.20, 1.90 (for
FeIII−OOH) and at g = 2.004 (for SQ) signal in the X-band
EPR underwent another intramolecular HAT (kH/kD = 2.2)
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from the semiquinone ring and decayed to form a FeIV�O
species with the concomitant formation of an oxidized
Quinone(Q). The appearance of ν(Fe�O) at 754 cm−1 in
the rR spectra, which was replaced by the Fermi doublet 729
and 698 cm−1 on using 18O2 and simultaneous decrease of the
above-mentioned two EPR signals clearly indicated the
formation of FeIV�O[Q]. RT FTIR data suggested that
after O2 addition to FeQH2 a quinone was formed as indicated
by a vibration at 1520 cm−1 instead of quinolinic band at 1316
cm−1 before O2 addition. The intermediate FeIV�O[Q] finally
decayed to FeIII−OH[Q] with a rhombically split axial g = 6
EPR signal (Scheme 5). Thus, out of the 4H+/4e− required for
ORR to produce H2O, 2e−/2H+ were obtained from iron (FeII
getting oxidized to FeIV) and remaining 2e−/2H+ were
supplied by the QH2 group appended to the porphyrin
ring.151 The [Fe−O2] species in synthetic analogues were
kinetically competent in abstracting HAT from QH2 with bond
dissociation free energy (BDFE) of 80 kcal/mol. However,
DFT calculations indicated that the H-bonding interaction
between ferric superoxide and hydroperoxide with −OH
groups of quinol played a vital role in this facile reactivity.
The effect of redox active quinol group (FeQH2, 27A) in the
second sphere was further compared with that of pendant
phenol (FePh, 27B) and 2,5-dimethoxyquinol derivative
(FeQMe2, 27C), and their O2 reduction activity was
investigated both in aqueous medium under heterogeneous
aqueous condition as well as in organic solution.152 RDE data
of these complexes over EPG electrode and their subsequent
K−L analysis revealed that all of these complexes 27A, 27B,
and 27C showed selective 4e−/4H+ ORR with second-order
rate constants of (7.09 ± 1) × 106 M−1 s−1, (1.97 ± 1) × 106
M−1 s−1, and (5.31 ± 2) × 107 M−1 s−1, respectively (Table 4).
Their corresponding cyclic voltammetry data over the EPG
electrode under anaerobic conditions as well as in the presence
of air-saturated pH 7 buffer solutions showed that for 27A and
27B E1/2 values of FeIII/II redox couples (−250 mV and −310
mV vs Ag/AgCl, respectively) matched with the EpORR
(defined as the peak potential of the ORR current) values,
indicating that reduction of FeIII to FeII was the most
thermodynamically uphill process in the catalytic cycle of
ORR for these two complexes. On the contrary, 27C showed
EPORR at 130 mV which was cathodically shifted from the
FeIII/II redox process, signifying that more driving force than
E1/2 was required for at least one step during the ORR. E1/2 of
the FeIII/II process and EpORR were further investigated at
different pH, and the pH dependence of E0 and EPORR had the
slope of 60 mV/pH for both 27A and 27C indicating a 1e−/
1H+ PCET for both. However, in the case of 27B, EPORR vs pH
had a slope of 30 mV/pH which was indicative of a 2e−/1H+
PCET step as the potential determining step of ORR.
RRDE data of these three complexes over the EPG electrode
(ET rate 106 s−1) agreed well with the K−L analysis of the
RDE data. These complexes followed the 4e−/4H+ ORR
pathway with only 4.0 ± 0.5, 3.3 ± 0.2, and 1.8 ± 0.5% PROS
for 27B, 27A, and 27C respectively. When the electron-transfer
rate was slowed down by using a SAM-attached Au electrode,
the PROS value remained almost similar to >95% selectivity
for 4e−/4H+ ORR in 27A and 27B. However, the PROS
increased gradually from 1.8% in EPG to 4.6% in C8−SAM,
and finally up to 10% (5-fold increase) over C16−SAM for 27C
(Table 4). Although these three complexes with second-sphere
distal residues showed preference for 4H+/4e− ORR and
almost similar rates, their difference in ORR onset potential
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Scheme 5. Proposed Mechanism of O2 Reduction by FeQH2 (27A) Mediated by Hydroquinone, Probed at −80 °C by Dey and
Co-workers151

Scheme 6. (A) Schematic Representation of a Mechanism of an ORR by FePh Involving a Disproportionation Reaction of
Ferric Superoxide Complex. (B) Schematic Representation of an ORR by FeQMe2 in Both Organic and Aqueous Medium152

and its pH dependence, selectivity (% PROS value) under slow
ET rate indicated that these three complexes followed different
mechanistic pathways to ORR. In the case of 27C, the
reduction of FeIII−O2, i.e., iron superoxide to hydroperoxide
step (FeIII−OOH), required an ET followed by a PT pathway
(ETPT) in aqueous medium. This was consistent with the
ORR onset potential being lower than the E1/2 of FeIII/II
process in aqueous medium (Scheme 6B). The pendant
methylated quinol group could restrain the hydrolysis of FeIII−
OOH species producing high 4H+/4e− selectivity under fast
ET flux (EPG). Rather, slow ET conditions (C16−SAM)
resulted in a higher %PROS for FeQMe2 (27C) as it required
12394
electrons to be supplied from electrode. The low reduction
potential of hydroquinone (∼0.46 V vs NHE) in neutral pH
allowed ORR by FeQH2 (27A) to progress via two consecutive
H atom transfer pathways (HAT) of the Fe−O2 species. Thus,
oxygen could be fully reduced to water once it bound to the
ferrous state to form Fe−O2 without any additional electron
from the external source as two electrons were supplied by the
ferrous porphyrin and another two electrons were supplied by
the distal hydroxyquinol group being converted to the FeIV�
O and quinone species, respectively, which mitigated the four
electrons required for selective 4e−/4H+ ORR (Scheme 5).
The FeIV�O and quinone formed during the electrochemical
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ORR might be reduced back to the ferrous quinol state on the

electrode at the potential where ORR was observed. Therefore,
27A should be intrinsically more selective toward 4e−/4H+
ORR even if ET rate from electrode was slowed. In organic
medium, consecutive HAT pathways during ORR involved
species like Fe−O2−QH2, FeIII−OOH−SQ, and FeIV−oxo−Q
intermediates which were all characterized using UV−vis, EPR,
and rR spectroscopy in organic medium, already discussed
previously (Scheme 5).151 On the other hand, phenol has a
higher reduction potential (∼0.8 V vs NHE) compared to
hydroquinone but has low pKa (pKa = 9.98), i.e., greater
acidity. This phenomenon helped FePh (27B) to make the
reduction of Fe−O2 to a FeIII−OOH species more facile via an
intramolecular PCET pathway, where proton was delivered Figure 28. (Left) Hangman carboxylic acid containing Co porphyrins
from the pendant phenol and the electron came from another (CoHPX) with substituted meso-phenyl groups (A−E). (Right)
Fe−O2 species present in solution (Scheme 6A). The DFT- Pentafluorotetraphenyl Co porphyrin [Co(C6H5)4] (F) studied as an
optimized structures of the reactant Fe−O2 and product FeIII− ORR catalyst by Nocera and co-workers.168
OOH of FeTPP, FePh, and FeQH2 showed that the O−O
bond length of Fe−O2 was shorter while that for FeIII−OOH
catalytic ORR activities were measured by RDE and RRDE
was longer for FeTPP relative to both FePh and FeQH2, which
techniques. The onset potentials of oxygen reduction varied
were responsible for the smaller displacement in O−O bond
between 550 and 600 mV vs NHE for these complexes, and
during the PCET process for the FePh/FeQH2 than FeTPP.
the peak maxima of the catalytic waves were found between
The calculated inner sphere reorganization energy (λinner‑sphere)
300 and 400 mV at low rotation rates. The number of
for a PCET process, which varied with the square of the
electrons (n) transferred to the oxygen reduction process was
displacement, became 3 kcal/mol lower in both FePh and
calculated by a K−L equation from the RDE data. The higher
FeQH2 than that of FeTPP making the process more favorable
amount of H2O2 production and number of electrons (n)
for the complexes containing pendant phenol/quinol groups involved in ORR for the series of Co porphyrins indicated that
capable of hydrogen-bonding interactions with the intermedi- with the shift in E1/2 of ORR toward a more cathodic potential
ate species. In addition, the prearranged favorable orientations (for the electron-rich substituents at the meso positions B, D,
of the second-sphere residues helped in H-bonding stabiliza- and E) the number of electrons (as obtained from K−L plot)
tion of the FeIII−OOH species to retain >95% selectivity for delivered to O2 decreased (Table 3). The results indicated that
the 4H+/4e− ORR even under slow ET flux from the electrode a competing 2e− reduction pathway was preferred at more
in neutral buffer medium. cathodic potentials (high ηORR). The CoHPX complex 28A
3.4. Role of Distal H-Bonding Interactions showed the highest selectivity (∼70%) for H2O production in
Natural heme enzymes utilize noncovalent secondary inter- the presence of electron-withdrawing pentafluorophenyl
actions like hydrogen bonding in the outer coordination sphere groups (C6F5). Moreover, the reactivities were compared
to stabilize O2 adducts as well as proton-transfer residues to with the electronically similar nonhangman analogue Co-
facilitate protonation of the intermediates involved in the (C6F5)4, 28F, which produced only 48% H2O. These
catalytic cycles of O2 activation and reduction. To understand experimental data suggested that the presence of hanging
the role of these hydrogen-bonding interactions and proton groups along with the electron-deficient porphyrin ligand
shuttles during the dioxygen activation in heme monoox- backbone might be important for the higher prevalence of H2O
ygenases, the challenging task of designing artificial metal- as the product of ORR.168 In particular, the intramolecular
loporphyrins with different hydrogen-bonding and proton- proton-transfer groups installed at the distal site were
transfer groups in the second coordination sphere had been demonstrated to assist facile O−O bond cleavage toward the
pursued and their reactivities were investigated for several 4e−/4H+ O2 reduction to form H2O.
years. Nocera and co-workers constructed a series of Another class of porphyrin, iron(III) meso-
(tetracarboxyphenyl)porphyrin chloride complexes (29C,
mononuclear cobalt hangman porphyrins functionalized with
29D) with ortho- and para-substituted pendant carboxylic
pendant carboxylic acid on a xanthene scaffold attached to the
acid groups, were examined for ORR by Mayer’s group (Figure
meso position of the porphyrin rings (Figure 28).189 The
29).12 The electrochemical oxygen reduction was carried out in
oxygen reduction properties of various meso substitution upon
organic medium (acetonitrile/DMF) in the presence of [DMF-
cobalt hangman porphyrins (CoHPX, A−E) was evaluated.168
H+][OTf−] acid source, and the 2-carboxyphenyl isomer 29C
The effect of the hanging group was evaluated in comparison
with a nonhangman analogue for the proton coupled
multielectron process in ORR, and the electronic properties
of the porphyrin macrocycle was also established with the
meso-phenyl substituents.
Electrochemical O 2 reduction was performed under
heterogeneous conditions in oxygen-saturated 0.5 M H2SO4
by immobilizing CoHPX (Figure 28A−E) on multiwall carbon
nanotubes (MWCNTs), and thin films were produced by
drop-casting CoHPX-MWCNTs solution on a glassy carbon Figure 29. Iron-porphyrin complexes (A−D) investigated as ORR
(GC) electrode which was used as the working electrode. The catalysts by Mayer and co-workers.12,175

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Table 5. Iron Porphyrins Used as ORR Catalysts under Homogeneous Conditions

catalyst solvent proton source E1/2 (mV) vs Fc+/Fc0 TOF (s−1) % H2O2 ref
+ −
17A DMF (DMF-H )Otf −530 2.7 × 101 <15 187
30A ACN (DMF-H+)Otf− −40 2.0 × 102 9 187
30A DMF (DMF-H+)Otf− −630 2.0 × 103 <15 187
30F DMF (DMF-H+)Otf− −486 1.5 × 101 <15 187
30B ACN (DMF-H+)Otf− −390 2.2 × 106 <15 187
30B DMF (DMF-H+)Otf− −611 2.5 × 103 <15 187
30C DMF (DMF-H+)Otf− −547 1.6 × 102 <15 187
30D DMF (DMF-H+)Otf- −536 1.8 × 102 <15 187
30E DMF (DMF-H+)Otf− −491 5.0 × 10° <15 187
30J ACN (DMF-H+)Otf− −326 6.5 × 104 <15 187
30I ACN (DMF-H+)Otf− −296 2.2 × 104 <15 187
30K ACN (DMF-H+)Otf− −280 2.2 × 102 <15 187
30G DMF (DMF-H+)Otf− −362 3.0 × 10° <15 187
29A water HOtf −250 vs NHE 6.0 × 102 5 171
29B water HOtf −150 NA 11−15 171
26A THF TFA NA NA 0 or 50 181
7A acetone TFA Me10Fc 4.0 × 101 minor pdt 27
12A acetone TFA Me10Fc 2.4 × 101 minor pdt 27
33B DMF (DMF-H+)Otf− −545 1.6 × 103 NA 193
33C DMF (DMF-H+)Otf− −502 3.96 × 103 NA 193
33D DMF (DMF-H+)Otf− −531 2.97 × 103 NA 193

