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Electrocatalytic O2 Reduction by Monolayer of Hemin: Role of pKa of Distal

and Proximal Oxygen of a FeIII-OOH Species in Determining Reactivity

Article  in  Chemical Communications · August 2014

DOI: 10.1039/C4CC03886J

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Electrocatalytic O2 reduction by a monolayer of

hemin: the role of pKa of distal and proximal
Cite this: DOI: 10.1039/c4cc03886j
oxygen of a FeIII–OOH species in determining
Received 23rd May 2014,
Accepted 10th August 2014
reactivity†
DOI: 10.1039/c4cc03886j Sohini Mukherjee, Sabyasachi Bandyopadhyay, Sudipta Chatterjee and
Abhishek Dey*
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Investigation of the proton coupled electron transfer steps involved
in electrocatalytic ORR by hemin modified electrodes allows the
determination of pKas of the proximal and distal oxygen atoms of a
rate determining FeIII–OOH species.
Iron porphyrins, synthetic analogues of heme, catalyze O2 reduction
reaction (ORR).1–4 The mechanism for electrocatalytic oxygen
reduction by these electrocatalysts has been investigated by several
groups. The reduction is known to proceed either by a 2e /2H+
pathway producing H2O2 or by a 4e /4H+ pathway producing
H2O.5,6 Different chemical attributes and structural motifs of these
catalysts influence their mechanism of action. Most investigations
in the past employed multi-layers of catalysts physiabsorbed on
electrodes.6 There are fewer reports of O2 reduction by site-isolated
monolayers of catalysts and the mechanisms of electrocatalytic ORR
are not yet explored in detail.2 Like homogeneous catalysis electro-
catalytic ORR is characterized by rate determining state(s) (rds) i.e.
steps having higher kinetic barriers. Additionally there is a potential
determining step (pds)7 corresponding to the step which requires
most thermodynamic driving force i.e. the step involving lowest
reduction potential during ORR. Mechanistically the O–O bond
cleavage of a FeIII–OOH species is significant in several biological
systems.8 In cytochrome P450 and peroxidases the O–O bond is
cleaved following a proton coupled electron transfer to this species
to generate the main oxidizing species in these oxidases known as
compound I.9,10 This requires the protonation of the distal oxygen of
the FeIII–OOH species as the protonation of the proximal oxygen
leads to the release of H2O2.3,11 However due to the instability of
these FeIII–OOH species in an aqueous environment, there is no
direct experimental estimation of the pKa of the bound hydroperoxo
ligand which clearly is the key in determining its reactivity.12
Recently the ORR by iron porphyrin complexes, physiadsorbed on
SAM, was investigated using a SERRS-RDE set up.13 The data
indicated the presence of low spin FeIII–OOH, FeIVQO and FeII
species on the electrode involved in steady state ORR. This suggested
that the decay of these three species was the rds of ORR. Hence, it is
desirable to investigate the mechanism of ORR on a single monolayer
of catalyst having a well defined spacing from the electrode.
In this communication we report the reactivity of a dilute site
isolated single monolayer of a heme based Fe porphyrin electrocata-
lyst (hemin-1Fe) installed at well defined distances from the electrode
using ‘‘click’’ chemistry (Scheme S1.3, ESI†). These electrodes are
characterized in situ using surface enhanced resonance Raman
spectroelectrochemistry (SERRS) and used to investigate the O2
reduction mechanism of the covalently attached species in buffered
solutions with and without externally added imidazole ligands at
different pH values. Analysis of the data using proton coupled
electron transfer (PCET) formalism allows determination of the pds
of the electrocatalytic ORR as well as the pKas of both the proximal
and distal oxygen atoms of a rate determining FeIII–OOH species.
The SERRS of the hemin-1Fe modified electrode held at an
oxidizing potential of 0 V shows the presence of a six coordinate
high spin (6-C-H.S) (n4 and n3 at 1370 and 1489 cm 1) FeIII
species in a pH 7 phosphate buffer and the presence of a six
coordinate low spin (6-C-L.S) (n4 and n3 at 1375 and 1507 cm 1)
FeIII species in a 100 mM imidazole (imz) pH 7 buffer (Fig. 1A).

Indian Association for the Cultivation of Science, Department of Inorganic Chemistry,

2A & 2B Raja S.C Mullick Road, Jadavpur, Kolkata, India. E-mail: icad@iacs.res.in;
Tel: +91-33-2473 4971-1366 Fig. 1 SERRS spectra of a hemin-1Fe modified electrode in both (A)
† Electronic supplementary information (ESI) available: Additional SERRS, electro- oxidized (at 0 V) and (B) reduced (at 0.5 V) states in pH 7 phosphate
chemical data and reaction pathway diagrams. See DOI: 10.1039/c4cc03886j buffer with and without 100 mM imidazole, respectively.

