pubs.acs.

org/accounts Article

Local Charge Distributions, Electric Dipole Moments, and Local

Electric Fields Influence Reactivity Patterns and Guide
Regioselectivities in α‑Ketoglutarate-Dependent Non-heme Iron
Dioxygenases
Sam P. de Visser,* Gourab Mukherjee, Hafiz Saqib Ali, and Chivukula V. Sastri*

Cite This: Acc. Chem. Res. 2022, 55, 65−74 Read Online

ACCESS Metrics & More Article Recommendations

CONSPECTUS: Non-heme iron dioxygenases catalyze vital processes for human health
related to the biosynthesis of essential products and the biodegradation of toxic
metabolites. Often the natural product biosyntheses by these non-heme iron dioxygenases
is highly regio- and chemoselective, which are commonly assigned to tight substrate-
binding and positioning. However, recent high-level computational modeling has shown
that substrate-binding and positioning is only part of the story and long-range electrostatic
interactions can play a major additional role.
In this Account, we review and summarize computational viewpoints on the high regio-
and chemoselectivity of α-ketoglutarate-dependent non-heme iron dioxygenases and how
external perturbations affect the catalysis. In particular, studies from our groups have
shown that often a regioselectivity in enzymes can be accomplished by stabilization of the
rate-determining transition state for the reaction through external charges, electric dipole
moments, or local electric field effects. Furthermore, bond dissociation energies in
molecules are shown to be influenced by an electric field effect, and through targeting a specific bond in an electric field, this can lead
to an unusually specific reaction. For instance, in the carbon-induced starvation protein, we studied two substrate-bound
conformations and showed that regardless of what C−H bond of the substrate is closest to the iron(IV)-oxo oxidant, the lowest
hydrogen atom abstraction barrier is always for the pro-S C2−H abstraction due to an induced dipole moment of the protein that
weakens this bond. In another example of the hygromycin biosynthesis enzyme, an oxidative ring-closure reaction in the substrate
forms an ortho-δ-ester ring. Calculations on this enzyme show that the selectivity is guided by a protonated lysine residue in the
active site that, through its positive charge, triggers a low energy hydrogen atom abstraction barrier. A final set of examples in this
Account discuss the viomycin biosynthesis enzyme and the 2-(trimethylammonio)ethylphosphonate dioxygenase (TmpA) enzyme.
Both of these enzymes are shown to possess a significant local dipole moment and local electric field effect due to charged residues
surrounding the substrate and oxidant binding pockets. The protein dipole moment and local electric field strength changes the C−
H bond strengths of the substrate as compared to the gas-phase triggers the regioselectivity of substrate activation. In particular, we
show that in the gas phase and in a protein environment C−H bond strengths are different due to local electric dipole moments and
electric field strengths. These examples show that enzymes have an intricately designed structure that enables a chemical reaction
under ambient conditions through the positioning of positively and negatively charged residues that influence and enhance reaction
mechanisms. These computational insights create huge possibilities in bioengineering to apply local electric field and dipole
moments in proteins to achieve an unusual selectivity and specificity and trigger a fit-for-purpose biocatalyst for unique
biotransformations.

■ KEY REFERENCES • de Visser, S. P.; Lin, Y.-T.; Ali, H. S.; Bagha, U. K.;
Mukherjee, G.; Sastri, C. V. Negative catalysis or non-
• de Visser, S. P. Second-coordination sphere effects on
selectivity and specificity of heme and non-heme iron Received: September 1, 2021
enzymes. Chem. - Eur. J. 2020, 26, 5308−5327.1 This Published: December 17, 2021
article reviews second-coordination sphere ef fects in enzymes
and assigns these as originating f rom substrate and oxidant
positioning, charge interactions, hydrogen-bonding interac-
tions, and salt bridges.

© 2021 American Chemical Society https://doi.org/10.1021/acs.accounts.1c00538

65 Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research pubs.acs.org/accounts Article

Figure 1. Examples of non-heme iron enzymes that bind iron to two histidine ligands and one carboxylate ligand. Crystal structure coordinates taken
from the 1MLW, 1OS7, and 2B1X PDB files.17−20

Scheme 1. Extract of the Catalytic Cycle of αKG-Dependent Non-heme Iron Dioxygenases and Three Examples of Oxidative
Transformations for Aliphatic C−H Hydroxylation (in CsiD), Oxidative Ring Closure (in HygX), and Desaturation/
Decarboxylation (in ScoE)

which is termed negative catalysis and enables enzymes

Bell−Evans−Polanyi reactivity by metalloenzymes: Ex-
amples from mononuclear heme and non-heme iron
oxygenases. Coord. Chem. Rev. 2021, 439, 213914.2 This
review highlights how enzymes often react selectively with
patterns opposite to the Bell−Evans−Polanyi reactivity,
unusual reactivity patterns for natural product biosynthesis.
• Mukherjee, G.; Satpathy, J. K.; Bagha, U. K.; Mubarak, M.
Q. E.; Sastri, C. V.; de Visser, S. P. Inspiration from
Nature: influence of engineered ligand scaffolds and
auxiliary factors on the reactivity of biomimetic oxidants.