Figure 30. (Left) Iron-porphyrin complexes used as ORR catalysts and (right) correlation of log(TOF) and effective overpotential (defined at
catalyst E1/2) for these catalysts.191

was found to be more selective toward 4e−/4H+ reduction of
O2 to water than the 4-carboxyphenyl isomer 29D. A
combination of techniques like RRDE and spectroelectro-
chemistry along with reactivity with H2O2 indicated the
formation of insignificant amounts of H2O2, and H2O was
established as the major product of oxygen reduction.
However, the 4-carboxyphenyl isomer did not show any
evidence for proton relay, likely because the carboxylic acid
pointing away from the iron center was a much less selective
catalyst. On the basis of a direct comparison between these two
isomers, the role of proton relay in the second coordination
sphere was proposed to be crucial to retain high selectivity
during ORR. Cyclic voltammetry (CV) measurements under
purely kinetic conditions at a high substrate/catalyst ratio and
low scan rate allowed the determination of TOF to be 200 s−1
for a soluble ORR electrocatalyst, 29C, for the first time.
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In addition to the iron porphyrin complexes 29C and 29D,
Mayer and co-workers compared the selectivity of oxygen
reduction with 2-pyridyl and 4-pyridyl derivatives (29A and
29B) of iron(III) meso-tetra(pyridyl)porphyrins as ORR
catalysts in acidic aqueous medium (0.25 M HCl/0.5 M
KCl) under homogeneous conditions.175 The RRDE data for
these two isomers revealed that 2-pyridyl-substituted chloride
complex produced no PROS (H2O2) and the triflate analogue
was 95% selective for 4e−/4H+ reduction of O2. The 4-pyridyl
chloride and triflate complexes produced almost 15% and 11%
H2O2, respectively, which was much higher than the 2-pyridyl
complex (Table 5). These water-soluble complexes showed
electrocatalytic oxygen reduction at reasonably high rates
(TOF ∼ 600 s−1). The onset potential of the 2-pyridyl
derivative, 29A, was 400 mV vs NHE, i.e., 800 mV more
negative than the thermodynamic potential of ORR. These
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pyridine moieties were envisaged to have a proton relay effect,
but the second sphere residues were far away from the metal
center as suggested by the pyH+−O bond distances (∼3.80 Å)
in the DFT-optimized structures of the Fe−O2 adduct.
To investigate O2 reduction with all of these complexes
under similar conditions (0.1 M HClO4), the iron(III) (meso-
tetraarylporphyrin) chloride complexes (29, A−D) were
immobilized on the electrode surfaces using three conventional
methods�(i) solution of catalyst in 0.5% nafion, drop casted
onto a GC working electrode (nafion membrane), (ii) a
suspension of catalyst solution in 0.5% Nafion with 2.5 mg/mL
vulcan carbon additive drop casted on GC (nafion/carbon),
and (iii) catalysts physiosorbed on edge-plane graphite
electrodes (EPG conditions)�and RRDE was performed in
order to determine the % H2O2 produced and selectivity of O2
reduction.190 The difference in reactivity upon changing the
medium indicated that the selectivity for O2 reduction was
influenced by the nature of the catalyst film on electrode,
although this conclusion needs spectroscopic validation of the
same kind. Nonetheless, the comparison among the four
catalysts in two isomeric forms clearly suggested that a
properly positioned proton relay (2-substituents) showed
better selectivity for the 4e−/4H+ pathway under similar
conditions. Since there were four potential relays in this set of
complexes, the effect of individual or single such group might
be indiscernible.
Later, Mayer and co-workers demonstrated that the
installation of such acid/base functional groups on meso-
phenyl rings of 30A and 30G had a negligible effect on the
turnover frequencies (TOFs) of the ORR. Rather, a correlation
was obtained for the TOF vs ηORR under homogeneous
conditions which could be regulated with the E1/2 of the
catalysts in a series of iron porphyrins by changing the solvent
(DMF, ACN) and acid [DMF-H+] concentrations as well
(Table 5).191 TOF was dependent on the O2 binding
equilibrium, rate-limiting protonation of ferric superoxide
intermediate (Fe−O2) instead of any electrochemical step.
These results were in agreement with the mechanism proposed
for FeFc4 by the Dey group. The reported TOFs vary over a
wide range (100−106) s−1 for these complexes, and the
log(TOF) vs ηORR correlations obtained under different
experimental conditions were linear (Figure 30, right). The
linear scaling relationship was analyzed on the basis of
computational studies where the free energy of O2 binding,
pKO2, and protonation of (Fe−O2) species were found to be
directly related to the reduction potential of the catalyst. The
electron-donating substituent shifted the E1/2 of the FeIII/II
redox couple to a more negative value by increasing the O2
binding affinity and the pKa of ferric superoxide that eventually
led to the linear increase in TOF, and the presence of an
electron-withdrawing substituent showed the opposite trend.
This might likely be the origin of overpotential correlations.
The 2-CO2H isomer (30A) in DMF exhibited 100 times
higher TOF than the 4-CO2H isomer (30F) at an extent of
∼140 mV higher ηORR. Again, catalyst 30A and its 2-CO2Me
derivative 30B showed the highest TOFs reported so far, which
were also very close in both ACN and DMF medium due to
their high ηORR having almost similar redox potentials (E1/2),
despite the fact that only 30A appeared to have proton relays.
However, the 2-pyridine-substituted catalyst 30G might exist
in some unique feature other than any other complex in the
series in DMF and followed the inverse trend of TOF vs acid
concentration. This was likely because of the protonation of
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the 2-pyridyl substituents upon addition of [DMF-H+] that
also shifted the E1/2 of FeIII/II to more positive values. As a
consequence, TOF for 30G decreased with an increase in the
amount of [DMF-H+]. The presence of potential proton relays
did not displace the pKO2 and the pKa of the catalyst from the
straight lines substantially, although that might suggest poor
proton translocation.
A series of iron(III) tetra(aryl)porphyrins (31, A−C) where
three meso positions bear a phenyl group and the remaining
meso position was modified by either a 2-pyridyl, 2-benzoic
acid, or 2-hydroxyphenyl group and were examined for oxygen
reduction by Warren and co-workers.192 The oxygen reduction
reaction was performed by drop-casting the catalysts onto BPG
and EPG electrodes under heterogeneous condition in acidic
aqueous medium (O2-saturated 1 M H2SO4). The incorpo-
ration of a single acid/base group at the meso position caused
significant improvement in electrocatalytic ORR activity
relative to the parent iron tetraphenylporphyrin (FeTPP).
The acidic pH was needed to confirm the protonation of the
functional groups that is essential for exhibiting a proton relay
effect.
All three complexes (Figure 31) showed oxygen reduction
with onset around 400 mV vs NHE, like Mayer’s
Figure 31. Asymmetric iron(III) tetra(aryl)porphyrins studied by
Warren and co-workers.192
Figure 32. Asymmetric cobalt(II) tetra(aryl)porphyrins studied by
Warren and co-workers.166,169
complexes,12,175 and these were comparatively stable even
after repeated voltammetry scans. The ηORR values for all these
complexes lie between 800 mV and 1 V, which were quite high
similar to the tetrapyridyl-substituted derivatives.175 Longer
steady current densities of these iron porphyrins, relative to
FeTPP (17A), indicated higher catalyst stability. Of these, the
current response for FeTPPy (31A) degraded by only 50%
during prolonged electrocatalysis and retained a stable current
density of ≥1 mA/cm2, suggesting that the catalyst was not
only more active but also more stable. In aqueous acidic
solutions, the pyridine group having lower pKa (pKa = 5.2)
might be a more suitable acid than phenol (pKa = 10)193,194 in
the absorbed catalyst layer on the electrode surface for
promoting proton relay. These results suggested that the iron
porphyrins functionalized with pyridine/carboxylic acids (31A
and 31C) could shuttle protons to the iron−oxygen
intermediates and enhanced stability of the molecular catalysts
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Figure 33. Synthetic iron porphyrins containing pendant bases in the second coordination sphere [(A) FeMPh, (B) FeL1, (C) FeL2, (D) FeL3,
and (E) Fe-MARG] studied by Dey and co-workers.197,198
immobilized onto the electrode surface by noncovalent
interactions.
The electrochemical properties of analogous CoTPPy, 32B,
immobilized on EPG were explored in 1 M H2SO4 under 1
atm O2 using a combination of techniques like CV, RDE, and
RRDE experiments.166 The presence of a single 2-pyridyl
group in 32B improved the O2 reduction kinetics and changed
the selectivity to 4e−/4H+ reduction (H2O) like those reported
by Anson in the 1990s,23,158,159 as opposed to 2e− reduction
(H2O2) commonly associated with unfunctionalized cobalt
porphyrins. The ORR onset potential was in between 600−700
mV which was about 50 mV higher than Nocera’s Co-
hangman porphyrins.168 No decay was observed in the
catalytic current during the first 1.5 h of electrolysis implying
that the catalyst to be as stable as 31A.192 The value of n
calculated from the RRDE experiment for the CoTPPy (32B)
adsorbed on EPG was 3.51 ± 0.02 (∼70 ± 3% H2O) at 0.4 V
applied potential (ηORR = 0.83 V), whereas unfunctionalized
CoTPP (32A) showed n = 2.6 ± 0.1 (∼27 ± 3% H2O) under
identical conditions (Table 3). The 70% selectivity for ORR to
H2O is similar to the selectivity exhibited by CoHPX (28A)
from Nocera. However, RRDE analysis of analogous iron
porphyrins provided n = 3.6 (∼80% H2O) for both 31A and
17A at 0.1 V applied potential (ηORR = 1.1 V). For the Fe
complexes investigated, the 2-pyridyl group did not change
selectivity but greatly improved stability. The observed
increase in selectivity for 4e−/4H+ ORR in 32B versus 32A
was specifically ascribed to the 2-pyridyl group and more
generally due to the presence of a proton donor or H-bonding
group. The presence of the 2-hydroxyphenyl group in complex
32C resulted in greater selectivity for the 4e− reduction of O2
to H2O relative to 32A but not as much as that of the pyridyl
group, 32B. At 0.50 V applied potential (ηORR = 0.73 V), the
calculated yield of H2O for 32C was almost half compared to
that for 32B. As is the case with the Mayer complexes,175 the 2-
pyridine group was unlikely to interact directly as a proton
relay with the active intermediates, although the pH-dependent
RRDE experiment showed that the protonated pyridyl group
could significantly affect the selectivity for H2O vs H2O2.
However, it should be noted that the Co porphyrins
functionalized with para-substituted analogues always pro-
duced higher H2O2 under similar experimental condi-
tions.23,195 The kinetics of ORR in aqueous medium (1 M
H2SO4) by 32B and 31A were also evaluated using the
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intercepts from K−L plots constructed from plateau currents
of the RDE data. The second-order catalytic rate constants
(kcat) for CoTPPy (32B) and FeTPPy (31A) were calculated
to be 2.9 × 107 and 1.4 × 107 M−1 s−1, respectively, implying
that the former achieved two times higher kcat at 230 mV lower
ηORR at pH 0 and these kcat values were much higher than
simple cobalt porphyrins196 and also very similar to those
obtained for iron porphyrins having basic functional groups,
reported earlier.197
To facilitate the rate-determining O−O bond heterolysis of
a FeIII−OOH species, as determined by in situ SERRS-RDE
formed during heterogeneous ORR, Dey and co-workers
incorporated pendant bases of different pKa values in a series of
simple mononuclear iron porphyrin complexes (Figure 33)
which were rationally designed, inspired by HRP, to achieve
efficient O−O bond activation and site-selective proton
transfer to effect facile and selective electrochemical reduction
of O2 to water.197,198 The complexes FeL1, FeL2, FeL3, and
Fe-MARG (Figure 33B−E) containing N,N-dibenzylaniline,
pyridine, and aliphatic primary and guanidine groups,
respectively, were physiadsorbed on EPG electrode and
analyzed for electrochemical ORR in neutral aqueous buffer
(pH = 7) medium.
All these catalysts showed electrocatalytic O2 reduction in
neutral pH 7 buffer with a substrate diffusion limited current at
an onset of −100 mV vs Ag/AgCl for FeL2 (33C), FeL3
(33D), and Fe-MARG (33E). The kinetics and selectivity of
O2 reduction were determined using K−L analysis from the
RDE data, and the kcat values obtained on the EPG electrode
surface for 33B, 33C, 33D, and 33E were (7.28 ± 0.02) × 106
M−1 s−1, (1.80 ± 0.07) × 107 M−1 s−1, (1.35 ± 0.09) × 107
M−1 s−1, and (4.2 ± 0.05) × 106 M−1 s−1, respectively (Table
4). The measured kcat values were >106 M−1 s−1, 1 to 2 orders
of magnitude higher than the values reported for unfunction-
alized iron−porphyrin complexes,174 heme/Cu model com-
plexes of Cc,O80,199,200 and similar to those reported for
sophisticated biosynthetic scaffolds.201 However, the role of
the pendant amines in enhancing the stability of FeIII−OOH
species against hydrolysis and subsequent PROS generation
was best evaluated under slow ET rates as established by
Collman and Chidsey.125,202
In an RRDE experiment, the amount of PROS produced was
quantified on the EPG working electrode as well as the Au
electrode surface attached to SAM of thiols having different
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Figure 34. (A) SERRS-RDE data of complex FeL3, 33D, over SAM-coated Ag electrode in the high-frequency region at oxidized (blue) and
steady-state conditions (red at −0.4 V, green at −0.5 V) in air-saturated pH 7 buffer under aerobic condition. The difference spectra from −0.5 to 0
V potential along with Lorentzian ν4 fits and Lorentzian ν2 fits showing different components are given in (B) and (C), respectively. All potentials
are expressed with respect to Ag/AgCl (saturated KCl) reference electrode.204