This journal is © The Royal Society of Chemistry 2014 Chem. Commun.


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The nature of the H.S FeIII species is found to be independent of pH
between pH 5 and 9 (S2.1, ESI†). The FeIII, FeII species are 6C LS
even at low pH (pH 5) in 100 mM imidazole buffer solution (S2.2,
ESI†). Upon changing the applied potential, on the disc, to 0.5 V
under completely anaerobic conditions, i.e., where the iron in the
Published on 22 August 2014. Downloaded by Indian Association for the Cultivation of Science on 02/09/2014 12:41:05.
heme is reduced, the presence of a mixture of five coordinate high
spin (5-C-H.S) FeII, intermediate spin (IS) FeII and 6-C-L.S FeII
species is observed at pH 7 (Fig. 1B) consistent with previous
reports on hemin.14 However, in 100 mM imidazole (pH 7) buffer,
the n4, n3 bands completely shift to 1360 and 1496 cm 1 (Fig. 1B),
respectively, indicating the formation of a pure six coordinate low-
spin FeII species at these potentials.14–17
The pH dependencies of electrocatalytic ORR by hemin-1Fe are
measured by linear sweep voltammetry (LSV). The electrocatalytic
oxygen reduction gradually shifts to a more negative potential as
the pH increases from 4 to 11. The plot of potential of inflection
(E(i)) of the ORR current (i.e. maxima of the 1st derivative) of oxygen
reduction against pH shows an B60 mV negative shift per unit
increase in pH in the pH range 4–11 consistent with a proton
coupled electron transfer (PCET) process18–20 (S3, ESI†) being the
pds. In the pH range 4–9, a similar B60 mV negative shift per unit
increase in pH is observed for the E(i) of hemin-1Fe when the buffer
contains 100 mM imidazole. The E(i) of electrocatalytic ORR can be
simulated by a PCET process over a large range of pH (Fig. 2) and
the pKa values of the species involved in the pds of the ORR process
are thus determined (Table 1).21 Thermodynamically, the 1e /1H+
redox couple behaves like a simple 1e redox couple with a pH
dependent reversible formal potential Er (given by eqn (1)).
Er = Em + (2.3RT/F)  log((Ka1/Ka2)1/2([H+] + Ka2)/([H+] + Ka1))
(1)
here Em = (E01 + E02)/2. Ka1, Ka2 are the acid dissociation constants
and E01, E02 are the formal potentials of the PET (proton electron
transfer) and EPT (electron proton transfer) pathways respectively.
In the most commonly accepted ORR mechanism (Scheme 1),
Fig. 2 PCET simulation of E(i) obtained from the LSV at different pH (A)
without imidazole (B) with 100 mM imidazole.
Table 1 Em, pKa1 and pKa2 or pKa obtained for the PCET or single protonation processes involved in electrocatalytic ORR
Phosphate buffer without any imidazole
Em (V) pKa1 pKa2
E1/2 (step i) 0.240  0.01 3.5  0.1 7.7  0.1
E(i) 0.300  0.01 3.5  0.1 10.2  0.1
PROS (step viii) — — — —
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steps (i), (iii), (iv) and (v) can, in principle, involve both protons and
electrons.13,22 Step (v) involves reduction of FeIVQO species to a
FeIII–OH which typically has a very large driving force (E1/2 B 1 V)23
as during electrocatalytic ORR, the potential of the electrode is held
below 0.2 V. Hence step (v) may not be the pds. Most likely, either
step (i), (iii) or (iv) is the pds i.e. origin of the PCET involved in ORR
by hemin-1Fe. The Tafel slope of the ORR current, which reflects
the rate of ORR, in this pH range varies between 100 to 164 mV per
decadoic increase in current (S6, ESI†). This indicates that the rds
in ORR likely involves a single electron transfer in this pH range.
The experiments performed in the absence of O2 in the buffer
allow investigation of the pH dependence of the reduction of FeIII
porphyrin to FeII porphyrin i.e. step (i) in Scheme 1. The CV of the
covalently modified electrode in an anaerobic buffer shows a quasi-
reversible FeIII/FeII (S7, ESI†). The plot of E1/2 against pH for both
the buffers with and without 100 mM imidazole shows that it
becomes more negative as the pH is increased from 3 to 8 and then
saturates (S8, ESI†). The data, which show a slope of 60 mV per pH
between pH 3 and 8, are indicative of a proton coupled electron
transfer (PCET) process and not protonation equilibrium. Simula-
tion of the plot of E1/2 against pH with a PCET model satisfactorily
yields the pKa values involved in the FeIII/FeII reduction process in
the buffers with or without 100 mM imidazole (Table 1). The