66 https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
ACS Catal. 2021, 11, 9761−9797.3 This review summa-
rizes biomimetic studies on the second-coordination sphere
ef fects in synthetic model complexes with the aim to obtain
unusual reactivity pathways and generate novel products.
1. INTRODUCTION
Second-coordination sphere effects have a major influence on
enzymatic reactivity and selectivity, where rate-determining
transition states are guided by charge distributions of the local
environment through charged residues, hydrogen bonding
interactions, and local electric field effects.1−3 These long and
mid-range electrostatic interactions include induced electric
dipole and electric field perturbations on an active site and
thereby affect electronic configurations and electron transfer
processes. In this Account, we will highlight several recent
discoveries on second-coordination sphere effects influencing
selectivity patterns in non-heme iron dioxygenases.
Oxygen-activating mononuclear iron enzymes are common in
biology and involved in important biosynthesis of natural
products that include hormones, for example, flavonols in plants
and serotonin in the human body, as well as antibiotics in
bacteria.4−11 In general, most of the reactions catalyzed by non-
heme iron dioxygenases are highly regioselective and stereo-
specific. In recent years, however, high-level computational
studies have shown that long-range electrostatic perturbations
play an important role in selectivity. Most mononuclear non-
heme iron dioxygenases bind iron to the protein in a facial ligand
orientation through interactions with two histidine ligands and
one carboxylate ligand in a so-called 2-His/1-Asp or 2-His/1-
Glu orientation (Figure 1).6,10 Therefore, the iron(II) in
octahedral symmetry leaves three coordination sites available
for other ligands, substrate, and cosubstrate to bind. The overall
chemical reaction of non-heme iron dioxygenases reduces
dioxygen by four electrons and oxidizes the substrate. The
source of these electrons varies among the non-heme iron
dioxygenases; for example, in the case of tetrahydrobiopterin
(BH4)-dependent aromatic amino acid hydroxylases and α-
ketoglutarate (αKG)-dependent dioxygenases,11,12 the cosub-
strate (BH4 or αKG) acts as the electron source. By contrast, in
isopenicillin N-synthase, dioxygen reacts with a substrate on an
iron(II) center through four sequential hydrogen atom
abstraction reactions that deliver the four electrons.13,14 Finally,
the Rieske dioxygenases have two [Fe2S2] clusters in the vicinity
of the active site that supply the electrons for the catalytic
mechanism.15,16 Examples of the active site and chemical
structures of tryptophan hydroxylase, taurine/αKG-dependent
dioxygenase (TauD), and the Rieske dioxygenase naphthalene-
1,2-dioxygenase as taken from the 1MLW, 1OS7, and 2B1X
protein databank (PDB) files are shown in Figure 1.17−20
Tryptophan hydroxylase has the characteristic 2-His/1-Glu
active site and binds BH4 cosubstrate in the vicinity of the iron,18
while TauD has a 2-His/1-Asp ligand system with αKG directly
bound to the iron(II) center as a bidentate ligand.19 The taurine
substrate is bound in the vicinity of the iron atom but is not
ligated to it. The Rieske dioxygenase structure has the iron
bound to a 2-His/1-Asp ligand structure with an Fe2S2 cluster of
another chain in close proximity.20 The Fe2S2 cluster is bound to
two Cys and two His residues of the other chain and is expected
to deliver electrons to the catalytic iron center. Consequently,
non-heme iron dioxygenases have differences in structure and
catalytic mechanism, but all generate a high-valent active oxidant
for the hydroxylation of unactivated C−H bonds. In this work,
67
pubs.acs.org/accounts Article
we will focus on the structure and reactivity of αKG-dependent
non-heme iron dioxygenases only as examples of how external
perturbations affect their activity and substrate conversion
processes.
Experimental and computational studies established details of
the catalytic cycle of αKG-dependent non-heme iron
dioxygenases,21−23 and the cycle is highlighted in Scheme 1.
The cycle proceeds in two stages, whereby in stage 1 substrate
(SubH), cosubstrate (αKG), and dioxygen bind into the active
site and the iron(II) is activated to form a high-valent iron(IV)-
oxo species concomitant with decarboxylation of αKG to form
succinate (Succ).2,4−9 In stage 2 of the catalytic cycle, the oxygen
atom transfer reaction from the iron(IV)-oxo species to the
substrate takes place. For several non-heme iron dioxygenases,
the iron(IV)-oxo species could be trapped and characterized
spectroscopically using resonance Raman, UV−vis absorption
and Mössbauer spectroscopy measurements.21,22 It has been
identified as the active oxidant of non-heme iron dioxygenases
and generally reacts through oxygen atom transfer to substrates,
thereby converting an aliphatic group into an alcohol (as shown
by the example of the carbon-induced starvation protein CsiD in
Scheme 1). Other oxidative transformations that have been
found for non-heme iron dioxygenases include the oxidative
ring-closure reaction as seen, for instance, in the hygromycin
biosynthesis enzyme (HygX) that forms an ortho-δ-lactone ring
and the oxidative decarboxylation of a Gly residue in a peptide
chain to form an isonitrile group in ScoE.24,25 Nevertheless, all
these oxidative transformations start with a hydrogen atom
abstraction step by an iron(IV)-oxo intermediate.
Structurally, the αKG-dependent non-heme iron dioxyge-
nases have an iron(II) resting state with the metal bound to the
protein through interactions with the side chains of two histidine
residues and a carboxylate group of either a Glu or Asp residue.
The two residues in the equatorial plane (one of the His and the
carboxylate ligand) in αKG-dependent non-heme iron dioxy-
genases (also seen in cysteine dioxygenase) are usually two
residues apart in the protein loop, that is, residue X and X + 2 in
the chain.1 An analysis of a selection of αKG-bound non-heme
iron dioxygenase structures from the protein databank shows the
αKG group to be bound as a bidentate ligand to iron(II) through
the carboxylate and keto groups.17 Furthermore, these structures
showed succinate to be held in a salt-bridge through its terminal
carboxylate group with the side chain of a conserved Arg residue
that is located 14 residues further down the chain from the axial
His residue (HisX13Arg) for several αKG-dependent non-heme
iron dioxygenases.1,26 This protein loop appears to be conserved
in many αKG-dependent non-heme iron hydroxylases and
positions αKG tightly in the binding pocket. Based on
computational studies a catalytic cycle for αKG-dependent
non-heme iron dioxygenases was established that starts with
dioxygen binding to the iron(II) center as an end-on iron(III)-
superoxo form trans to the axial histidine residue, which removes
the final water ligand of iron and initiates a reaction between