chain lengths resulting in differing the rate of ET from the
electrode to the catalyst.123,125,203 As the kET was lowered by
104−6 times from EPG (fast ET transfer) to C16−SAM ((slow
ET transfer flux), the percentage of ROS generated also
increased from 2% to 15% in these complexes (Table 4) which
indicated that the selectivity for 4e−/4H+ O2 reduction was
mostly retained even under slow ET conditions. Thus, the
selectivity exhibited by this series of complexes was in stark
contrast to all of the previous reports of mononuclear iron
porphyrin complexes (e.g., FeTPP, FePf, FeEs4) without any
additional redox centers (e.g., distal Cu, Fc, or phenol), which
produced about 50−100% PROS in C16−SAM (Table 4),
which often led to rapid catalyst degradation.174,202 These iron
porphyrins were also chemically attached to the ImdC11SH-
functionalized Au electrode using a mixed thiol SAM
combining a linker ImdC11SH and a diluent C8SH (Imz
SAM), where the imidazole terminal of the SAM acted as an
axial ligand to produce “push” effect to the attached catalyst
during O2 activation.198 The RRDE data indicated that Imz
SAM bound 33D and 33E recorded only 2.9 ± 0.1 and 4.9 ±
0.1% PROS, respectively (Table 4), which were almost half of
the PROS produced in their respective C8−SAM though the
ET rate (103 s−1) was the same in both cases. These data
clearly suggested that the combination of “push” of imidazole
and “pull” of amine/guanidine functional group were very
effective in allowing very selective ORR.204 These four
mononuclear porphyrin complexes were over 95% selective
for 4e−/4H+ O2 reduction to H2O like the FeCu CcO models.
Despite having a suitably positioned H-bonding guanidine
moiety in the distal superstructure as in HRP, Fe-MARG
(33E) produced 18% PROS over C16SH-modified Au
electrode which was higher relative to other porphyrins
12399
(33C, 33D) having pendant pyridine and primary amine
moieties. Two structural variants of 33E, o-
monomethylguanidinotetraphenyliron(III)−porphyrin (Fe-
MeMARG), and o-monophenylguanidinotetraphenyliron-
(III)−porphyrin (Fe-PhMARG) were synthesized, and their
reactivities were compared with 33E.198 The 1H NMR spectra
of the free ligand Me-MARG and Ph-MARG showed that the
resonances for the methyl CH’s of the guanidine were at 1.35
ppm and that for the aromatic CH’s of guanidine were at 5.8,
6.2, and 6.4 ppm respectively. This data demonstrated that
both methyl and phenyl protons were shielded by the aromatic
porphyrin groups, which could only happen if the methyl and
phenyl groups were poised just on top of the porphyrin ring.
This phenomenon made the distal site much more hydro-
phobic, having an impact in the selectivity of 4H+/4e− oxygen
reduction. The % PROS value for Fe-MeMARG and Fe-
PhMARG dropped to 7.9 ± 0.1% and 9.2 ± 0.4% over the
C16−SAM-modified Au electrode, respectively, which further
dropped to 2.6 ± 0.1% and 1.3 ± 0.1% over the Imz SAM-
modified Au electrode, respectively (Table 4). Nonetheless,
the in situ mechanistic investigation seemed necessary to shed
light into the differences in selectivity observed for the
complexes 33D and 33E.
In subsequent work, Dey et al. employed the previously
developed SERRS-RDE approach to find out the mechanism of
ORR at neutral pH, catalyzed by the iron porphyrins
immobilized over the SAM-coated electrode.204 In the case
of FeL3 (33D), i.e., the porphyrin with a distal amine group,
two intermediate species were accumulated during the catalytic
turnover while the resting state of the SERRS data over C8−
SAM showed the presence of a HS FeIII species (Figure 34A).
The difference spectra (data at −0.5 V potential, resting
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Figure 35. SERRS-RDE data of complex FeL3, 33D, over SAM-coated Ag electrode (A) in the low-frequency region at oxidized (blue) and at
steady-state conditions (red, at −0.4 V) in air-saturated pH 7 buffer. (B) Overlay of the difference spectra from −0.4 to 0 V in 16O2 and 18O2. (C)
Overlay of the difference spectra from −0.4 to 0 V in air-saturated pH 7 and pD 7 buffer, respectively. * represents the characteristic phosphate
buffer peak.204

Figure 36. (A) SERRS-RDE data of complex Fe-MARG, 33E, over C8-SAM in the high-frequency region at oxidized (blue at 0 V) and steady-state
conditions (red at −0.3 V, green at −0.5 V) in air-saturated pH 7 buffer under aerobic conditions. The difference spectra from −0.5 to 0 V
potential along with Lorentzian ν4 fits and ν2 fits showing different components are given in (B) and (C), respectively. * represents the plasma
line.204

oxidized) confirmed that the major species under steady state
was a LS FeII species having a ν4 band at 1356 cm−1 and a ν2
band (characteristic of the spin state marker) at 1547 cm−1
(Figure 34B,C, green line) and another minor LS FeIII species
12400
with ν4 and ν2 bands at 1366 and 1563 cm−1 (Figure 34B,C,
brown line), respectively, was also observed. In the low
frequency region, a new band gained intensity at 824 cm−1 in
16
O2 saturated pH 7 buffer during ORR (Figure 35A), and this
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Figure 37. SERRS-RDE data of complex Fe-MARG, 33E, over C8-SAM (A) in the low-frequency region at oxidized (blue) and at steady-state
conditions (red at −0.3 V, green at −0.5 V) in air-saturated pH 7 buffer. (B) Overlay of the data in 16O2- and 18O2-saturated buffer and (C) overlay
of the data in air-saturated pH 7 and pD 7 buffer, respectively. * represents the characteristic phosphate buffer peak.204

band was reproducibly shifted to 765 cm−1 in 18O2 saturated
buffer and to 815 cm−1 in pD7 buffer (Figure 35B,C). The 824
cm−1 vibrations, with ν16/18O2 = 59, were thus identified as the
ν(O−O), originating from a LS FeIII−OOH species in
agreement with the ν4 and ν2 bands from the high-frequency
region. Unfortunately, a further low-frequency region of the
spectrum did not show the corresponding Fe−O vibration.
Judging by the intensities of the observed vibrations, it was
concluded that although LS−FeIII−OOH species were
accumulated over the electrode under catalytic turnover for
33D, the population of the species was substantially lower
compared to that of 17A and 17B. The low population of the
FeIII−OOH species likely jeopardized the identification of the
weak ν(Fe−O) stretch in SERRS-RDE under steady-state
conditions for 33D.
Alternatively, Fe-MARG (33E) showed substantially differ-
ent spectra containing a mixture of species in the SERRS-RDE
data under steady state when the resting state comprised only
HS FeIII species (ν4 at 1362 cm−1 and ν2 at 1554 cm−1)
(Figure 36A). The ν4 and ν2 marker bands in the difference
spectra showed the development of a HS FeII species as the
major component havinga ν4 band at 1346 cm−1 and ν2 band
at 1542 cm−1 along with a minor population of high valent
FeIV�O species with ν4 and ν2 bands at 1372 and 1569 cm−1,
respectively, under catalytic turnover conditions (Figure
36B,C). In the low-frequency region, a gradual increase of an
820 cm−1 vibration was observed when the cathodic potential
was applied in an air-saturated pH 7 buffer (Figure 37A). Data
collected in the 18O2-saturated pH 7 buffer indicated that the
820 cm−1 band in 16O2 was shifted to 798 cm−1 in 18O2 (Figure
37B). This vibration was assigned as the ν(Fe−O) of a FeIV�
O species, which was also suggested by the ν4 and ν2 bands at
1372 and 1569 cm−1. The experimentally observed ν16/18O2
12401
shift of 22 cm−1 was slightly less for 33E compared to that of
the theoretical value (36 cm−1) owing to the fact that Fe−O
vibration in FeIV�O species is coupled to other out-of-plane
motions of the porphyrin ring. Again, an 3 cm−1 H/D shift
from 820 to 817 cm−1 was also observed for this ν(Fe−O) of
33E in pD 7 phosphate buffer (Figure 37C), which suggested
that the FeIV�O unit might be hydrogen bonded to the
pendant protonated guanidine of Fe-MARG. Therefore,
spectro-electrochemical analysis revealed that for 33E on
C8−SAM the LS FeIII−OOH intermediate was not observed at
all and only high valent FeIV�O and unreacted FeII species
were accumulated under steady state.
The results obtained using the SERRS-RDE technique
clearly revealed that simple mononuclear iron porphyrins
showed the accumulation of an LS FeIII−OOH species, which
in turn was prone to hydrolysis and subsequently produced
more PROS during the ORR catalytic cycle.145,204 The
production of PROS swayed the selectivity toward 2e−/2H+
ORR. On the contrary, the presence of pendant hydrogen-
bonding groups in iron porphyrins which stayed protonated at
neutral pHs significantly reduced the population of the rate-
determining LS FeIII−OOH species under steady state for 33D
and eliminated the accumulation of LS FeIII−OOH species for
33E. This phenomenon could be better explained based on the
faster heterolytic cleavage of the O−O bond of FeIII−OOH
species which was directly correlated with the enhanced ORR
rates and 4H+/4e− selectivity in these complexes.
Homogeneous ORR electrocatalysis were also investigated
for 33B, 33C, and 33D in the presence of strong acid, [DMF-
H+]OTf−, in O2-saturated DMF solution.197 The first-order
rate constants of O2 reduction (TOF) were quantified using
the foot-of-the-wave analysis, and those kcat values for 33C
(FeL2) and 33D (FeL3) were significantly higher at the same
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Figure 38. Optimized structures of six-coordinate low-spin (A) [FeIIITPP-OOH]; (B) [FeIIIL1-OOH]H+; (C) [FeIIIL2-OOH]H+; (D) [FeIIIL3-
OOH]H+; (E) [FeIIIMARG-OOH]H+ with hydroperoxide and water as axial ligands and protonated distal basic residues in PCM model
considering water as solvent. Color codes: carbon, gray; hydrogen, white; nitrogen, blue; oxygen, red; and iron, bluish gray.197

Figure 39. DFT-optimized structures of water-bound LS ferric hydroperoxide (FeIII−OOH) species for both FeL3 and Fe-MARG. (A) H-bonded
geometry with a distal oxygen atom is energetically more stable by 2.35 kcal/mol than the structure where H-bonding is primarily with the proximal
oxygen atom for FeL3. (B) H-bonded geometry where H-bonding is primarily with the proximal oxygen atom is energetically more stable by 1.70
kcal/mol than the structure where H-bonding is with the distal oxygen atom for Fe-MARG.204

ηORR with respect to the other catalysts (Table 5). Thus, the
iron porphyrins having distal basic groups (33C, 33D) slightly
deviated from the linear correlation of log(TOF) vs ηORR as
demonstrated by Mayer. Hence, the enhancement of rates at a
particular ηORR observed here reflected other factors than solely
the primary coordination sphere.
DFT calculations were performed to understand the effect of
hydrogen-bonding interactions with the basic groups for the
spectroscopically observed models of low spin ferric super-
oxides (FeIII−O2) and ferric hydroperoxides (FeIII−OOH)
with water as axial ligand. These amine groups have pKa very
12402
close to or higher than 7 and were expected to remain
protonated at pH 7.0 buffer or in acidic DMF.194 The
theoretically determined absolute pKa values of these pendant
bases of ferric hydroperoxide species in water were found to be
higher by 4−5 units than that of free bases because of strong
hydrogen-bonding interactions with the distal oxygen atom of
the bound superoxide or hydroperoxide anion.197 The
optimized structures of the Fe−O2 species with protonated
distal bases showed only slight elongation of the O−O bond
lengths (>1.30 Å) due to H-bonding interactions with the
distal oxygen atom. A significant elongation of O−O bond
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Figure 40. Molecular structures of the four atropisomers (A−D) of the [FeIII(o-TMA)]5+ cation.209