differences in pKa1 and pKa2 values estimated in buffer solution
with and without imidazole are likely due to the imidazole binding
to the hemin-1Fe in the oxidized and reduced form as indicated by
the SERRS data. These values are quite different from those
involved in the electrocatalytic ORR process i.e. E(i) signifying that
the FeIII–OH to FeII–H2O reduction is not the pds of ORR. Thus
either step (iii) or step (iv) (Scheme 1) is the pds of the ORR
catalyzed by hemin-1Fe.
Reaction pathway diagrams can be considered for step (iii) and
step (iv) (Scheme 2A and B). Very importantly for either step (iii) or
(iv) to be the pds, both the pKa1 and pKa2 obtained from the PCET
analysis of Ei must be less than that of free superoxide in water,
B4.8,24–26 (Scheme 2A) or of the free H2O2, 11.89,27,28 (Scheme 2B),
respectively, as metal binding lowers the pKa of the bound ligand
from its value in solution. For the electrocatalytic ORR mechanism,
the pKa1 of 3.5 is lower and pKa2 of 10.2 is higher than that of
the free superoxide in water, B4.8. Thus step (iii) (Scheme 2A) is
not responsible for the PCET behaviour of the ORR observed
i.e. step iii is not the pds of the ORR. Both of these experimental
pKa values are however lower than the pKa of free H2O2, 11.89
(Scheme 2B). Thus the PCET behaviour of the ORR is consistent
with a pds which involves the protonation of the FeIII–OOH species
followed by the O–O bond cleavage and finally leading to the
formation of FeIVQO species. Using the same argument for ORR
in the presence of imidazole, where the SERRS data indicate that
Phosphate buffer with imidazole
Em (V) pKa1 pKa2 pKa
0.27  0.01 3.3  0.1 7.7  0.1 —
0.32  0.01 4.25  0.05 9.65  0.05 —
— — 5.35  0.05
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Scheme 1 Mechanism of O2 reduction by iron porphyrin.
Scheme 2 Reaction pathway diagram presenting (A) the conversion of
FeIII–O2  to FeIII–OOH (B) the conversion of FeIII–OOH to FeIVQO.
the ferric and ferrous species are low spin, the pKa1 and pKa2 values
of E(i) (4.25 and 9.65, Table 1) are consistent with a PCET decay of
FeIII–OOH (step iv. in Scheme 1) to FeIVQO, being the potential
determining step in this case as well. Note that a six coordinate
heme FeIII–OOH species with either water or imidazole trans axial
ligand will be low spin even if the resting ferric or ferrous
complexes are not.13
The amount of PROS generated, due to incomplete reduction of
O2 to H2O2 and O2 , by the monolayer of hemin-1Fe varies with
pH. PROS analysis indicates that the catalyst produces 8.5  1.5%
H2O2 during O2 reduction in pH 7 buffer without any imidazole
and it steadily decreases from 12% at pH 5 to 2  1.5% as the pH
is increased to 8 (S9.A, ESI†). This indicates that the 4e /4H+
reduction of O2 to H2O dominates at higher pH values producing
less PROS. An almost similar trend is followed in the case of buffer
with 1 mM (S9.B, ESI†) or 100 mM imidazole (S9.A, ESI†) with
the only difference that the PROS in 100 mM imidazole buffer are
2–4 times less than those measured in buffer containing no
imidazole (S9.A, ESI†) and are B10 times less than the previously
reported value for a catalyst bearing a covalently bound imidazole
ligand.1 The pH dependence of the PROS generation by the
electrocatalyst hemin-1Fe in buffers with no imidazole, cannot be
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Fig. 3 (A) Simulation of experimental points for PROS at phosphate buffer
of different pH with 100 mM imidazole (pKa model fit). (B) Overlay of both
E1/2 and PROS with pH in buffer (50 mV s 1 scan rate, 300 rpm rotation
speed, Ag/AgCl reference electrode and Pt counter electrode).
fitted with single protonation equilibrium. However, in the
presence of 100 mM imidazole in the buffer the pH dependence
can be fitted easily with a single protonation pathway with a pKa of
5.35  0.05 (Fig. 3A). This indicates that in the case of buffer with
100 mM imidazole, the PROS production doesn’t involve any
electron transfer; it only involves protonation.
Mechanistically production of PROS reflects the rate of hydro-
lysis of the oxygen bound species, like FeIII–O2  (or FeII–O2) or
FeIII–OOH. PROS can be produced in three different ways – by a
single protonation of either FeIII–O2  (step viii. in Scheme 1)
species or of FeIII–OOH species (step vi. in Scheme 1) or else it