dioxygen and αKG that leads to its decarboxylation to form
succinate (Succ), CO2, and an iron(IV)-oxo species.23,27,28 The
iron(IV)-oxo species reacts with aliphatic groups by hydrogen
atom abstraction from a substrate (SubH) to form an iron(III)-
hydroxo species, which then relays the OH group to the
substrate via a radical rebound step to form an alcohol product
(SubOH). This reaction is often regio- and enantioselective, and
for instance, in the carbon starvation protein D (CsiD), a
glutarate substrate is converted into (S)-2-hydroxyglutarate as
the dominant product.29,30
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
Figure 2. Regio- and enantioselective reaction processes catalyzed by some non-heme iron dioxygenases. The values give DFT calculated homolytic
bond dissociation energies (BDEs) of various C−H bonds in the substrates in the gas phase (in kcal mol−1) as taken from refs 26, 39, 43, 47, and 48.
It has been proposed that the enzymatic selectivity originates
from a preorganized stabilization of the transition state due to
electrostatic interactions within the protein.31 Furthermore, it
also has been realized that the dielectric constant varies
throughout a protein and is not a constant value, and hence
this may affect the local charge distributions.32 Most of these
hypotheses originate from computational studies, while few
experimental reports have managed to provide evidence and
support of such findings. In particular, the measurement of the
electric field effect inside a protein is a daunting task. Moreover,
the acid dissociation constant (pKa) of enzymatic residues is
strongly influenced by the electric field around the basic amino
acid residues, for example, Lys and Arg. The protonated form of
these residues is more stabilized by a negative potential. In
recent computational studies, the effect of the binding of ions,
Na+, K+, Cl− etc., to the periphery of a P450 isozyme was
studied.33 The work showed that these long-range electrostatic
interactions of the bound cations and anions affected the redox
potential of the enzyme active site dramatically. As such, charge
distributions and local electric fields can shift dissociation
constants, pKa values, and redox potentials of charged residues
and cofactors in biological systems. Vibrational Stark spectros-
copy has been used to measure changes to protein function and
properties as a function of an applied electric field,34−37 in which
an immobilized small molecule or a large metalloprotein is
exposed to an external electric field and the resulting directional
effect on its infrared spectrum is observed.
Many αKG-dependent non-heme iron dioxygenases react
highly regio- and enantioselectively with substrate, and this often
happens as part of the biosynthesis of a natural product such as
an antibiotic or a hormone.4−9 In many of those biosynthesis
enzymes, the substrate is a natural amino acid, such as arginine,
proline, or glutamate, which acts as the template for the synthesis
of the natural product and is hydroxylated in the proc-
ess.29,30,38−43 Rate constant comparisons of substrate hydrox-
ylation reactions by biomimetic metal-oxo oxidants showed
them to correlate with the homolytic bond dissociation free
energy (BDE) of the sacrificial C−H bond of the substrate.44−46
Several examples of the substrates and reaction products
obtained for these biosynthesis enzymes are given in Figure 2
68
pubs.acs.org/accounts Article
with the respective BDE values of calculated C−H bond
strengths.26,39,43,47,48 Thus, prolyl-4-hydroxylase (P4H) chemo-
and enantioselectively hydroxylates the C4-position of a proline
residue in a peptide chain to form R-4-hydroxyproline, an
essential component of collagen strands that gives it its structure
and function.38 A series of computational studies on P4H,
however, actually showed that the C5−H bonds in proline are
significantly weaker than the C4−H bonds and hence C5-
hydroxylation should be the dominant reaction pathway.39,40 In
particular, the homolytic bond dissociation energies (BDEs) of
the C4−H and C5−H bonds were calculated to be 92.4 and 85.4
kcal mol−1, respectively. However, P4H activates the strong C4−
H bond rather than the weaker C5−H bond and hence reacts
through negative catalysis, where the thermodynamically
favored reaction channel of C5-hydroxylation is blocked by the
enzyme in favor of an alternative reaction pathway.2 How
enzymes achieve negative catalysis has been the topic of many
studies,2 and in this Account, we will show some examples of
how electrostatic interactions through charged residues and
local electric fields can perturb reaction channels and guide
reaction selectivities. Quantum mechanics/molecular mechan-
ics (QM/MM) on P4H showed the reaction selectivity to
originate from tight binding of the substrate in the substrate
binding pocket through positioning by aromatic residues and
hydrogen bonding interactions of a Tyr residue toward the oxo
group.40 This hydrogen bonding network guides the reaction to
C4-activation of the substrate and blocks access to the C5- and
C3-positions of the substrate.
Recent computational studies from our groups also
investigated the binding and activation of charged substrates
by αKG-dependent non-heme iron dioxygenases and show that
the local charge distributions, local dipole moments, and local
electric field perturbations affect substrate activation mecha-
nisms dramatically; hence enzymatic turnover is not only the
result of substrate-binding and positioning.47−49 Figure 2 shows
gas-phase calculated BDE values of a selection of substrates
utilized by αKG-dependent non-heme iron dioxygenases in
nature. Thus, there are several examples of αKG-dependent
non-heme iron dioxygenases where a chemo- or regioselective
reaction mechanism is performed by an arginine-activating
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
Figure 3. Active site cluster model of HygX and hydrogen atom abstraction barriers calculated from the reactant complex (5Re) for abstraction of the
tertiary C−H bond or the alcohol O−H group and the effect on the barrier height of the inclusion of the Lys137 residue in the model. Energies are in kcal
mol−1; data taken from ref 49.
enzyme.41,42 In particular, L-Arg activating enzymes include the
viomycin biosynthesis enzyme VioC, the naphthyridinomycin
biosynthesis enzyme NapI, and the arginine dihydroxylating
enzyme OrfP that acts as part of streptolidine biosynthesis.26 As
shown from the relative gas-phase homolytic BDEs of the C3−H
and C4−H bonds of an isolated arginine molecule, they are close
in energy, and consequently the activation of arginine should
give a mixture of products for both bond activation pathways. As
these arginine-activating enzymes are highly selective, this
means that they must block the unwanted reaction channels
from being activated. In the next sections, we will discuss what
modeling of VioC and OrfP has concluded on how these
enzymes react so selectively.