length (1.52 Å) was obtained for [FeIIIL1-OOH]H+ which is of the iron porphyrin complexes mimicking the distal basic
much lower compared to other complexes as the protonated residues in the enzyme active sites (Histidine, arginine and
tertiary amine residue is further away from the bound lysine) emulated the “pull effect”, which is the primary
hydroperoxide species relative to FeL2, FeL3, and Fe-MARG determinant in modulating the facile rates and high selectivity
(Figure 38). The hydrogen-bonding interactions from of ORR. However, the direct observation of O−O bond
protonated nitrogen bases to the distal oxygen atoms were heterolysis of a putative FeIII−OOH species was not directly
substantially strengthened for 38C, 38D, and 38E in the series, demonstrated (see section 5).
and O−O bond lengths were elongated to 1.88 and 1.86 Å in 3.5. Role of Electrostatic Interactions
[FeIIIL2-OOH]H+ and [FeIIIL2-OOH]H+, respectively. The
bond length elongation was maximum (1.90 Å) in the case of There has been a growing interest in including properly
Fe-MARG (38E) that appeared to highly activate the O−O oriented charged functional groups as electrostatic motifs in
bond of ferric hydroperoxide species the most for further the second coordination sphere to improve molecular
cleavage in the next step of ORR. This predicted faster O−O reactivity, catalysis, and electrocatalysis. Based on an earlier
bond heterolysis in 33E was consistent with the SERRS-RDE report of Saveant and Robert on CO2 reduction with highly
data where accumulation of FeIII−OOH species was not efficient electrocatalytic activity using positively charged iron
observed in the steady state unlike FeL3 (33D), during the porphyrins,205 Mayer et al. described electrocatalytic ORR
catalytic turnover.204 Two different possible H-bonding process using the same polycationic iron porphyrin, [FeIII(o-
orientations to the proximal and distal oxygen atoms of TMA)]5+ bearing four positively charged o-trimethylanilinium
bound hydroperoxide ligand were considered for the low-spin [N(CH3)3+] groups with buffered weak acids as an example of
FeIII−OOH structures in 38E. These calculations predicted a exceptional homogeneous electrocatalyst (Figure 40).206 The
ΔG of 1.7 kcal/mol at rt corresponding to an equilibrium ORR was investigated in CH3CN solvent using the iron
constant of around ∼17 favoring the orientation where the H- porphyrin catalyst in a series of buffers. The strongest acid
bonding was with the proximal oxygen of FeIII−OOH; i.e., the source, ([DMF-H+]OTf−; pKa = 6.1) produced TOFmax = 8.5
ratio of proximal H-bonding conformation and distal H- s−1 and ηORR = 1.16 V (Table 6). In contrast, acetic acid
bonding confirmation was ∼17 (Figure 39B). Alternatively, for (AcOH, pKa = 23.5) gave distinctly improved catalysis: a faster
38D, the energy difference was 2.35 kcal/mol, favoring the TOFmax (170 s−1) at less than half ηORR (0.54 V) which is ∼104
distal H-bonding conformation which transformed to an
equilibrium constant of ∼1/50 for the same ratio (Figure Table 6. Electrocatalytic ORR Parameters of Fe(o-TMA) in
39A). This was consistent with the hydrolysis of FeIII−OOH, Different Buffers206
leading to the formation of PROS under slow ET conditions
E1/2 (mV) vs ηORR TOFmax
for 33E even though it had a favorably disposed proton transfer buffer pKa Fc+/Fc0 (V) (s−1) log(TOFmax)
residue. On the other hand, 38D geometry stabilized the
none −0.295
orientation with H-bonding to the distal oxygen atom and thus
[DMF-H+]/ 6.1 −0.25 1.16 8.5 0.91
primarily allowed O−O heterolysis. Overall, the optimized DMF
geometries of all these iron porphyrin complexes showed that TFAH/TFA− 12.6 −0.349 0.88 3.2 0.51
the proton donor groups are oriented in a way which allowed [Lut-H+]/Lut 14.1 −0.23 0.68 0.07 −1.17
selective delivery of the protons needed to the distal oxygen SalOH/ 16.7 −0.536 0.82 12 1.08
atom of the hydroperoxide species, activating it for heterolytic SalO−
O−O bond cleavage (Figure 38). All the experimental results BzOH/BzO− 21.5 −0.653 0.67 63 1.80
confirmed that these pendant bases in the second coordination AcOH/AcO− 23.5 −0.651 0.54 170 2.23

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times faster at similar ηORR than any earlier reported molecular
catalyst for the homogeneous ORR.25,207 While using other
acids with the decreasing order of acid strength from
trifluoroacetic acid (TFAH), salicylic acid (SalOH), to benzoic
acid (BzOH), similar improvements in both TOFmax and ηORR
were obtained (Table 6). This result contradicted previously
derived log(TOFmax)/ηeff relationships, which always predicted
that a lower ηORR might give a slower rate, as observed for
another series of iron tetraphenylporphyrins.191 In due
consideration of these results, it was concluded that a single
scaling relationship was not adequate for predicting the
combined changes in log(TOFmax) vs ηORR plot and a vector
sum of two different scaling relationship was developed.206
Mayer and his co-workers examined how covalently
positioned charged groups affected substrate binding and
other key pre-equilibrium during ORR in another work by
comparing the αβαβ isomer of polycationic iron porphyrin
complex, [FeIII(o-TMA)]5+ ,with the analogous unsubstituted
complex, FeTPP.208 Developing electrostatic effects to control
chemical reactivity and catalysis might depend on a
fundamental understanding of how electrostatics impact the
thermodynamics of individual chemical steps in a reaction.
Despite the fact that [FeIII(o-TMA)]5+ was found to be a
remarkable molecular electrocatalyst for O2 reduction with
high rates at low ηORR, the key electrostatic effect has no role
for the O2 binding to the ferrous porphyrin but rather the pre-
equilibrium binding of acetate (the anionic conjugate base of
the buffer), is enhanced.206 The charged groups facilitated the
binding of an acetate ligand, which caused a cathodic shift in
the E1/2 of the acetate form, [FeII(o-TMA)(AcO)]3+. Acetate
binding to the FeII(o-TMA)4+ was directly enhanced by
electrostatic interactions with the cationic-porphyrin ligand,
with its acetate binding constant (KAcO) being 65 times higher
than that of the neutral FeIITPP under low ionic strength in
absence of electrolyte. These results provided valuable
references in the literature to identify and quantify the effects
of intramolecular electrostatic interactions for ligand binding
and small molecule activation.
In a following work, Mayer and his co-workers studied
electrocatalytic ORR using all the four possible atropisomers of
Fe(o-TMA) (Figure 40) and determined how the positioning
of the tetra-cationic groups affected catalysis by identifying the
similarities and differences that exist among the isomers.209
These charged [N(CH3)3+] groups were effective in facilitating
multiproton multielectron reactions like ORR that involved
charge redistribution or high-energy charged intermediates or
transition states in molecular electrocatalysis. The E1/2 values
for each of the FeIII/FeII redox couples of Fe(o-TMA)
atropisomers were significantly positive than the same reported
for neutral iron tetraphenylporphyrins.210 The electrochemical
oxygen reduction by the four atropisomers were examined
under the similar homogeneous conditions (CH3CN contain-
ing 0.1 M H2O, 1:1 buffered acetic acid/acetate (AcOH/
AcO−) that was employed for the mixture of the isomers
producing the best catalysis data. All of these four isomers
exhibited reversible FeIII/II redox processes under anaerobic
conditions with the cathodically shifted E1/2 values between
−0.595 V (αααα) and −0.644 V (αβαβ) in the presence of
AcOH/AcO− buffer, which were replaced by a large
irreversible current in the presence of O2 indicating catalytic
turnover. The isomers catalyzed O2 reduction with faster rates
at low ηORR, and their catalytic rate constants (TOFmax) range
from 1 to 60 s−1 with the ηeff values differed only by 50 mV
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with and without an acetate ligand. The αβαβ isomer (40D)
was most reactive with the highest TOFmax, while the αααα
(40A) had the lowest TOFmax and the other two isomers lay in
between them.
The four atropisomers showed a roughly linear relationship
between log(TOFmax) and ηORR. The most important factor
controlling the relative values of both TOFmax and ηORR for
these systems was the catalyst E1/2 of FeIII/II under electro-
catalytic conditions. These relationships are likely to be
directly linked to the O2 binding and the barrier for rate-
limiting proton transfer to the ferric superoxide species which
have dependence on the catalyst E1/2. In general, catalysts with
more negative reduction potentials (E1/2 values) are more
nucleophilic and have higher binding affinity toward O2 and
result in more basic Fe−O2 intermediates, facilitating their
protonation (rds) and rapid turnover. The slope of the
log(TOFmax)/E1/2 relationship for the Fe(o-TMA) atro-
pisomers (34 ± 7 mV per decade) was nearly two times
steeper than the previously reported slope (55 ± 2 mV per
decade) for a series of substituted neutral iron porphyrin
catalysts.191 The steeper slope indicated a more sensitive
relationship between the catalyst E1/2, the free energy of O2
binding, the distal oxygen atom basicity of the corresponding
superoxide intermediate, and the barrier to superoxide
protonation. The positioning of the charged groups had
multiple effects and extended beyond one specific step of the
catalytic cycle and further characterization of the nature of
these intermediates should shed further light on this. These
data highlighted the significance of net change in charge
density upon ligand binding rather than orientation, on the
thermodynamics and kinetics of multistep molecular electro-
chemical transformation.
Warren and co-workers explored the strategies of using the
pendant groups with secondary interactions that have been
utilized for iron porphyrin ORR electrocatalysts to improve the
performance of the cobalt porphyrins. In this work, the authors
investigated electrochemical ORR of a series of Co porphyrins
with modifications in the 2-position of one of the phenyl
groups containing −NH2, −N(CH3)2, and −N(CH3)3+ groups
(Figure 32D−F) to probe systematically the relative
importance of proton relay effect and electrostatic interactions
of ancillary groups in catalysis.169 All these complexes were
drop casted over the EPG electrode in order to perform pH
dependence heterogeneous ORR, and they showed a definite
increase in current near the CoIII/II couple. The CV data of the
CoIII/II couple for Co porphyrins (32D−32F) showed pH
dependence that might be attributed to the protonation/
deprotonation of axially bound water. In addition, the
protonation state of CoTPPNH2 (32D) and CoTPPNMe2
(32E) depended on the pH at which the experiments were
conducted. Considering the pKa values of aniline (pKa = 4.6)
and N,N-dimethylaniline (pKa = 5.2) in water as models, 32D
and 32E were expected to remain fully protonated at pH 0 and
deprotonated at pH 7. However, the trimethylanilinium group
in CoTPPNMe3+ (32F) being positively charged, was
independent of the pH in the medium. Investigations using
CV and RRDE showed that the presence of a cationic −
N(CH3)3+ group proximal to the Co center in 32F, improved
selectivity for the 4H+/4e− reduction of O2 was observed
across a wide pH range from pH= 0 to 7 (Table 3). In contrast,
32D and 32F were more selective for reduction of O2 to H2O
at low pH when the pendant nitrogenous bases were
protonated, but the production of H2O2 was increased at
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Figure 41. Crystal structures and computer models of biosynthetic CcO mimics. (A) Overlay of the crystal structures of native bovine CcO (black)
and designed E-CuBMb (cyan); (B) overlay of the crystal structures of bovine CcO (black) and E-F33Y-CuBMb (cyan); (C) overlay of the
computer model (yellow) and crystal structure (cyan) of E-F33Y-CuBMb; (D) overlay of the E-CuBMb crystal structure (cyan) and the computer
model of E-G65Y-CuBMb (yellow).218,219 Reproduced with permission from ref 219. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA.