can be produced by outer sphere e transfer from the reduced FeII–
H2O species to the oxygen dissolved in the bulk solution.29 The
overlay of the PCET behaviour for the reduction of FeIII–OH and
PROS shows remarkable similarity (Fig. 3B) implying that the PROS
is produced from the reduced FeII–OH2 species by outer sphere
electron transfer from FeII–H2O species to the oxygen, also reported
for a low spin thiolate bound iron porphyrin complex.29 However
in buffer solutions containing imidazole, the pH dependence of
E1/2 for FeIII/FeII reduction (PCET with pKa1 B 3.3  0.1, pKa2 B
7.7  0.1), E(i) for O2 reduction (PCET with pKa1 B 4.25  0.05,
pKa2 B 9.65  0.05) and PROS production (pKa B 5.35  0.05)
are all different indicating that PROS is neither produced by the
potential determining step of ORR nor by the outer sphere reduction
of O2. The PROS, in a buffer containing imidazole, is likely produced
by the protonation at the proximal oxygen of the FeIII–OOH species
(step (vi), Scheme 1) which does not involve a simultaneous electron
transfer. Hence, the pKa of 5.35  0.05 for the PROS production step
is the pKa of the proximal oxygen of a FeIII–OOH species in the buffer
containing 100 mM imidazole. Note that PROS production by step
(viii) is unlikely as it involves protonation of electron deficient
proximal oxygen bound to the FeIII metal centre.
The analyses of these electrochemical data suggest that the
pKa1 of E(i) is the pKa for the protonation of the distal oxygen of
FeIII–OOH species in the pds and is 3.5. Similarly the pKa of the
distal oxygen of an imidazole bound FeIII–OOH species is 4.25.
Thus the replacement of axial H2O with imidazole raises the
pKa of the distal oxygen of the trans-OOH ligand by B1 unit
(Table S2, ESI†). Using a co-relation between pKa and proton
affinity (PA) in the gas phase30 (DpKa = DPA/1.37) it can be
estimated that the PA of the distal oxygen of a imidazole bound
FeIII–OOH is 1.027 kcal mol 1 (Table S2, ESI†) more than that of
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Communication
a H2O bound FeIII–OOH species. This suggests that imidazole,
being a stronger donor than H2O, increases the PA of the trans-
OOH ligand. A higher PA leads to a higher pKa of the distal
oxygen of the FeIII–OOH species with a trans imidazole ligand.
Similarly the analysis of the PROS data reveal that the pKa of
Published on 22 August 2014. Downloaded by Indian Association for the Cultivation of Science on 02/09/2014 12:41:05.
the proximal oxygen of the potential determining FeIII–OOH
species is 5.35 which is B1 unit greater than that of the distal
oxygen (4.25). Thus the proximal oxygen has 1.507 kcal mol 1
PA higher than the distal oxygen.
In summary, Au electrodes bearing azide functionalized thiols
have been constructed with alkyne modified hemin-1Fe by covalent
attachment using ‘‘click’’ chemistry. Analyses of the electrochemical
data obtained for the hemin-1Fe modified electrode suggest that a
PCET decay of FeIII–OOH to FeIVQO is likely to be the pds of the
electrocatalytic ORR mechanism both for a water or imidazole axial
ligand. Heme FeIII–OOH species with a trans imidazole ligand has
been proposed to be key intermediates in peroxidases, CcO as well
as in non-heme iron systems.31–34 To date there has been no
experimental estimation of the pKas of these oxygen atoms or their
relative PAs. The data and analysis reveal that the pKa of the distal
oxygen atom of a heme FeIII–OOH species is 3.5 and 4.25 with an
axial H2O and imidazole ligand, respectively. In most known
enzyme active sites this intermediate undergoes protonation of
the distal oxygen followed by O–O bond heterolysis. The data
obtained there indicate that the proximal O centre has a higher
pKa i.e. it is easier to protonate.35–37 The DpKa of 1.1 (pKa of proximal
oxygen – pKa of distal oxygen) for the imidazole bound hemin-1Fe,
favouring the protonation of the proximal oxygen, translates into a
1.51 kcal mol 1 higher proton affinity of this proximal oxygen
relative to the distal oxygen. This additional energy is likely to be
provided by the hydrogen bonding between the conserved arginine
and histidine residues in the distal pocket of peroxidases and
forms the very basis of the Poulos–Kraut mechanism of O–O bond
heterolysis in peroxidases.38–40
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