Another enzyme that activates a natural amino acid is the
carbon starvation-induced protein D (CsiD), which enantiose-
lectively hydroxylates glutarate to form S-2-hydroxyglutarate
products, while no R-2-hydroxyglutarate is observed.29,30 In the
gas phase, the calculated homolytic C−H bond strengths of the
pro-R and pro-S C2−H bonds of glutarate are virtually the same
(Figure 2), which would not explain the enantioselectivity of the
reaction. It was reasoned, therefore, that substrate-binding and
positioning trigger selectivity. Computational modeling, how-
ever, shows that regardless of substrate positioning and whether
the pro-R or pro-S bond is closest to the iron(IV)-oxo species,
the products formed are S-2-hydroxyglutarate only.43 Thus, the
studies investigated two substrate-binding models, whereby in
one of them, the pro-R C2−H bond was closest to the iron(IV)-
oxo species, while in the other model, the pro-S C2−H bond was
closest. Despite differences in substrate-binding and positioning
for both models, the lowest energy barrier was for the pro-S C2−
H hydrogen atom abstraction.43 It was shown that the active site
incurs a relatively large dipole moment on the substrate that
affects the individual C−H bond strengths and makes the pro-R
C−H bond stronger, so that the reaction is guided toward the S-
2-hydroxyglutarate reaction channel through electrostatic
perturbations.43
The final example shown in Figure 2 is for the 2-
(trimethylammonio)ethylphosphonate dioxygenase (TmpA),
which selectively hydroxylates the C1−H bond of the substrate
to trigger the glycine betaine biosynthesis.50 Interestingly, the
69
pubs.acs.org/accounts Article
calculated weakest C−H bond of 2-(trimethylammonio)-
ethylphosphonate in the gas phase is the C2−H bond and,47
therefore, TmpA reacts through negative catalysis where the
thermodynamically favored pathway is blocked in favor of an
alternative reaction channel, namely, C1−H bond hydroxyla-
tion.2 Below, we give details of our computational work on
TmpA and how we believe this selectivity is obtained. The bond
dissociation energies of the substrates shown in Figure 2
highlight that in most of these αKG-dependent non-heme iron
enzymes, it is not the weakest C−H bond of the substrate that is
being activated. To understand the origin of this reversal of
selectivity and how non-heme iron dioxygenases react through
negative catalysis, our groups have done a series of computa-
tional studies in the mechanism of substrate activation. These
studies have shown how polar residues and local electric fields
affect substrate positioning and selectivity patterns.1−3
Previous computational studies on a variety of enzymes have
shown that charged groups, hydrogen bonding interactions, and
even local electric field effects can influence the electronic
properties of the transition metal center, as well as its reactivity
patterns in metalloenzymes.51−56 In one of the first examples, it
was shown that the electronic configuration of cytochrome P450
compound I is influenced by hydrogen bonding interactions to
the axial ligand or external electric field perturbations.51,57 Thus,
P450 compound I is an iron(IV)-oxo heme cation radical species
with an axially ligated cysteinate group.58−60 Using a minimal
cluster model of a truncated heme and thiolate as the axial
ligand, the ground state was shown to have three unpaired
electrons in either an overall doublet or quartet spin state with
two metal-oxo type orbitals singly occupied, while the third was
a mixed thiolate−heme orbital labeled a2u.57 Enlarging the
model with hydrogen bonding interactions toward the thiolate
changed the character of this singly occupied a2u orbital and gave
it more heme contribution.57,60−62
Experimental studies on P450 variants with the axial
hydrogen-bonding network disrupted indeed confirmed spec-
troscopic changes to the enzyme active site.63−65 This could be
further accentuated by the addition of an external electric field
along the Fe−S bond.51 Similar ground state property changes
were seen when a computational model of cytochrome c
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
Figure 4. (a) Calculated bond dissociation energies of selected C−H bonds of 2-(trimethylammonio)ethylphosphonate substrate in TmpA enzymes
in the gas phase and with an external electric field along the y-axis (EY) applied. Values in kcal mol−1 from ref 47. The field direction is as defined in
Gaussian, with the arrows denoting the positive direction. (b) Calculated C1- versus C2-hydroxylation in the large model, the model truncated by the
protein but not its charges, and the fully truncated protein without charges.
peroxidase compound I was perturbed either by an electric field
effect or through the addition of point charges at a distance of 10
Å from the reaction center.66 In particular, the electronic
configuration of compound I through external perturbations was
seen to shift from a heme radical to a Trp radical depending on
the direction of the field or whether a positive or negatively
charged residue was present.66 Since then, many examples of
enzymatic systems where either local electric fields or local
charge distributions affect structure and activity have been
found.51−56 In particular, recent computational studies on a
range of non-heme iron biosynthesis enzymes showed them to
react through negative catalysis, where a specific enantio- or
regioselective reaction pathway takes place that is guided and
triggered by either charged residues or a local electric field.2
2. CHEMOSELECTIVITY IN ENZYMES GUIDED BY
CHARGED AMINO ACID RESIDUES
Enzymatic structures contain many polar groups to stabilize the
protein and fix its three-dimensional structure and its
interactions with either a membrane, a partner protein, or the
solvent. In addition, charged amino acid residues interact with
metal ions and charged groups of substrate molecules and hence
often are involved in positioning substrate and oxidant in an
active site. As mentioned above, αKG cosubstrate typically
interacts with the side chains of one or more Arg residues that fix
it in its position. Recent computational studies of our group,
however, show that these charged residues also induce local
dipole moments and local electric fields that affect bond
strengths and the electronic properties of catalytic reaction
centers.1,2 The hygromycin biosynthesis enzyme HygX catalyzes
the oxidative ring closure between two oligosaccharide groups to
form an ortho-δ-lactone ring as part of antibiotics biosyn-
thesis.67,68 A substrate-devoid but αKG-bound crystal structure
70
pubs.acs.org/accounts Article
was resolved17,69 that identified HygX as an αKG-dependent
non-heme iron dioxygenase with 2-His/1-Glu iron coordina-
tion. As the enzyme reacts fast, no intermediates of the catalytic
cycle could be trapped; hence a computational study was
performed.49 A large 303 atom cluster of HygX was created of an
iron(IV)-oxo species bound to the protein and with succinate