higher pH. In particular, at pH 0, the selectivity of CoTPPy
(32B, pKa of pyridine = 5.3) was quite similar to those of
CoTPPNH2 (32D) and CoTPPNMe2 (32E) because of the
comparable pKa values (∼5) of all the proton relays, despite
the different distances between the ionizable nitrogen and the
metal. The Co porphyrin analogue without any secondary
effects, CoTPP (32A), catalyzed primarily the 2e− reduction of
O2 at all pH, consistent with the other reports.23,195 The
second-order rate constants of O2 reduction for CoTPPNMe3+
(32F) were determined to be pH-independent and also
significantly high (∼106 M−1 s−1) throughout the entire pH
range at low ηORR. It was proposed that instead of hydrogen
bonding, electrostatic stabilization of anionic intermediates by
conjugate acids of nitrogen bases, or trimethylanilinium
(cationic) groups was important in determining selectivity
for the Co porphyrin ORR catalysts.
4. BIOSYNTHETIC MODELS OF CCO
Using a biosynthetic approach, stable naturally occurring
proteins were used as scaffolds for creating mimics of several
metalloenzymes. There are few examples where biosynthetic
models that structurally and functionally mimic CcO have
been reported.29,211
4.1. Role of Distal Cu
It has been difficult to obtain and handle large membrane-
bound proteins, HCO for practical O2 reduction. On the other
hand biosynthetic models of HCO’s created via site directed
mutagenesis in myoglobin proteins are easier to handle.
Myoglobin being a small globular protein that serves as an O2
storage in nature, is easy to obtain in large quanitities.212−214 Yi
Lu and his co-workers designed and engineered copper
binding sites in the active site of cytochrome c peroxidase215
and sperm whale myoglobin (swMb).216 The CuBMb mutant
of swMb was created by introducing two non-native histidine
residues through Leu29His and Phe43His mutations which
along with the native distal His64 residue formed the Cu-
12405
binding site in the distal pocket that closely mimics the
binuclear center in CcO, and this was the first reported
protein-based model for CcO (Figure 41).216 The reversible
oxygen-binding property of WTswMb is well-known, and the
incorporation of the CuB binding site in swMb provided ample
scope to investigate the effect of copper ion on O 2
reduction.213,217 Exposure of the CuII-free reduced CuBMb to
O2 resulted in partial conversion to the Fe−O2 adduct. The
mutations introducing two histidine residues in the distal
pocket with no bound metal ions, reduced the O2 binding
affinity of the protein compared to the native WTswMb.
Binding of AgI and ZnII to the three histidine distal sites,
increased the O2 binding affinity. These results strongly
suggested that the presence of CuB center is important for O2
binding forming a stable Fe−O2 adduct and increases the O2
binding affinity of the protein, albeit with a lower affinity than
the wild-type protein.213
WTMb is known to reduce O2 upon exposure to air in the
presence of a reductant and induce heme degradation to
verdoheme and then FeIII−biliverdin-like heme oxygenase. It
was shown previously that WTMb is incapable of O2 reduction
to H2O in the presence of subequivalent amounts of catalase
despite the addition of excess CuII ions and reductants like
ascorbate and TMDA (N,N,N′,N′-tetramethyl-p-phenylenedi-
amine), implying exogenous peroxides must be involved in
coupled oxidation unlike heme oxygenase (HO). To further
probe the role of CuB center during O2 reduction, the reaction
of reduced CuBMb was followed with UV−vis absorption
kinetics. The spectral changes suggested that the CuB ion in
CuBMb facilitated the reduction and then converted the heme
in CuBMb to verdoheme whereas oxy-WTMb did not show
any spectral changes. No O2 reduction was observed in other
control experiments, e.g., either the CuB metal was absent in
CuBMb mutant or the position was occupied by redox-inactive
ZnII or AgI metal. The CuBMb would likely to go through the
peroxy-heme intermediate during O2 reduction similar to HO
as suggested by similarities in the reaction products.213 WTMb
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only reversibly binds to dioxygen without reducing it and
P450, HO shows 2e− reduction of O2, whereas HCO reduces
oxygen by 4e− to water. It was proposed that the first step in
the catalytic cycle of HCO, P450, OH, and CuBMb mutant of
swMb involves the formation of two-electron reduction of
oxygen to peroxide intermediate (Scheme 7).46,212,220,221 Thus,
Scheme 7. Proposed Reaction Mechanism of CuBMb with
O2213
interestingly, these enzymes perform different reactions even
after going through the same putative peroxy-heme inter-
mediate. Introducing the Cu-binding site into myoglobin
transformed it from a simple oxygen carrier into a copper
dependent heme oxygenase at pH 8,213,214,222 which degraded
the heme cofactor to verdoheme. Since one of the key features
in the catalytic active site of native CcO is the presence of
tyrosine residue covalently linked to the histidine ligand bound
to the CuB center,57 the lack of it might have been detrimental
to the reactivity of these biosynthetic models. Furthermore,
proton delivery coupled to the O2 reduction is necessary to
avoid a HO reaction pathway that would lead to heme
degradation and loss of HCO activity.
4.2. Role of Tyrosine Residue
Lu et. al reported two mutants, F33Y-CuBMb and G65Y-
CuBMb (Figure 42), introducing a tyrosine residue at two
different positions close to the histidines of the copper-binding
Figure 42. Oxygen consumption rates in the presence of EDTA and
varying amounts of copper in the absence (blue) or presence (red) of
catalase and SOD. Error bars indicate standard deviation. Reproduced
with permission from ref 219. Copyright 2012 Wiley-VCH Verlag
GmbH & Co. KGaA.
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site of CuBMb to understand the role of the Tyr244 residue for
O2 reduction in the CcO-active site and examined their
reactivities toward O2 reduction.219 The absorption spectros-
copy was measured for both E-F33Y-CuBMb and E-
G65YCuBMb in the presence of excess reductant [ascorbate
with TMPD as a mediator] in air saturated buffer solutions,
and the results suggested reduction of O2. The rates of O2
reduction were measured quantitatively using an O2 electrode
in the presence of reductant to monitor the concentration of
O2 over time, following similar methods reported for native
HCO.223 To identify the reduced product (O2−, O22−, or
H2O), superoxide dismutase (SOD) and catalase were added
which selectively reacted with superoxide and peroxide,
respectively, producing O2 as one of their products. By
comparing the rates of O2 consumption in the absence and
presence of SOD and catalase, the portion of O2 reduction that
is due to water formation versus superoxide or peroxide
formation could be calculated (see Figure 42).
Not surprisingly, most of the O2 consumption by WTswMb
was due to PROS formation, consistent with autoxidation, and
these rates remained unaffected in the presence of Zn2+, Ag+,
or Cu2+ ions. Again, introducing two histidine residues
(CuBMb) into the distal pocket of WTswMb resulted in
substantial inhibition of superoxide/peroxide formation
relative to WTswMb but did not significantly contribute to
water formation and resulted in a decrease in the overall rate of
O2 consumption. In contrast, introducing a tyrosine next to the
one of the histidine ligands at either position 33 or 65 in F33Y-
CuBMb and G65Y-CuBMb, respectively, resulted in an increase
in water formation and overall rate of O2 consumption. The
rate of O2 reduction to water by E-G65Y-CuBMb was
measured to be 28 min−1, which is about 150-fold of native
HCOs.224 The mutants E-F33Y-CuBMb and E-G65Y-CuBMb
attained more than 505 and 1056 turnovers, respectively. The
crystal structures of E-CuBMb, E-F33Y-CuBMb, and WTswMb
indicated that the distal pocket containing two histidine
residues and an additional tyrosine residue had the capability
of stabilizing two and three water molecules in the distal site,
respectively (Figure 43), in comparison to one water molecule
in the distal pocket of WTswMb. Hence, WTswMb has only
one hydrogen bond donor site (H64) available to interact with
the bound oxygen, and two and three more hydrogen-bond-
donating residues are available in E-CuBMb and E-F33Y-
CuBMb, respectively.219 On the basis of the results, the authors
proposed that the tyrosine and its associated hydrogen
bonding network activated the ferric-superoxo species to
accept another electron forming ferric peroxide which
underwent the PCET process to produce a ferryl intermediate
for the complete reduction.
The formation of a Tyr• was directly observed in the EPR
spectroscopy during the reactions of 1 equiv of H2O2- and O2-
saturated buffer with the oxidized and reduced protein,
respectively, providing a strong evidence for the active role
of a tyrosine residue in the enzymatic reaction mechanism.225
The EPR data, both X and Q-bands and hyperfine splitting on
an anisotropic radical signal at g ∼ 2, matched with those of the
Tyr244 radical in HCO.129,226 This Tyr•, being transient and
unstable, disappeared completely within ∼100 ms, which is
why it difficult to obtain under turnover conditions.
In a subsequent work, the detailed EPR and X-ray
crystallographic data revealed that an extensive H-bonding
network existed in the active site of oxy-F33YCuBMb that
helped in promoting proton delivery to the active site and
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Figure 43. Water networks in the distal pockets of (A) E-F33Y-CuBMb and (B) E-CuBMb crystal structures. Reproduced with permission from ref
219. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA.

Figure 44. (A) Proposed mechanism of O2 reduction by F33YCuBMb in comparison with WTMb. (B) Crystal structure of the oxy-F33Y-CuBMb
(1.27 Å resolution; PDB ID: 5HAV) with two water molecules in the active site pocket (left). Cryo-reduction method for formation and trapping
of (hydro)peroxo intermediates (X = active site His or Tyr residue) (center). The EPR spectra of oxy-F33Y-CuBMb cryoreduced at 77 K showing a
ferric-hydroperoxo (H-bonded to an active site water, green) which decays upon stepwise annealing (right). Reproduced from ref 227. Copyright
2016 American Chemical Society.

facilitated the reduction process, thus inducing oxidase activity
in a biochemical model of HCO.227 It had been already
established that cryo-reduction of the parent Fe−O2 complex
at 77 K followed by stepwise annealing revealed the
intermediates formed during O2 reduction pathway in heme
proteins. 228−232 Oxy-F33YCu B Mb upon cryo-reduction
showed two rhombic EPR signals corresponding to the ferric
peroxo (g = 2.24, 2.13, and 1.95) and ferric hydroperoxo (g =
2.29, 2.17, and 1.96) intermediates, and these species were
again confirmed by the 1H ENDOR measurements. Then the
decomposition of the hydroperoxo species at high temperature
resulted in O−O bond cleavage and an EPR-silent ferryl
species (compound II), and this intermediate underwent
further cryoreduction at 77 K generating the prominent low-
spin rhombic EPR signal (g = 2.52, 2.15, and 1.90), assigned as
ferric hydroxo species (Figure 44B, right). In contrast, the
cryoreduced oxy-WTMb was unable to deliver a proton and
showed a dominant rhombic signal (g = 2.22, 2.13 and 1.96)
which was identified to be a ferric-peroxo species.232,233 High-
12407
resolution (1.27 Å) X-ray crystallographic data were obtained
for the oxy-F33Y-CuBMb showing a H-bond network of two
water molecules linking the two engineered residues (Tyr33
and His29) to the terminal O atom of the Fe−O2 species
(Figure 44B, left). These were not present in the crystal
structure of oxy-WTMb234 which was incapable of O2
reduction. Altogether, the spectroscopic and crystallographic
evidence suggested the importance of H-bonding interactions
that would transform a simple O2 carrier protein, WTMb to
F33Y-CuBMb which could activate O2 and reduced it to H2O
(Figure 44A), and this was the first successful design of
artificial enzyme system known so far.
Similarly, Karlin and Solomon’s combined work based on
the synthetic heme−peroxo−copper models also supported the
role of a tyrosine residue.137,138 In these systems, introduction
of external phenol formed a stable adduct with the bridging
peroxo complex which mimicked the active site structure of
HCOs. From the kinetic study and theoretical calculation, it
was evident that PhOH played an important role for the H-
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Scheme 8. Construction of Electrode-Bearing Biosynthetic Modelsa

a
Reproduced with permission from ref 201. Copyright 2018 American Chemical Society.

bond assisted homolytic O−O bond cleavage via a PCET could be directly transferred to the haem from the electrode to
mechanism. The energy barrier for the reductive O−O bond facilitate O2 reduction.237
cleavage decreased in the presence of either H-bonding In air-saturated buffer solutions, the bioinspired electrode
interactions or proton transfer from the phenolic O−H bond. loaded with the Mb mutant G65YCuBMb showed a large
In a recent study, Dey et. al demonstrated the role of a electrocatalytic O 2 reduction current at pH 7. The
redox-active quinol group in ORR mechanism using synthetic G65YCuBMb protein predominantly catalyzed a 4e−/4H+
model porphyrin.151 They reported that even a simple ferrous reduction of O2 to H2O as RRDE data indicated the formation
porphyrin complex (27A) containing a pendant quinol group of only 6% PROS during ORR. The G65YCuBMb biosynthetic
showed complete O2 reduction to water in terms of oxidation Mb scaffold-based bioelectrode was found to be remarkably
of quinol to quinone in the absence of any other additional stable for the ORR.236 Any SAM-bearing covalently attached
electron and proton source. The ferric superoxide species of O2-reducing electrocatalysts reported so far have not been
27A underwent the first HAT resulting in a ferric peroxide stable enough to allow these dynamic electrochemical
which could perform another HAT from the semiquinone experiments to determine the kinetic parameters. The kcat
group and generated ferryl species along with the residual was evaluated to be 1.98 × 107 M−1 s−1, yielding a TON in the
quinone group. Therefore, these results might contribute to order of 104 from RDE data and their K-L analysis (Table 7).
establish the importance of redox-active H-bond donor sites in
model systems like the residues present in enzyme active sites. Table 7. Biosynthetic CcO Models as ORR Catalysts199,201
In the F33Y-CuBMb model, the presence of tyrosine and its
kcat (× 107) TON PROS
specific positioning had been shown to play an important role catalysts (M−1 s−1) (104) (%) SKIE
in directing the product formation from the complete
CuBMb ND ND 6 ± 1 ND
reduction of O2 with the minimum release of PROS similar G65YCuBMb 1.98 × 107 >0.65 6 ± 1 16.00 ± 1.0
to the activity of terminal oxidases, with more than 1000 V68ECuBMb 2.2 × 107 >2.8 4 ± 1 4.30 ± 0.5
turnovers. Although the designed proteins were less active than V68E/I107E CuBMb 0.86 × 107 >0.5 7 ± 1 2.43 ± 0.07
terminal oxidases, it is noteworthy that oxygen reduction to synthetic CcO model 1.2 × 10−2 ND ∼8 ND
water was achieved by a much smaller protein, myoglobin, with (FeCu analogue)
only three mutations of the distal pocket.219,235 The rate of
electron transfer to the active site remained a major issue in
achieving efficient catalysis in these systems. The catalytic ORR rate by G65YCuBMb exceeded those
reported for the best artificial synthetic analogues as well as
4.3. Electrocatalytic ORR by Biosynthetic Models of CcO
native CcO itself. Efforts on attaching native CcO on electrode
To address the slow ET rates, the biosynthetic model of CcO similar to bioelectrodes resulted in very sluggish O2 reduction
containing the distal CuB and tyrosine residue was immobilized due to improper alignment of this membrane-bound protein
on a bioinspired electrode developed by Dey and co-workers on the electrode, which prevented efficient electron transfer to
following their earlier method employed for wTMb.236 The the active site.238,239 As a result, a CcO-functionalized SAM-
electrode bearing the biosynthetic model, G65YCuBMb, was covered Au electrode showed <1 μA electrochemical O2
constructed in situ where the native haem cofactor in Mb was reduction current at −300 mV, whereas this bioelectrode
replaced by a modified hemin cofactor bearing an alkyne group produced ∼100 μA current at similar potentials.240
(hemin-yne, Scheme 8) and reconstitution of apoprotein was To understand the mechanism of facile and selective O2
done in situ with the hemin-yne group tethered to the reduction catalyzed by the G65YCuBMb biochemical model,
functionalized SAM of thiols on Au electrode so that electrons the SERRS-RDE technique was applied. With the help of this
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Scheme 9. Comparison between the Mechanism of Native CcO in Solution and the Biosynthetic G65YCuBMb Model on the
Electrode236
technique, in situ spectroscopic detection of reactive
intermediates can be done over the electrode surface during
electrocatalytic ORR under steady state. When a cathodic
potential was applied in an oxygenated buffer, the SERRS-RDE
data clearly showed the characteristic features of low spin heme
and ferryl species. The lack of signals of high spin ferrous and
high spin ferric species indicated that the O2 binding was more
facile for this mutant having the design principle of Mb which
had 10 times faster binding rate than native CcO and the ET to
FeIII resting state was also very facile at these potentials as may
be expected due to direct attachment of the Hemin-yne to the
electrode. The clear increase in intensities of bands having ν3
and ν2 at 1508 and 1591 cm−1, respectively, and 18O2-sensitive
bands in the low-frequency region corresponding to the ferryl
species (FeIV�O) entailed the O−O bond cleavage faster for
the formation than its decay under steady state. A Gedanken
steady-state turnover experiment with CcO, where the electron
transfer to the active site was very fast (that is, in the
hypothetical situation where ET from Cyt c to CcO is not the
rds), the species that would accumulate during turnover, based
on the Babkock−Wikstrő m mechanism were the FeII, FeII−O2,
FeIII−O22−, FeIII−OOH, FeIV�O, and FeIII−OH for which the
rates of formation of these species were greater than their rates
of decay (Scheme 9).241,242 Out of these, the FeII−O2, FeIII−
O22−, and FeIII−OOH species would have rR signatures of low
spin heme, FeIII−OH would have rR signature of high spin
heme and the FeIV�O would have signature unique to heme
ferryl species.4,242,243 The in situ SERRS-RDE data were
consistent with such an expectation.
The overall rate-limiting step of native CcO in solution is the
dissociation of hydroxide from FeIII−OH, which is the end
product of O2 reduction reaction, to get back to the active
resting ferric state with a first-order rate constant of 500
s−1.91,242,243 The potential determining step of ORR by
G65YCuBMb was 166 mV more negative than E1/2 for the
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resting high spin ferric state and was likely to be the reduction
of the FeIII−OH species. Thus, immobilized of an electrode,
one could circumvent the kinetic barrier associated with the
dissociation of the hydroxide by directly reducing it to ferrous
at more negative potentials. This direct ET to the ferric
hydroxide species, which is an intermediate in the catalytic
cycle of CcO, is an ET shunt analogous to peroxide shunt in
cytochrome P450. Thus, the rds of ORR by these biosynthetic
models was expected to be the protonation of the FeIII−O22−
species with a first-order rate of 5000 s−1 (Scheme 9). Here,
too, the tyrosine residue present in the biosynthetic model of
CcO (Y65 in G65YCuBMb mutant) could help in both proton
and electron transfer during ORR and substantially reduced
the amount of PROS relative to CuBMb, which is analogous to
G65YCuBMb without the Y65 residue and resulted in higher
TONs.
The oxygen-reducing enzyme CcO and nitric oxide
reductase (NOR) both belong to the HCO superfam-
ily.4,244,245 In spite of having similarities in the active-site
structures, they have distinct reactivities likely due to the
differences in distal metal and/or the key second sphere
residues.216,219,246,247 For example, the CcO active site
contains a CuB and a conserved and cross-linked Tyr244
residue while the active site of NOR has FeB and two
conserved glutamate residues. Lu and co-workers successfully
designed and engineered the active site of NOR where FeB was
introduced in the distal site instead of CuB and the Val68
residue of Mb was mutated with Glutamate residue resulting in
the mutant V68ECuBMb.247 Introduction of another glutamate
residue mutating the I1e107 residue in the distal side of heme
in Mb resulted in a mutant I107ECuBMb.246 In order to create
myoglobin based perturbed models of CcO, the FeB metals in
the distal site of V68ECuBMb and V68E/I107ECuBMb
biosynthetic models of NOR were replaced with Cu and
proton transfer glutamate residues were introduced instead of
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the redox active tyrosine residues. The glutamate residue Scheme 10. Comparison between the Mechanisms of
containing mutants provided a unique opportunity to probe Oxygen Reduction of the Two Biosynthetic Models while
the potential role of proton transfer in the O2 reduction Attached on Top of the Electrodea
process in general and specifically in CcO.
The electrochemical O2 reduction was investigated for these
two mutants by Dey et al. with a combination of H2O/D2O
solvent kinetic isotope effect and in situ SERRS-RDE to unveil
the main role played by the local acidic residues in determining
the selectivity and kinetics of O2 reduction as well as in
modulating the rds of 4e−/4H+ ORR.201 The heterogeneous
ORR were performed by reconstitution of apoproteins of the
mutants to the hemin−yne-modified functionalized SAM
surfaces like G65YCuBMb. The LSV data of these electrodes
bearing mutants showed large irreversible electrocatalytic O2
reduction current in air saturated pH 7 buffer. The kinetics of
ORR and selectivity were determined with RDE and K-L
analysis and thereby, the biosynthetic models, (V68ECuBMb
and V68E/I107ECuBMb) exhibited second-order rate con-
stants of >107 M−1 s−1 as high as for the biosynthetic CcO
model G65YCuBMb (Table 7). For V68E/I107ECuBMb, the
slightly lower value of the kcat was due to the presence of 37%
LS FeIII component which was unable to take part in ORR.
The presence of the additional proton donor residues near the
active site did not compromise the selectivity of 4e−/4H+ ORR
as these V68ECuBMb and V68E/I107ECuBMb mutants
produced minimum amount (4−7%) of PROS. The kcat
obtained in deuterated buffer allowed estimation of H2O/
D2O solvent kinetic isotope effect (SKIE) which, in turn,
reflected the involvement of proton transfer in the rds. The
SKIE of O2 reduction for G65YCuBMb was 16.0 which was
reduced to 4.3 in V68ECuBMb (Table 7). Thus, the presence
of proton transfer glutamate residue in V68ECuBMb reduced
a
the SKIE. The introduction of an additional glutamate residue Reproduced from ref 201. Copyright 2018 American Chemical
in V68E/I107ECuBMb mutant further reduced the SKIE to Society.
∼2.4. The loss of preorganized proton transfer channel due to
mutation of proton transfer residues was also proposed to be
responsible for high SKIE in heme enzymes like Cytochrome protonation steps in V68ECuBMb relative to G65YCuBMb.
P450 and peroxidases.248,249 Hence, the large H/D isotope Thus, the distal glutamate residues in the biosynthetic models,
effect ∼16 associated with G65YCuBMb indicated a rate- V68ECuBMb and V68EI107ECuBMb, behaved as proton
determining protonation step and the lack of an efficient transfer residues similar to those present in natural enzyme
proton transfer channel to the active site. resulting in facile and selective 4e−/4H+ ORR and also lowered
For G65YCuBMb mutant, the protonation of LS ferric the H2O/D2O SKIE value of kcat.201
peroxide was likely to be the rds as indicated by the 4.4. Role of Distal Arginine Residue in Amylin Peptide
accumulation of LS ferric peroxide species in the SERRS- Dey et al. assembled naturally occurring amylin (Ay) peptide
RDE data and was also supported by the large SKIE. The on top of Au electrodes with thiol groups of two cysteine
detailed electrochemical study of ORR for all of the mutants residues (Cys2 and Cys7) present in its 1−19 domain (total 37
showed that they reduce oxygen at comparable rates with the amino acid residues are present) to form a stable AySAM
only variation in their H2O/D2O SKIE values (2.4−16) (Table assembly.250 The assembly was bound to heme with the
7). However, the same rds for G65YCuBMb, V68ECuBMb, and His18 residue present in the 1−19 domain of amylin to
V68E/I107ECuBMb having zero, one, and two preorganized construct a potential biochemical scaffold which was shown to
proton transfer residues, respectively, would result in a be an excellent O2 reduction catalyst by Dey and co-workers.
variation in the kcat which was not the case suggesting that Wild-type (WT) heme-AySAM and its Arg11ASN mutant
the rate-determining steps were different depending on the (AySAMR11N) bound heme assembly were at first fully
nature of the second-sphere residues of these mutants. On the characterized using CV, SERRS, and atomic force microscopy
basis of the electrochemical and SERRS-RDE data, the (AFM) spectroscopy. Formation of a monolayer of site-
mechanism of ORR by the mutants other than G65YCuBMb isolated heme−AySAM active sites was concluded from low
also had been proposed. The SERRS-RRDE data of surface coverage value (9 × 1012 molecules/cm2) in CV data,
V68ECuBMb, the construct with the highest rate, showed and heme binding was ensured from the SERRS data,
only HS FeIII species under steady state, and neither a LS FeIII indicating the presence of a six coordinate FeIII−HS species
peroxide nor a FeIV�O species were observed unlike in on the surface. RDE data with the above-mentioned
G65YCuBMb (Scheme 10). Since, decay of both species bioinspired electrode systems and their subsequent K−L
required protons and the fact that these species did not analysis revealed that both heme−AySAMWT and AySAMR11N
accumulate during steady state was consistent with faster followed selective 4H+/4e− O2 reduction pathway with kcat
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values (1.8 ± 0.6) × 107 M−1 s−1 and (2.7 ± 0.4) × 105 M−1
s−1, respectively. RRDE was applied to detect PROS formation
for these two systems, which is about 9 ± 0.5% for heme−
AySAMWT and 6.6 ± 1% for the R11N mutant. This particular
construct with Ay attached to the Au electrode via two Cys−
Au linkages formed a very stable monolayer; otherwise, the kcat
value could not be calculated for monolayers of catalysts. A
facile H+ transfer pathway from the Arg11 residue was
operative, which was the key factor behind higher kcat in
ORR by the WT Ay compared to R11N mutant. The
difference in PROS was also due to the presence of the acidic
Arg11 residue, which although not directly involved in bonding
with heme provided an H-bonding interaction from the second
sphere. In this report, naturally occurring stable peptide
scaffold with heme binding was shown to be 100 times more
facile in ORR compared to simple mononuclear synthetic
mimics.
5. ROLE OF SECOND-SPHERE DISTAL RESIDUES IN
PEROXIDE ACTIVATION
The oxygen reduction cycle involves the formation of a ferric
hydroperoxide species (FeIII−OOH), also known as com-
pound 0, which is generally a very short-lived intermedi-
ate.251,252 It is formed by two-electron reduction of molecular
O2 followed by protonation in many heme metalloenzymes like
Cyt P450 and nitric oxide synthase. The same intermediate can
be generated by adding H2O2 directly to the resting ferric state
of heme enzymes.253 The compound 0 intermediate plays a
crucial role for the formation of high valent iron(IV)−oxo
porphyrin cation radical [(FeIV�O)Por*+], commonly known
as compound I, intermediate which is the active oxidant in
monooxygenase, peroxygenase and peroxidases.253−255 The
formation of compound I from compound 0 involves the
heterolytic cleavage of the O−O bond in this FeIII−OOH
species. Factors that can affect heterolytic O−O bond cleavage
of metal hydroperoxide species have been, thus, of great
interest.256−259 Investigations using monofunctional perox-
idases, catalases, cyt P450 monooxygenases, and peroxygenases
and their site-directed mutants revealed the importance of both
first- and second-coordination sphere residues in the active-site
structures that are essential for the generation of high-valent
catalytic intermediates via heterolysis of the O−O bond
present in compound 0.47,55,260−264 The generation of
compound 0 and the subsequent O−O bond cleavage to
form compound I in peroxidases are facilitated by residues at
the distal pocket acting as acid−base catalysts, as proposed first
by Poulos and Kraut based on structural and mechanistic
investigations.256,265−267 Similarly, the O−O bond heterolysis
of compound 0 in cytP450 is aided by a threonine residue in
the distal site directing the proton necessary to the FeIII−OOH
unit which is further activated by the “push” effect of the
electron rich axial thiolate ligation.37,268−270 Over the last
several decades, several synthetic metal-porphyrin complexes
were designed for the investigation of the “push-pull” effect
during the O−O bond cleavage followed by the formation of
compound I using various oxidants.271−275
Groves and Watanabe first reported the reaction of
chloro(5,10,15,20-tetramesitylporphyrinato)iron(III)
[FeIII(TMP)(Cl)] with peroxyacids in organic medium
(CH2Cl2) at low temperature and monitored O−O bond
cleavage step by direct observation of the conversion of
acylperoxoiron(III) porphyrins to form high-valent spe-
cies. 272,276 The corresponding manganese analogue
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[MnIII(TMP)OH] reacted the same way with peroxyacids to
generate a (acylperoxo)MnIII TMP adduct followed by the
formation of the oxo-manganese(V) intermediate which was
well characterized.277,278 It was suggested that the nature as
well as rate of the O−O bond cleavage would have been
affected upon changing the substituents in peroxyacids. In
order to understand the “push effect” on the O−O bond
cleavage reaction of the FeIII−OOH species, Watanabe et al.
examined the reactivity of a series of (acylperoxo)iron(III)−
porphyrins by changing substituents at the meso positions of
the porphyrin ring (Figure 45) as well as introducing an
Figure 45. Structures of meso-phenyl-substituted iron porphyrin
complexes (A−E) employed in this study.280
imidiazole axial ligand.279,280 It was demonstrated that the O−
O bond cleavage mechanism can be homolytic or heterolytic
and it could be controlled by changing the solvent polarity
from CH2Cl2 (heterolysis) to toluene (homolysis).272,276,281
Generally the O−O bond cleavage step is the rds in these
reactions and the first-order rate constants (khetero) for the
heterolytic O−O bond cleavage of these substituted porphyr-
ins were evaluated (Table 9). The complexes having electron-
donating substituents at the meso positions of a porphyrin ring
were found to enhance the rates of O−O bond cleavage
consistent with the “push effect” exerted by the porphyrin
ligand, whereas electron-withdrawing groups decelerated the
formation of compound I. In addition to that the nature of
substituents on the peroxy acids affected the rate of heterolysis
and the (acylperoxo)iron(III)−porphyrin complexes decom-
posed faster for the p-nitroperbenzoate derivatives than the m-
chloro derivatives. The most electron rich iron(III)-porphyrin
(45A) exhibited the highest khetero values among all other
substituted analogous porphyrins using both the peroxy acids
under similar experimental conditions. These investigations
laid the foundation of later work in the area where second-
sphere residues were introduced.
In order to understand the O−O bond activation, Nocera
and his co-workers constructed a class of metal-porphyrin
complexes, hangman porphyrins, where −COOH functional
groups were positioned above the redox-active metal center of
iron porphyrins for the facile proton delivery to the metal-
bound ligand in a face-to-face arrangement within a single
molecular architecture.184,282 The hanging carboxylic acid or
ester group was included on a rigid xanthene (HPX) or
dibenzofuran (HPD) scaffold at the meso position of the
trimesitylporphyrin platform to develop a set of HPX and HPD
complexes containing transition metals (Fe, Mn) (Figure
46A−K).283
The likely role of protonation assisted O−O bond activation
was examined with a set of complexes where both the proton
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donor ability (carboxylic acid versus ester) and orientation