and bound substrate (Figure 3a). The model included all polar
groups in the substrate and oxidant binding sites as well as
several water molecules. Next, the oxidative ring closure was
studied that formally results in two sequential hydrogen atom
abstraction steps from the tertiary C−H bond of the substrate
(atom in red in Figure 3) and the alcohol O−H bond (atom in
blue in Figure 3). Using the large cluster model, the two barriers
were close in energy with a small preference for hydride transfer
from the alcohol group that had a barrier of only 2.4 kcal mol−1,
while the hydrogen atom abstraction from the C−H bond was
7.5 kcal mol−1. When the positively charged Lys residue was
removed from the model, the recalculated barriers both went up
and particularly those for the C−H abstraction. Thus, the active
site Lys group forms a hydrogen bonding interaction with the
iron(IV)-oxo group as well as with the substrate. Its charge
distributions clearly have a major impact on the structure and
thermodynamics of the reaction mechanism in HygX.
3. ELECTRIC FIELD AFFECTING SUBSTRATE BOND
STRENGTHS
The TmpA enzyme binds 2-(trimethylammonio)-
ethylphosphonate and selectively hydroxylates it at the C1-
position.50 However, for an isolated 2-(trimethylammonio)-
ethylphosphonate molecule in the gas phase a homolytic
BDEC1−H = 96.1 kcal mol−1 was calculated, while the C2−H
bond energy is significantly less, namely, BDEC2−H = 91.5 kcal
mol−1.47 Therefore, based on the relative values of the BDEs for
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
Figure 5. (a) Electric field gradient in the VioC structure as analyzed from the 6ALM pdb file. (b) Electric dipole vector identified in the optimized
geometry of the iron(IV)-oxo species of VioC for a 245 atom cluster model with electric dipole moment in Debye. (c) Electric field effect calculations
on an isolated arginine molecule and its effect on the C3−H and C4−H BDEs as reported in ref 48.
the C1−H and C2−H bonds, a regioselective C2-hydroxylation
would be expected; however, the products distributions only
find C1-hydroxylation instead. Previously, for a large test set of
chemical compounds, we showed that the bond dissociation
energy is linearly related to the change in polarizability along that
chemical bond and consequently bond energies may be affected
by electric field perturbations.70 Furthermore, experimental and
computational studies using electric field effects showed drastic
changes in charge distributions and consequently on the
properties of chemical systems.34−37,51−56,71−73 To test whether
external perturbations could affect substrate bond energies in
enzymes, we took the optimized geometry of the 2-
(trimethylammonio)ethylphosphonate substrate of TmpA and
the substrate with a hydrogen atom removed and calculated the
BDEs under external electric field perturbations with a field
along the molecular x-, y-, and z-axis of the substrate.47 Figure 4
shows the calculated BDEs when fields along the positive or
negative y-axis are applied. As can be seen, a reversal in the
ordering of C−H bond strengths is obtained through a field
effect along the molecular y-axis, that is, along the C1−H bond.
Thus, under a positive electric field along the y-axis, the C1−H
bond is strengthened and the C2−H bond is weakened, and as
such, it should give a regioselective C2−H hydroxylation. By
contrast, an electric field along the negative y-direction gives the
opposite trends and will favor C1−H hydroxylation. Clearly,
charged residues in the protein can mimic these electric field
effects and affect reaction selectivities. These studies imply that
the local charges in the substrate-binding pocket can influence
the regioselectivity of substrate activation. To test this, the large
cluster model (242 atoms) was truncated by removing all
protein residues except those directly bound to iron and the
positively charged Arg residue was replaced with a Li+ cation.47
The truncated model still predicted regioselective C1-hydrox-
ylation over C2-hydroxylation by a similar amount. However,
when the charges were completely removed from the model and
the substrate entities, the regioselectivity of the reaction changed
to C2-hydroxylation. Therefore, the charge distributions in the
71
pubs.acs.org/accounts Article
substrate-binding pocket of TmpA appear to create a local
dipole or electric field effect that weakens the C1−H bond
strength or strengthens the C2−H bond strength so that a
regioselective C1−H hydroxylation is possible.
Another enzyme for which external perturbations were shown
to influence selectivity patterns is the arginine-activating
viomycin biosynthesis enzyme VioC.48 It regioselectively
activates the C3−H bond of a free arginine substrate despite
the fact that the homolytic C3−H and C4−H bond energies are
virtually the same (see Figure 2 above).48 For this enzyme, an
initial electrostatic analysis of the PDB structure was performed
using the 6ALM PDB file, Figure 5.17,48 It was shown that an
electric field gradient was located along the arginine substrate,
thanks to a combination of positively charged amino acid
residues, for example, Arg334, as well as negatively charged
groups, from, Asp222, Asp223, and Asp268. Here, the net dipole
moment is oriented from the positively charged residues to the
negatively charged ones. A DFT cluster study on the substrate
activation of arginine was performed using a 245 atom cluster
model that contains the iron with its first coordination sphere of
ligands as well as the substrate and its binding pocket. The
optimized geometry of this iron(IV)-oxo model complex of
VioC had a dipole moment aligned with the electric field
gradient of the protein and clearly affected local charge
distributions (Figure 5b). In particular, a local electric field
perturbation on an isolated substrate was shown to affect C−H
bond strengths. As shown in Figure 5c, a field along the C3−H
bond in the positive direction weakens the C3−H bond
dramatically, whereas the C4−H bond is weakened with a field
of opposite direction. Consequently, a local electric field along
the C3−H bond can affect substrate catalysis and direct the
reaction to either C3−H hydroxylation or C4−H hydroxylation
depending on the location of the charged residues in the
substrate-binding pocket.
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
4. CONCLUSION
In this Account, we highlight recent computational studies on
the effect of polar groups in enzymatic active sites and how they
affect the structure and reactivity. In particular, positively and
negatively charged residues, such as Arg, Lys, Asp, and Glu side
chains, incur long-range charge−dipole interactions that can
perturb the structure and reactivity of a catalytic reaction center
buried inside a protein. We show that in many non-heme iron
dioxygenases charged residues lining the substrate-binding
pocket incur local electrostatic perturbations and local electric
fields that influence substrate-positioning, as well as C−H bond
strengths. For instance, models on the HygX non-heme iron
enzyme with and without the active site Lys137 residue showed a
change in reactivity when the Lys was removed. Further studies
on various non-heme iron dioxygenases identified an electric
field gradient in the protein structure guiding a regioselective
reaction mechanism. These modeling studies predict how
enzymes can be engineered in order to trigger a chemo- and
regioselective reaction mechanism. As such, there is huge
potential in biotechnology and bioengineering to use non-heme
iron dioxygenases for interesting selective reaction mechanisms.
■ AUTHOR INFORMATION
Corresponding Authors
Sam P. de Visser − Manchester Institute of Biotechnology and
Department of Chemical Engineering and Analytical Science,
The University of Manchester, Manchester M1 7DN, United
Kingdom; Department of Chemistry, Indian Institute of
Technology Guwahati, 781039 Assam, India; orcid.org/
0000-0002-2620-8788; Email: sam.devisser@
manchester.ac.uk
Chivukula V. Sastri − Department of Chemistry, Indian
Institute of Technology Guwahati, 781039 Assam, India;
Email: sastricv@iitg.ac.in
Authors
Gourab Mukherjee − Department of Chemistry, Indian
Institute of Technology Guwahati, 781039 Assam, India;
Present Address: G.M.: School of Chemistry, The
University of Edinburgh, David Brewster Road, Edinburgh,
EH9 3FJ, United Kingdom; orcid.org/0000-0002-7150-
5570
Hafiz Saqib Ali − Manchester Institute of Biotechnology and
Department of Chemical Engineering and Analytical Science,
The University of Manchester, Manchester M1 7DN, United
Kingdom; orcid.org/0000-0001-5770-5376
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.accounts.1c00538
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
C.V.S. acknowledges the Department of Science and Technol-
ogy (SERB), India, for research funding through grant code
CRG/2019/000387. The Punjab Endowment Fund (PEEF) in
Pakistan is acknowledged for a Ph.D. scholarship to H.S.A.
Notes
The authors declare no competing financial interest.
72
pubs.acs.org/accounts Article
Biographies
Sam P. de Visser is a Reader at the University of Manchester with
research interests related to the inorganic reaction mechanisms of heme
and non-heme iron enzymes and biomimetic model complexes. His
group uses a broad spectrum of computational methods.
Gourab Mukherjee obtained his Ph.D. from IIT Guwahati, India, in
2019 under the direction of Prof. C. V. Sastri. He was a visiting
researcher at the University of Manchester in the de Visser group in
2018−2019. Currently he is a postdoctoral researcher at The University
of Edinburgh, working on artificial metalloenzymes.
Hafiz S. Ali recently obtained his Ph.D. from the University of
Manchester under supervision of Dr. Richard Henchman and Sam de
Visser. He subsequently moved to the University of Edinburgh as a
postdoctoral researcher. His research interests are focused on the
development and implementation of newly and previously developed
computational methods to study reaction kinetics of heme and non-
heme iron enzymes and artificial metalloenzymes.
Chivukula V. Sastri is a Professor of Chemistry at the Indian Institute
of Technology Guwahati, India. He completed his Ph.D. from the
University of Hyderabad in 2004. After postdoctoral research in South
Korea and the U.S.A., he became a group leader at IIT Guwahati in
2008. His research interests include bioinspired models of non-heme
enzymes and their reactivity studies.
■ ACKNOWLEDGMENTS
S.P.d.V. thanks IIT Guwahati for a visiting professorship.
■ ABBREVIATIONS
αKG, α-ketoglutarate; BDE, bond dissociation energy; CsiD,
carbon starvation induced protein D; DFT, density functional
theory; P4H, prolyl-4-hydroxylase; PDB, protein databank file;
QM/MM, quantum mechanics/molecular mechanics; VioC,
viomycin biosynthesis enzyme
■ REFERENCES
(1) de Visser, S. P. Second-coordination sphere effects on selectivity
and specificity of heme and non-heme iron enzymes. Chem. - Eur. J.
2020, 26, 5308−5327.
(2) de Visser, S. P.; Lin, Y.-T.; Ali, H. S.; Bagha, U. K.; Mukherjee, G.;
Sastri, C. V. Negative catalysis or non-Bell-Evans-Polanyi reactivity by
metalloenzymes: Examples from mononuclear heme and non-heme
iron oxygenases. Coord. Chem. Rev. 2021, 439, 213914.
(3) Mukherjee, G.; Satpathy, J. K.; Bagha, U. K.; Mubarak, M. Q. E.;
Sastri, C. V.; de Visser, S. P. Inspiration from Nature: influence of
engineered ligand scaffolds and auxiliary factors on the reactivity of
biomimetic oxidants. ACS Catal. 2021, 11, 9761−9797.
(4) Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S.
K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y. S.; Zhou, J. Geometric
and electronic structure/function correlations in non-heme iron
enzymes. Chem. Rev. 2000, 100, 235−349.
(5) Abu-Omar, M. M.; Loaiza, A.; Hontzeas, N. Reaction mechanisms
of mononuclear non-heme iron oxygenases. Chem. Rev. 2005, 105,
2227−2252.
(6) Bruijnincx, P. C. A.; van Koten, G.; Klein Gebbink, R. J. M.
Mononuclear non-heme iron enzymes with the 2-His-1-carboxylate
facial triad: recent developments in enzymology and modeling studies.
Chem. Soc. Rev. 2008, 37, 2716−2744.
(7) de Visser, S. P., Kumar, D., Eds.; Iron-containing enzymes: Versatile
catalysts of hydroxylation reactions in nature; Royal Society of Chemistry
Publishing, Cambridge, U.K., 2011.
(8) White, M. D.; Flashman, E. Catalytic strategies of the non-heme
iron dependent oxygenases and their roles in plant biology. Curr. Opin.
Chem. Biol. 2016, 31, 126−135.
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research
(9) Herr, C. Q.; Hausinger, R. P. Amazing diversity in biochemical
roles of Fe(II)/2-oxoglutarate oxygenases. Trends Biochem. Sci. 2018,
43, 517−532.
(10) Kal, S.; Que, L., Jr. Dioxygen activation by nonheme iron
enzymes with the 2-His-1-carboxylate facial triad that generate high-
valent oxoiron oxidants. JBIC, J. Biol. Inorg. Chem. 2017, 22, 339−365.
(11) Hausinger, R. P. FeII/alpha-ketoglutarate-dependent hydrox-
ylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 2004, 39, 21−
68.
(12) Bugg, T. D. H.; Ramaswamy, S. Non-heme iron-dependent
dioxygenases: unravelling catalytic mechanisms for complex enzymatic
oxidations. Curr. Opin. Chem. Biol. 2008, 12, 134−140.
(13) Baldwin, J. E.; Bradley, M. Isopenicillin N synthase: mechanistic
studies. Chem. Rev. 1990, 90, 1079−1088.
(14) Lundberg, M.; Siegbahn, P. E. M.; Morokuma, K. The
mechanism for isopenicillin N synthase from density-functional
modeling highlights the similarities with other enzymes in the 2-His-
1-carboxylate family. Biochemistry 2008, 47, 1031−1042.
(15) Wackett, L. P. Mechanism and applications of Rieske non-heme
iron dioxygenases. Enzyme Microb. Technol. 2002, 31, 577−587.
(16) Bugg, T. D. H. Dioxygenase enzymes: catalytic mechanisms and
chemical models. Tetrahedron 2003, 59, 7075−7601.
(17) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.
N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank.