Figure 46. Hangman porphyrins based on xanthene (A−I) and
dibenzofuran (J, K) scaffolds studied by Nocera and co-workers.283,284
(xanthene versus dibenzoufuran) of the pendant distal
functionality was varied. These iron hangman porphyrins
were evaluated for, catalase like, disproportionation of H2O2
under biphasic buffered condition (CH2Cl2/H2O, pH 7) in the
presence of 1,5-dicyclohexylimidazole, axial ligand to the metal,
at 25 °C. A simple porphyrin analogue, FeIII(TMP)(Cl) (45C)
having similar steric effect and electronic structure due to the
presence of mesityl groups, was used as a control. The complex
46B, containing a xanthene spacer and carboxylic acid group,
showed the highest activity toward H2O2 disproportionation
with 10−100 times higher TON obtained for O2 production
than any other catalysts examined (46C, 46J, and 45C). The
catalytic activity decreased significantly by replacing xanthene
with dibenzofuran spacer with the pendant carboxylic acid
functionality remaining the same or by replacing only the
carboxylic acid with a methyl ester keeping the xanthene
scaffold intact. The addition of 1 equiv of either benzoic acid
or methyl benzoate externally to 45C, as a source of
intermolecular proton, failed to induce catalytic catalase
activity. The catalytic epoxidation of olefins e.g., styrene, cis-
cyclooctene, using hydrogen peroxide as a terminal oxidant,
was carried out using manganese HPX and HPD derivatives as

Scheme 11. (Top) Formation of Compound I (b) and Compound II (c) Intermediates from Acylperoxo−FeIII(HPX-CO2R)
(a) by the Reaction of 46C with m-CPBA and PPAA, Respectively. (Bottom) Proposed Reaction Mechanism of H2O2
Activation by (HPX-CO2H)FeIII(OH)274

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catalysts (46H, 46I, and 46K) and MnIII(TMP)Cl as
control.283 The results for epoxidation reactivity of the
manganese derivatives followed the same general trend
observed for catalase reactions catalyzed using the correspond-
ing iron analogues. The results showed that Complex 46H
bearing a protic carboxylic acid group on a xanthene scaffold
was most active, affording styrene epoxide (yield 70%) and cis-
cyclooctene oxide (yield 92%) with TONs almost twice as high
as those obtained for the ester derivative, 46I. Under similar
conditions, both the HPD complex 46K and MnIII(TMP)Cl
produced significantly less epoxide products upon reaction
with either of the two substrates. Altogether, activity
enhancement observed for the xanthene-bridged platform
with a pendant carboxylic acid group (HPX-CO2H) indicated
a clear advantage of a geometrically more favorable proton
source in reactivity. Increasing the distance between the proton
donor −COOH group and the metal in the porphyrin cavity
by using a dibenzofuran scaffold instead of the xanthene
scaffold resulted in reactivity comparable to the control TMP
despite the presence of a pendant protic hydrogen bonding
group. Structure−function relationships of the HPX and HPD
complexes in both catalase-like H2O2 disproportionation and
peroxide-mediated olefin epoxidations revealed that both the
nature of the hydrogen-bonding group and its spatial
orientation are crucial for the efficient activation of O−O
bond of putative metal peroxides.
In a subsequent work, a series of hangman iron porphyrins
based on xanthene scaffold (HPX) containing pendant groups
with a wide range of different acid−base functionalities (pKa
ranging from ∼2 to 25 in water) was synthesized (Figure
46).284 The kinetics of H2O2 disproportionation suggested that
the initial rate constants for catalase activity followed the
acidity of the pendant group such that the complex 46A with
strongly acidic phosphonic acid functional group, showed the
highest rate constant. Thus, HPX complexes (46A and 46B)
bearing the most acidic pendants (phosphonic acid and
carboxylic acid) exhibited similar initial rate constants that
were 3 orders of magnitude higher than that of the complex
45C. The complex 46A decomposed rapidly during the
disproportionation reaction, but complex 46B retained its
activity. It was proposed that hydrogen-bonding groups with
higher pKa values were less competent in donating the protons
required for the heterolytic O−O bond cleavage, while the
conjugate base of the more acidic phosphonic acid
functionality (pKa < 2) was incapable for accepting protons
during the catalytic cycle. However, the overall catalase activity
was maximum for 46B, iron hangman porphyrin bearing a
carboxylic acid group (pKa ∼ 4.2) which exhibited the highest
TON where an optimal balance might have been achieved for
acid−base catalysis needed for O−O bond heterolysis.
Nocera et al. also directly investigated the O−O bond
cleavage of peroxyacids resulting in high-valent oxo inter-
mediates to decipher the influence of a proton donor group
using iron hangman porphyrins (P)FeIII(OH) having carbox-
ylic acid (P = HPX-CO2H, 46B) and methyl ester (P = HPX-
CO2Me, 46C) by following the kinetics of formation of
compound I species.274 Both of these complexes upon reaction
with m-CPBA in CH2Cl2 underwent heterolytic O−O bond
cleavage generating compound I intermediate at cryogenic
temperature (−40 °C) having characteristic absorption
features (Scheme 11, top). The rate constants for the O−O
bond heterolysis were determined from the stopped-flow
kinetics data and the rates of compound I formation which
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were not significantly perturbed by changing the acid
functional group as the rates of O−O heterolysis in the
hangman scaffold, bearing either the −COOH or the
−COOMe substituents, were similar and comparable to the
parent FeTMP analogue (45C). However, changing the
organic medium to an even more nonpolar solvent, toluene,
the reaction of 45C and m-CPBA at −20 °C resulted in the
formation of ferric N-oxide species via a homolytic O−O bond
cleavage where high-valent compound II (TMP)FeIV�O was
initially formed and subsequent attack on the porphyrin
pyrrole N atom.272 The formation of the ferric N-oxide
product formation was significantly decreased in the presence
of hangman ester scaffold of 46C under the same reaction
conditions, and no ferric N-oxide product was observed at all
for the hangman −COOH analogue 46B. The generation of
compound II from peracid entailed homolysis of the O−O
bond of the peroxide intermediate. While compound II could
be directly obtained from the reactions of 46C with
phenylperoxyacetic acid (PPAA) at −40 °C in toluene
(Scheme 11, top), no compound II could be observed for
46B having pendant −COOH group suggesting that hangman
architecture with properly oriented acid functional group
favored the heterolytic O−O bond cleavage to generate
compound I over competing homolytic cleavage (1e− path-
way) (Scheme 11, bottom).
In addition to the hangman porphyrins from Nocera, several
Mn porphyrins with positively changed meso substituents were
reported to have encouraging reactivities.285 The first-order
rate constant of O−O bond heterolysis was obtained as 66 ±
12 s−1 with activation parameters estimated as ΔH⧧ = 4.2 ±
0.2 kcal mol−1 and ΔS⧧ = −36 ± 1 cal mol−1 K−1. The positive
charge on the substituent on the meso carbons of porphyrin
ring exerted strong electron-withdrawing effect which resulted
in ∼200 mV higher MnIII/II reduction potential relative to
other Mn porphyrins.286,287 The positively charged porphyrin
meso substituent was also proposed to aid in O−O heterolysis
via electrostatic effect.285
In a recent work, Dey et al. developed a structural as well as
a functional model of peroxidase (Fe-MARG), containing a
covalently attached guanidine moiety in the second sphere of
porphyrin ligand (Figure 33E), which mimicked the Arg38
residue present in the active site of HRP.288 The role of Arg38
in HRP had been established since the early 1980s, but
guanidine functional groups were not modeled in synthetic
systems. This was the first instance where the guanidine
residue was incorporated in a synthetic model porphyrin that
showed peroxidase-like activity affecting the O−O bond
heterolysis of FeIII−OOH intermediate compound 0 by the
reaction of H2O2, being used as a green oxidant. To investigate
the peroxidase assay in a molecular catalyst, 3,3′,5,5′-
tetramethylbenzidine (TMB) was used as a model sub-
strate.289,290 The reaction kinetics for the catalytic oxidation
of TMB to diimine formation by Fe-MARG (33E) using H2O2
oxidant and a trace amount of acetic acid in a 5% v/v H2O−
acetonitrile mixture, was monitored using absorption spectros-
copy. The rate of TMB oxidation was much faster in 33E than
other analogous porphyrins like FeTPP (17A) and FePf (20A),
which clearly indicated the role of pendant guanidinium group
in accelerating catalytic substrate oxidation. In addition, 33E
catalyzed a variety of substrates like [tetramethylbenzidine
(TMB), o-phenylenediamine (OPD), and 2,4,6-tri-tert-butyl-
phenol (TBPH)] oxidations291,292 using other oxidant like m-
CPBA. Thus, the suitably poised guanidine moiety in the distal
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Table 8. Comparison of Kinetic Parameters of Oxidation of Organic Substrates Using H2O2
catalyst KMH2O2 (mM) kcat (s−1)
Fe(III)-MC6 44 ± 2 370 ± 10
E2L(TD)-MC6 31 ± 2 780 ± 60
Protein-86 n.d ∼280
Protein-K5 ∼217
CTMs-C45 94 1200
μ-peroxidase 21
reconstituted-Mb 7.4 ± 0.5 1.1 ± 0.1
antibody−heme complex 24 6.56
peroxidase (HRP, MPO) 3.7 0.01−2100*
FeIIICl-MARG (33E) 4.76 ± 0.5 25
site of simple TPP analogue made Fe-MARG (33E) an
indiscriminate peroxidase mimic. Additionally, complex 33E
showed a Michaelis−Menten-type saturation-kinetics over a
reasonable range of TMB and H2O2 concentration, which is
very rarely observed for any artificial catalyst as a catalyst−
substrate [CS] complex285,293,294 analogous to enzyme−
substrate complex [ES]. The two most relevant kinetic
parameters KMH2O2 and the catalytic efficiency (ηH2O2) were
determined to be 4.76 ± 0.5 and 3.4 ± 0.4 mM, respectively.
The KM values of both TMB and H2O2 were also similar to
that of native HRP (Table 8).295,296 The catalytic rate toward
TMB oxidation of Fe-MARG is 25 ± 5 s−1 and the catalytic
efficiency (ηH2O2) is 5.2 × 103 M−1 s−1, which are lower than
that of the natural enzyme but comparable to or even higher
than that of most of the artificial proteins and engineered
myoglobin reported (Table 8).294,297−301 In addition, the
nonsequential multiple substrate binding was observed for this
complex, having low KM for both TMB and H2O2 just like
natural enzymes, suggesting a “ping-pong” mechanism in a
molecular catalyst.295 The reactive intermediate formed on the
reaction of 33E with peroxides was compound I as evident
from typical absorption features. The second sphere guanidine
group present in 33E remained protonated at neutral pHs and
assisted in facile heterolytic cleavage of FeIII−OOH species
leading to the compound I formation. The guanidinium group
being a strong hydrogen bond donor and could also provide
auxiliary binding site to the substrates used here, e.g., TMB,
OPD, and TBPH (Scheme 12). Overall, this model complex,
Fe-MARG, utilized the “ping pong” mechanism for substrate
oxidation with KMH2O2 and kMsubstrate values similar to that of
native enzyme and exhibited competitive inhibition kinetics
(with azide), thus emulating enzymatic activity on inclusion of
second sphere guanidine residue.288,302 These results clearly
suggested that the presence of distal guanidinium group
transformed the simple iron porphyrin into a miniature
enzyme both qualitatively as well as quantitatively.
Dey et al. used a series of ferric iron porphyrin complexes
(FeL2, FeL3, and FeMPh) having pendant basic groups
(pyridine, primary amine, and control) covalently attached to
the amine group of the meso-phenyl ring in the TPP
architecture (Figure 33), in order to investigate the rates of
O−O bond cleavage and to characterize the ensuing
intermediates formed.303 In the active site of peroxidases,
histidine residue plays a vital role with the “pull effect”
transferring proton to the distal oxygen atom of compound 0
for the heterolytic O−O bond cleavage. To date, the role of the
histidine residue remains to be successfully modeled in any
synthetic heme-based model system. These model complexes
FeL2 (33C) and FeL3 (33D) containing pendant pyridine and
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ηH2O2 = kcat/KMH2O2 (M−1 s−1) ref
8.4 ± 0.6 × 103 290
25 ± 3 × 103
500 296
125
1.3 × 104 294
680 297
1.4× 102 293
274 296
4.6 × 106 291, 292
5.2 × 103 284
Scheme 12. Plausible mechanism of FeIIICl-MARG catalysed
substrate oxidation. Note that the Cl− ion is likely to be
exchanged by OH− under the reaction conditions (5%v/v
H2O/CH3CN).288
amine functional group, respectively, were rationally designed
to emulate the distal histidine residue in peroxidases. A
structurally analogous iron porphyrin without basic moiety,
FeMPh (33A), containing a pendant benzyl group in the distal
site had been employed as control. The formation of a high
valent iron−oxo intermediate was observed in the reaction of
these iron(III)−porphyrin complexes with m-CPBA at low
temperature using stopped-flow kinetics. This intermediate had
been trapped and unequivocally identified at a compound I
species by 9-GHz EPR spectroscopy at 4K. The broad (2000
G) and axial EPR spectrum of an exchange-coupled
oxoferrylporphyrin radical species, the [(FeIV�O)Por*+]
with gO5eff = 3.80 and g||eff = 1.99, was observed upon reaction
of 33D (FeL3) with m-CPBA. The formation of this reactive
intermediate was much faster for 33C and 33D than that of the
benzyl analogue, 33A. The khetero values of O−O bond
heterolysis were determined to be more than 2 orders of
magnitude faster in 33C and 33D with respect to 33A which
provided definitive proof of the advantage of having pendant
nitrogenous bases during O−O heterolytic cleavage. The khetero
values for compound I formation for these two complexes were
much higher than some other previously reported model
complexes (Table 9).274,280 In contrast, no compound I
intermediate was observed during the reaction of parent
FeTPP (17A) with m-CPBA under identical conditions rather
it showed a slow formation of isoporphyrin, a species
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Table 9. Rate of O−O Heterolysis and the Corresponding Scheme 13. Proposed Mechanism of the Catalytic Cycle for
Activation Parametersa the Formation of High-Valent Intermediates Starting from
Iron(III) Porphyrins in the Presence of m-
temp ΔG⧧
catalyst kheterolysis (s−1) (°C) (kcal/mol−1) ref Chloroperbenzoic Acid (Oxidant) and One-Electron
45A 16.6 × 10 −3
−80 12.78 276
Donor/Substrate (S)303
45B 4 × 10−3 −80 13.33 276
45D 3.94 × 10−3 −40 16.19 276
45E 0.5 × 10−4 −80 14.14 276
45C (6.5 ± 1)×10−3 −40 15.96 ± 0.1 270
46B (6 ± 3)×10−3 −40 15.99 ± 0.2 270
46C (1 ± 0.2)×10−2 −40 15.76 ± 0.1 270
33C 21.67 ± 1 −30 12.72 ± 0.02 299
33D 69.6 ± 2 −30 12.15 ± 0.01 299
33A (8.5 ± 1)×10−2 −30 15.08 ± 0.05 299
HRP 200−500 25 13−14 295, 302
H42A/H42 V (5−50)×10−4 25 21 ± 1 53, 303
a
m-CPBA was used as the oxidant for all of the catalysts except for
those used in refs 53, 295, and 303 (where hydrogen peroxide is the
oxidant).