Nucleic Acids Res. 2000, 28, 235−242.
(18) Wang, L.; Erlandsen, H.; Haavik, J.; Knappskog, P. M.; Stevens,
R. C. Three-dimensional structure of human tryptophan hydroxylase
and its implications for the biosynthesis of the neurotransmitters
serotonin and melatonin. Biochemistry 2002, 41, 12569−12574.
(19) O’Brien, J. R.; Schuller, D. J.; Yang, V. S.; Dillard, B. D.;
Lanzilotta, W. N. Substrate-induced conformational changes in
Escherichia coli taurine/α-ketoglutarate dioxygenase and insight into
the oligomeric structure. Biochemistry 2003, 42, 5547−5554.
(20) Gakhar, L.; Malik, Z. A.; Allen, C. C. R.; Lipscomb, D. A.; Larkin,
M. J.; Ramaswamy, S. Structure and increased thermostability of
Rhodococcus sp. naphthalene 1,2-dioxygenase. J. Bacteriol. 2005, 187,
7222−7231.
(21) Price, J. C.; Barr, E. W.; Tirupati, B.; Bollinger, J. M., Jr; Krebs, C.
The first direct characterization of a high-valent iron intermediate in the
reaction of an α-ketoglutarate-dependent dioxygenase: a high-spin
Fe(IV) complex in taurine/α-ketoglutarate dioxygenase (TauD) from
Escherichia coli. Biochemistry 2003, 42, 7497−7508.
(22) Proshlyakov, D. A.; Henshaw, T. F.; Monterosso, G. R.; Ryle, M.
J.; Hausinger, R. P. Direct detection of oxygen intermediates in the non-
heme Fe enzyme taurine/α-ketoglutarate dioxygenase. J. Am. Chem.
Soc. 2004, 126, 1022−1023.
(23) Borowski, T.; Bassan, A.; Siegbahn, P. E. M. Mechanism of
dioxygen activation in 2-oxoglutarate-dependent enzymes: A hybrid
DFT study. Chem. - Eur. J. 2004, 10, 1031−1041.
(24) McCulloch, K. M.; McCranie, E. K.; Smith, J. A.; Sarwar, M.;
Mathieu, J. L.; Gitschlag, B. L.; Du, Y.; Bachmann, B. O.; Iverson, T. M.
Oxidative cyclizations in orthosomycin biosynthesis expand the known
chemistry of an oxygenase superfamily. Proc. Natl. Acad. Sci. U. S. A.
2015, 112, 11547−11552.
(25) Harris, N. C.; Born, D. A.; Cai, W.; Huang, Y.; Martin, J.; Khalaf,
R.; Drennan, C. L.; Zhang, W. Isonitrile formation by a non-heme
iron(II)-dependent oxidase/decarboxylase. Angew. Chem., Int. Ed.
2018, 57, 9707−9710.
(26) Ali, H. S.; Henchman, R. H.; de Visser, S. P. What determines the
selectivity of arginine dihydroxylation by the nonheme iron enzyme
OrfP? Chem. - Eur. J. 2021, 27, 1795−1809.
(27) Quesne, M. G.; Latifi, R.; Gonzalez-Ovalle, L. E.; Kumar, D.; de
Visser, S. P. Quantum mechanics/molecular mechanics study on the
oxygen binding and substrate hydroxylation step in AlkB repair
enzymes. Chem. - Eur. J. 2014, 20, 435−446.
(28) Latifi, R.; Minnick, J. L.; Quesne, M. G.; de Visser, S. P.; Tahsini,
L. Computational studies of DNA base repair mechanisms by nonheme
iron dioxygenases: Selective epoxidation and hydroxylation pathways.
Dalton Trans. 2020, 49, 4266−4276.
73
pubs.acs.org/accounts Article
(29) Knorr, S.; Sinn, M.; Galetskiy, D.; Williams, R. M.; Wang, C.;
Müller, N.; Mayans, O.; Schleheck, D.; Hartig, J. S. Widespread
bacterial lysine degradation proceeding via glutarate and L-2-
hydroxyglutarate. Nat. Commun. 2018, 9, 5071−5075.
(30) Herr, C. Q.; Macomber, L.; Kalliri, E.; Hausinger, R. P. Glutarate
L-2-hydroxylase (CsiD/GlaH) is an archetype Fe(II)/2-oxoglutarate-
dependent dioxygenase. Adv. Protein Chem. Struct. Biol. 2019, 117, 63−
90.
(31) Warshel, A. Energetics of enzyme catalysis. Proc. Natl. Acad. Sci.
U. S. A. 1978, 75, 5250−5254.
(32) Warwicker, J.; Watson, H. C. Calculation of the electric potential
in the active site cleft due to alpha-helix dipoles. J. Mol. Biol. 1982, 157,
671−679.
(33) Dixit, V. A.; Warwicker, J.; de Visser, S. P. How do metal ions
modulate the rate-determining electron transfer step in cytochrome
P450 reactions? Chem. - Eur. J. 2020, 26, 15270−15281.
(34) Varadarajan, R.; Zewert, T. E.; Gray, H. B.; Boxer, S. G. Effects of
buried ionizable amino acids on the reduction potential of recombinant
myoglobin. Science 1989, 243, 69−72.
(35) Waegele, M. M.; Culik, R. M.; Gai, F. Site-specific spectroscopic
reporters of the local electric field, hydration, structure, and dynamics of
biomolecules. J. Phys. Chem. Lett. 2011, 2, 2598−2609.
(36) Kim, H.; Cho, M. Infrared probes for studying the structure and
dynamics of biomolecules. Chem. Rev. 2013, 113, 5817−5847.
(37) Fafarman, A. T.; Sigala, P. A.; Schwans, J. P.; Fenn, T. D.;
Herschlag, D.; Boxer, S. G. Quantitative, directional measurement of
electric field heterogeneity in the active site of ketosteroid isomerase.
Proc. Natl. Acad. Sci. U. S. A. 2012, 109, E299−E308.
(38) Gorres, K.; Raines, R. T. Prolyl 4-hydroxylase. Crit. Rev. Biochem.
Mol. Biol. 2010, 45, 106−124.
(39) Karamzadeh, B.; Kumar, D.; Sastry, G. N.; de Visser, S. P. Steric
factors override thermodynamic driving force in regioselectivity of
proline hydroxylation by prolyl-4-hydroxylase enzymes. J. Phys. Chem. A
2010, 114, 13234−13243.
(40) Timmins, A.; Saint-André, M.; de Visser, S. P. Understanding
how prolyl-4-hydroxylase structure steers a ferryl oxidant toward
scission of a strong C-H bond. J. Am. Chem. Soc. 2017, 139, 9855−9866.
(41) Yin, X.; Zabriskie, T. M. VioC is a non-heme iron, α-
ketoglutarate-dependent oxygenase that catalyzes the formation of
3S-hydroxy-L-arginine during viomycin biosynthesis. ChemBioChem
2004, 5, 1274−127.
(42) Dunham, N. P.; Mitchell, A. J.; Del Río Pantoja, J. M.; Krebs, C.;
Bollinger, J. M., Jr.; Boal, A. K. α-Amine desaturation of D-arginine by
the iron(II)- and 2-(oxo)glutarate-dependent L-arginine 3-hydroxylase,
VioC. Biochemistry 2018, 57, 6479−6488.
(43) Han, S. B.; Ali, H. S.; de Visser, S. P. Glutarate hydroxylation by
the carbon starvation-induced protein D. A computational study into
the stereo- and regioselectivity of the reaction. Inorg. Chem. 2021, 60,
4800−4815.
(44) Mayer, J. M. Proton-coupled electron transfer: A reaction
chemist’s view. Annu. Rev. Phys. Chem. 2004, 55, 363−390.
(45) Sastri, C. V.; Lee, J.; Oh, K.; Lee, Y. J.; Lee, J.; Jackson, T. A.; Ray,
K.; Hirao, H.; Shin, W.; Halfen, J. A.; Kim, J.; Que, L., Jr.; Shaik, S.;
Nam, W. Axial ligand tuning of a nonheme iron (IV)-oxo unit for
hydrogen atom abstraction. Proc. Natl. Acad. Sci. U. S. A. 2007, 104,
19181−19186.
(46) Shaik, S.; Kumar, D.; de Visser, S. P. A valence bond modeling of
trends in hydrogen abstraction barriers and transition states of
hydroxylation reactions catalyzed by cytochrome P450 enzymes. J.
Am. Chem. Soc. 2008, 130, 10128−10140.
(47) Lin, Y.-T.; Ali, H. S.; de Visser, S. P. Electrostatic perturbations
from the protein affect C-H bond strengths of the substrate and enable
negative catalysis in the TmpA biosynthesis enzyme. Chem. - Eur. J.
2021, 27, 8851−8864.
(48) Ali, H. S.; Henchman, R. H.; Warwicker, J.; de Visser, S. P. How
do electrostatic perturbations of the protein affect the bifurcation
pathways of substrate hydroxylation versus desaturation in the
nonheme iron-dependent viomycin biosynthesis enzyme? J. Phys.
Chem. A 2021, 125, 1720−1737.
https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74
Accounts of Chemical Research pubs.acs.org/accounts Article