isoelectronic with compound I and an intermediate in heme

degradation of heme oxygenase cycle, that has characteristic
bands at 810 and 900 nm.304,305 The formation of isoporphyrin
was not only very minor but also sluggish in 33C and 33D,
indicating that the pendent basic groups avoid degradation of
the metallopophyrins upon their reaction with peroxyacids.
The energy barrier for O−O heterolysis was found to be
very low (12−13 kcal/mol) for 33C and 33D (Table 9)
relative to previously investigated iron porphyrin complexes
including the hangman porphyrins. The activation parameters
for compound I formation of FeL2 were determined to have
ΔH⧧ = 2.34 ± 0.2 kcal/mol) and ΔS⧧ = −44.60 ± 1 cal mol−1
K−1. The remarkably low enthalpic barrier and large negative
entropy of activation indicated a very facile O−O bond
cleavage step. The corresponding activation energy barrier
ΔG⧧ for HRP (∼13−14 kcal/mol) was higher than those
achieved in 33C and 33D (Table 9). Additionally, the three
times faster rate and 0.5 kcal/mol lower ΔG⧧ of 33D with an
aliphatic amine (pKa ∼ 16.9 in CH3CN) than 33C with a
pyridine group (pKa ∼ 12.33 in CH3CN) suggested that the
O−O bond heterolysis was more favored by a pendent amine
with higher basicity than the peroxyacid. Therefore, these two
residues, in principle, easily acted as efficient acid−base catalyst
capable of translocating proton from proximal oxygen atom of
bound m-CPBA to the distal oxygen atom that eventually
facilitated the heterolytic O−O bond cleavage (Scheme 13).
Note that mutation of the His42 residue in native HRP led to
several orders of magnitude decrease in the rate of compound I
formation via O−O bond heterolysis,55,299,306,307 which was
reasonably captured by 2 orders of magnitude decrease in the
rate of compound I formation for 33A relative to 33C and
33D. Thus, the “pull effect” operating by the distal histidine
residue in peroxidases was successfully mimicked in synthetic
models including pendent basic groups (analogous to histidine
residue) in the second coordination sphere of the iron
porphyrin. The proposed enhancement in O−O bond
heterolysis in FeL2, FeL3 and Fe-MARG based on in situ
SERRS-RDE data obtained during electrocatalytic ORR,
indicated the same.
DFT calculations provided further insights into the effect of
pendent basic groups covalently attached to iron porphyrins in
relation to such O−O bond cleavage. The distal oxygen atoms
12415
of FeIII−OOH species of these complexes (33C and 33D)
were involved in strong H-bonding interactions with the
protonated pendant bases and the O−O bond distances are
elongated to 1.88 and 1.86 Å in their corresponding DFT-
optimized structures of ferric hydroperoxide species of 33C
and 33D, respectively (Figure 38).197 A molecular orbital
(MO) diagram was constructed to elucidate the bonding in a
putative compound 0 intermediate of 33C and 33D having
pendant pyridine and amine functional groups. The substantial
activation of the O−O bond of a FeIII−OOH species with H-
bonding by a protonated base was the root of the “pull effect”
proposed in peroxidase active sites.46,253,308 The enhanced
back-bonding from the Fe 3d orbitals was facilitated by the
lowering of the O−O σ* orbital energies due to hydrogen
bonding interactions with the pendent basic groups of [FeIIIL2-
OOH]H+ (38C) and [FeIIIL3-OOH]H+] species (38D). This
strong H-bonding polarized the electron density of the O−O
bond toward the distal OH group reducing the overlap
between the two O2p orbitals as a result of which the O−O
bond was weakened and the O−O σ* orbital energy was
lowered. The DFT calculated wave functions showed that the
Fe dπ character in the O−O σ* orbitals significantly increased
from 4.86% in the non H-bonded system to 36.89% and
38.69% in FeL3 and FeL2, respectively (Figure 47). Thus, the
“pull effect” might be interpreted as pulling the energy of the
O−O σ* orbital down substantially by hydrogen bonding to a
protonated basic residue to allow better back-bonding into the
O−O σ* orbital from filled Fe dπ orbital of the metal. The O2p
coefficient increased in the Fe dπ orbital from 3.62% in
FeIIITPP-OOH to 18.05% in [FeIIIL3-OOH]H+ and 17.55% in
[FeIIIL2-OOH]H+, signifying increased covalent donation
from the proximal oxygen to the vacant Fe dπ orbital, i.e.,
strong π bonding (Figure 47). A similar increase in σ covalency
was observed in the Fe dz2 orbital. These were consistent with
the shortening of Fe−O bond upon elongation of the O−O
bond converting the reactant FeIII−OOH to the product
[(FeIV�O)Por*+].
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Figure 47. Calculated (BP86) ground-state MO diagram of FeIIITPP-OOH (no H-bonding residue), [FeIIIL3-OOH]H+ and [FeIIIL2-OOH]H+
(protonated H-bonding residue). The relative energies are normalized with respect to the nonbonding dxy orbital. Only the β orbitals are shown.
The orbital contributions of the occupied dxz orbital and the unoccupied O−O σ* orbital are shown. The contributions of dz2 and the dxy orbitals of
the Fe that σ and π bonds, respectively, to the hydroperoxide are also shown. The nonbonding dxy and the dx2‑y2 are not shown for clarity.303

Inclusion of pendent basic groups in iron porphyrins
mimicking the distal basic residues in the active site structure
of peroxidases, enabled facile O−O bond heterolysis of
peroxyacid lowering the activation energy barrier for the
generation of high-valent compound I intermediate and in
doing so allow the “pull effect” exhibited by the secondary
coordination environment of the heme cofactor of heme
hydroperoxidase enzymes to be modeled.
6. CONCLUSION
Inclusion of second-sphere residues in the design of small-
molecule artificial bioinspired complexes has produced some
remarkable advancements in our understanding of how these
weak interactions play vital roles in attenuating the reactivity of
metallo-enzymes and has accelerated the community along
toward generating functional mimics of these naturally
occurring elegant catalysts. The nature of second-sphere
residues varies substantially and so does the effect it has on
the reactivity of the active site. A detailed understanding of the
nuances introduced by the second-sphere residues in the
geometric and electronic structure of the reactive species in the
active site is prerequisite to successful implementation of these
design principles in small molecules. The dramatic improve-
ments in O−O bond cleavage in small molecule catalysts on
switching pendant carboxylic acids with pendant ammines
(mimics histidine and arginine in HRP) as well as the clear
advantage of a properly positioned phenol in affecting O−O
bond cleavage are good examples of that. The examples
discussed here, while limited to O2 and H2O2 activation by
heme active sites, succinctly demonstrate how these synthetic
12416
and biosynthetic models provide convenient platforms for
understanding the finer attributes of metalloenzyme activity
outside the protein matrix. In addition to understanding the
reactivity, some recent examples have mimicked the catalytic
function of the enzymes as well which has been a long-term
goal of the bioinspired community. It is perhaps not too
premature to state that judicious inclusion of second sphere
interactions in synthetic models likely hold the key for the
transition from structural modeling to functional modeling, a
much needed change in characterization of the bioinspired
community.
AUTHOR INFORMATION
Corresponding Author
Abhishek Dey − School of Chemical Science, Indian
Association for the Cultivation of Science, Kolkata 700032,
India; orcid.org/0000-0002-9166-3349;
Email: abbeyde@gmail.com
Authors
Sarmistha Bhunia − School of Chemical Science, Indian
Association for the Cultivation of Science, Kolkata 700032,
India
Arnab Ghatak − School of Chemical Science, Indian
Association for the Cultivation of Science, Kolkata 700032,
India
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrev.1c01021
https://doi.org/10.1021/acs.chemrev.1c01021
Chem. Rev. 2022, 122, 12370−12426
Chemical Reviews pubs.acs.org/CR Review

Author Contributions PT proton transfer

†
S.B. and A.G. contributed equally. Q quinone
RDE rotating disc electrochemistry
Notes
RDS rate-determining step
The authors declare no competing financial interest. rR resonance Raman
Biographies RRDE rotating ring disc electrochemistry
SAM self-assembled monolayer
Sarmistha Bhunia received her B.Sc. (2013) from Midnapore college SERRS surface-enhanced resonance Raman spectroscopy
and M.Sc. (2015) from the Indian Association for the Cultivation of SERRS-RDE surface-enhanced resonance Raman spectros-
Science. She joined as a Ph.D. scholar in the same institute under the copy coupled to rotating disc electrochemistry
supervision of Prof. A. Dey in 2015. Her current research focuses on SKIE solvent kinetic isotope effect
synthesis and reactivity of iron porphyrin complexes having second SQ semi quinone
sphere distal residues. TBPH 2,4,6-tritert-butylphenol
Arnab Ghatak received his B.Sc. (2014) from Presidency University, TMB tetramethylbenzidine
Kolkata, and M.Sc. (2016) from Indian Institute of Technology TOF turn over frequency
Kharagpur. He joined the Indian Association for the Cultivation of TON turn over number
Science as a Ph.D. scholar under the supervision of Prof. A. Dey in Tyr• tyrosyl radical
2016. His current research focuses on the spectroscopic investigation Fc* decamethyl ferrocene
of the mechanism of oxygen reduction reaction catalyzed by iron
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