(49) Ali, H. S.; Henchman, R. H.; de Visser, S. P. Mechanism of McCulloch, K. M. The structure of the bifunctional everninomicin
oxidative ring-closure as part of the hygromycin biosynthesis step by a biosynthetic enzyme EvdMO1 suggests independent activity of the
nonheme iron dioxygenase. ChemCatChem 2021, 13, 3054−3066. fused methyltransferase-oxidase domains. Biochemistry 2018, 57,
(50) Rajakovich, L. J.; Pandelia, M.-E.; Mitchell, A. J.; Chang, W.-c.; 6827−6837.
Zhang, B.; Boal, A. K.; Krebs, C.; Bollinger, J. M., Jr A new microbial (69) McCulloch, K. M.; McCranie, E. K.; Smith, J. A.; Sarwar, M.;
pathway for organophosphonate degradation catalyzed by two Mathieu, J. L.; Gitschlag, B. L.; Du, Y.; Bachmann, B. O.; Iverson, T. M.
previously misannotated non-heme-iron oxygenases. Biochemistry Oxidative cyclizations in orthosomycin biosynthesis expand the known
2019, 58, 1627−1647. chemistry of an oxygenase superfamily. Proc. Natl. Acad. Sci. U. S. A.
(51) Shaik, S.; de Visser, S. P.; Kumar, D. External electric field will 2015, 112, 11547−11552.
control the selectivity of enzymatic-like bond activations. J. Am. Chem. (70) de Visser, S. P. On the relationship between internal energy and
Soc. 2004, 126, 11746−1174. both the polarizability volume and the diamagnetic susceptibility. Phys.
(52) Shaik, S.; Ramanan, R.; Danovich, D.; Mandal, D. Structure and Chem. Chem. Phys. 1999, 1, 749−754.
reactivity/selectivity control by oriented-external electric fields. Chem. (71) Fried, S. D.; Boxer, S. G. Measuring electric fields and
Soc. Rev. 2018, 47, 5125−5145. noncovalent interactions using the vibrational Stark effect. Acc. Chem.
(53) Hill, N. S.; Coote, M. L. Internal oriented electric fields as a Res. 2015, 48, 998−1006.
strategy for selectively modifying photochemical reactivity. J. Am. Chem. (72) Cisneros, G. A.; Karttunen, M.; Ren, P.; Sagui, C. Classical
Soc. 2018, 140, 17800−17804. electrostatics for biomolecular simulations. Chem. Rev. 2014, 114, 779−
(54) Yu, S.; Vermeeren, P.; Hamlin, T. A.; Bickelhaupt, F. M. How 814.
oriented external electric fields modulate reactivity. Chem. - Eur. J. 2021, (73) Sowlati-Hashjin, S.; Matta, C. F. The chemical bond in external
27, 5683−5693. electric fields: Energies, geometries, and vibrational Stark shifts of
(55) Kirshenboim, O.; Frenklah, A.; Kozuch, S. Switch chemistry at diatomic molecules. J. Chem. Phys. 2013, 139, 144101.
cryogenic conditions: quantum tunnelling under electric fields. Chem.
Sci. 2021, 12, 3179−3187.
(56) Ramanan, R.; Waheed, S. O.; Schofield, C. J.; Christov, C. Z.
What is the catalytic mechanism of enzymatic histone N-methyl
arginine demethylation and can it be influenced by an external electric
field? Chem. - Eur. J. 2021, 27, 11827−11836.
(57) Ogliaro, F.; Cohen, S.; de Visser, S. P.; Shaik, S. Medium
polarization and hydrogen bonding effects on Compound I of
cytochrome P450: what kind of a radical is it really? J. Am. Chem. Soc.
2000, 122, 12892−12893.
(58) Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I.
Structure and chemistry of cytochrome P450. Chem. Rev. 2005, 105,
2253−2277.
(59) Green, M. T. C-H bond activation in heme proteins: the role of
thiolate ligation in cytochrome P450. Curr. Opin. Chem. Biol. 2009, 13,
84−88.
(60) Dubey, K. D.; Shaik, S. Cytochrome P450: The wonderful
nanomachine revealed through dynamic simulations of the catalytic
cycle. Acc. Chem. Res. 2019, 52, 389−399.
(61) Louka, S.; Barry, S. M.; Heyes, D. J.; Mubarak, M. Q. E.; Ali, H. S.;
Alkhalaf, L. M.; Munro, A. W.; Scrutton, N. S.; Challis, G. L.; de Visser,
S. P. The catalytic mechanism of aromatic nitration by cytochrome
P450 TxtE: Involvement of a ferric-peroxynitrite intermediate. J. Am.
Chem. Soc. 2020, 142, 15764−15779.
(62) Ali, H. S.; Henchman, R. H.; de Visser, S. P. Lignin
biodegradation by a cytochrome P450 enzyme: A computational
study into syringol activation by GcoA. Chem. - Eur. J. 2020, 26, 13093−
13102.
(63) Suzuki, N.; Higuchi, T.; Urano, Y.; Kikuchi, K.; Uekusa, H.;
Ohashi, Y.; Uchida, T.; Kitagawa, T.; Nagano, T. Novel iron porphyrin-
alkanethiolate complex with intramolecular NH···S hydrogen bond:
synthesis, spectroscopy, and reactivity. J. Am. Chem. Soc. 1999, 121,
11571−11572.
(64) Yoshioka, S.; Tosha, T.; Takahashi, S.; Ishimori, K.; Hori, H.;
Morishima, I. Roles of the proximal hydrogen bonding network in
cytochrome P450cam-catalyzed oxygenation. J. Am. Chem. Soc. 2002,
124, 14571−14579.
(65) Mak, P. J.; Yang, Y.; Im, S.; Waskell, L. A.; Kincaid, J. R.
Experimental documentation of the structural consequences of
hydrogen-bonding interactions to the proximal cysteine of a
cytochrome P450. Angew. Chem., Int. Ed. 2012, 51, 10403−10407.
(66) de Visser, S. P. What affects the quartet-doublet energy splitting
in peroxidase enzymes? J. Phys. Chem. A 2005, 109, 11050−11057.
(67) Li, S.; Liu, Q.; Zhong, Z.; Deng, Z.; Sun, Y. Exploration of
hygromycin B biosynthesis utilizing CRISPR-Cas9-associated base
editing. ACS Chem. Biol. 2020, 15, 1417−1423.
(68) Starbird, C. A.; Perry, N. A.; Chen, Q.; Berndt, S.; Yamakawa, I.;
Loukachevitch, L. V.; Limbrick, E. M.; Bachmann, B. O.; Iverson, T. M.;

74 https://doi.org/10.1021/acs.accounts.1c00538
Acc. Chem. Res. 2022, 55, 65−74