Epoxidation Catalyzed by the Nonheme Iron(II)- A nd 2-Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate

Article abstract of DOI:10.1021/jacs.0c00484

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- A nd 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, M?ssbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O₂ addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.

Full text of DOI:10.1021/jacs.0c00484

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Article
Epoxidation Catalyzed by the Non-heme Iron- and 2-
Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic
Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate
Jikun Li, Hsuan-Jen Liao, Yijie Tang, Jhih-Liang Huang, Lide Cha, Te-Sheng Lin, Justin L.
Lee, Igor V Kurnikov, Maria G Kurnikova, Wei-chen Chang, Nei-Li Chan, and Yisong Guo
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c00484 • Publication Date (Web): 04 Mar 2020
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Epoxidation Catalyzed by the Non-heme Iron- and 2-Oxoglutarate-
Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen
Atom Transfer by a Ferryl Intermediate
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Jikun Li,a,‡ Hsuan-Jen Liao,b‡ Yijie Tang,a Jhih-Liang Huang,c Lide Cha,c Te-Sheng Lin,b Justin L. Lee,a
Igor V. Kurnikov,a Maria G. Kurnikova,a,* Wei-chen Chang,c,* Nei-Li Chan,b,* Yisong Guo a,*
a Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States
b Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
c Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States
ABSTRACT: Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the
studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed
by non-heme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the non-heme
iron- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogs with different para substituents
ranging from electron-donating groups (e.g. methoxy) to electron-withdrawing groups (e.g. trifluoromethyl) were used to probe the
mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray
crystallography, density functional theory calculations and molecular dynamic simulations collectively revealed the following
mechanistic insights: 1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding
mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad,
instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); 2) The decay rate
constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step,
a reactivity behavior that is drastically different from non-heme Fe(IV)-oxo synthetic model complexes; 3) The OAT step most likely
proceeds through a step-wise process with the initial formation of C(benzylic)-O bond to generate an Fe(III)-alkoxide species, which
is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.
Introduction
and quinolone alkaloid by AsqJ (Scheme 1).22 While both
pathways require the formation of an iron(IV)-oxo (or ferryl)
intermediate to initiate epoxidation, only recently such a species
has been experimentally elucidated by us in the AsqJ catalyzed
epoxide installation.28 AsqJ, together with H6H, CAS, LolO,
and DdaC, belongs to the iron(II) and 2-oxoglutarate (Fe/2OG)
dependent enzyme superfamily, whose members catalyze a
broad array of transformations and are involved in many
important natural product biosyntheses.29 Notably, AsqJ
catalyzes stepwise desaturation and epoxidation in the
biosynthesis of a quinolone-type fungal alkaloid, 4’-methoxy-
viridicatin (Scheme 1).22
The epoxide (oxirane) functional group is widely distributed in
natural products with a broad spectrum of biological activities,
including antibacterial, antifungal, antiviral, and antitumor
activities.1 Furthermore, installation of epoxide moiety is an
important and useful synthetic strategy to achieve structural and
functional versatility in organic synthesis.2–6
In nature, two main strategies are utilized to install epoxide,
namely formal dehydrogenation by cleaving a C-H and an O-H
bond on two adjacent carbons, and an oxygen atom insertion,
a.k.a. oxygen atom transfer (OAT), reaction onto an olefin
moiety of the substrate (Scheme 1).1,7 For both strategies,
molecular oxygen (O2) and in some cases H2O2, is utilized as an
oxidant, a source of oxygen, or playing a dual role.1,8–17 Usually,
the epoxidation is initiated by a highly reactive intermediate
based on flavin,1 thiolate-heme,13–16 non-heme di-iron,8,10–12 or
non-heme mononuclear iron.18–22 For non-heme mononuclear
iron enzymes, epoxidation through the dehydrogenation
pathway has been reported in the biosyntheses of scopolamine
by hyoscyamine 6β-hydroxylase (H6H),18 fosfomycin by 2-
hydroxyl propyl phosphonate epoxidase (HppE),19 clavulanic
acid by clavamate synthase (CAS),23–26 and loline by LolO.27
Alternatively, epoxidation through the oxygen atom insertion is
reported in the biosynthesis of Nβ -epoxy-succinamoyl-DAP-
Val by DdaC,20 pentalenolactone by PenD (PntD) and PtlD,21,28
Mechanistic
understanding
of
enzyme-catalyzed
epoxidation through the oxygen atom insertion onto an olefin
moiety is mainly derived from the studies on thiolate-heme
containing epoxidases, such as choloroperoxidases (CPOs) and
cytochrome P450s.14 Briefly, the C-O bonds of an epoxide can
be installed in a concerted fashion or a stepwise manner via a
discrete radical or cation intermediate. A concerted mechanism
is supported by the observation of retention of the olefin
stereochemistry reported in P450 epoxidases. The stepwise
mechanism with the involvement of a putative cation
intermediate is implicated by the formation of the carbonyl
product through a hydride or a chloride migration to the
adjacent carbon.30–32 A cation-triggered phosphono group
migration has also been reported for the non-heme iron enzyme
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HppE in the presence of a nonnative substrate.9 The possible
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involvement of a radical intermediate is suggested by the
formation of alkylated porphyrin side product via alkylation of
a pyrrole nitrogen of the porphyrin heme framework by the
intermediate substrate radical species in some P450
epoxidases.33–35 It is generally accepted that the key
intermediate in olefin epoxidation of CPOs and P450s is an
iron(IV)-oxo porphyrin-cation-radical species, Compound I. To
explain the energetic feasibility of these two competing
mechanisms (concerted v.s. stepwise), computational studies on
P450 Compound I catalyzed epoxidation suggest that the
doublet- (S = 1/2) and quartet-spin (S = 3/2) states of
Compound I are energetically close and both are suitable for
epoxidation.34–39 The utilization of the doublet spin state of
Compound I could result in a concerted epoxidation while the
use of the quartet spin state intermediate is proposed to proceed
Compared to significant research efforts in elucidating
plausible epoxidation mechanisms employed by thiolate-heme
containing epoxidases, the molecular basis of non-heme-iron-
enzyme-catalyzed epoxidation is much less studied. Inspired by
the literature precedents, several possible reaction pathways (a
step-wise mechanism via a cation intermediate, pathway i, or a
radical intermediate, pathway ii, and a concerted mechanism,
pathway iii) can be envisioned and are depicted in Scheme 2.
Herein, we use AsqJ as an example to study detailed reaction
mechanism of non-heme-iron-enzyme-catalyzed epoxidation
by using a multi-faceted approach, including LC-MS analysis
of the enzymatic reactions, transient-state kinetics,
spectroscopic characterizations using Mössbauer and electron
paramagnetic resonance (EPR), X-ray crystallography, density
functional theory (DFT) calculations and molecular dynamics
(MD) simulations. AsqJ native substrate (2-OMe) and several
para-substituted analogs (2-H, 2-F and 2-CF3) were used in this
study to probe the reactivity and plausible epoxidation
mechanism. The transient-state kinetics and Mössbauer results
showed that the the decay rate constant of the Fe(IV)-oxo
intermediate is largely insensitive to the para substitutions on
the enzyme substrate. This behavior is in contrast with the OAT
reactivity exhibited by non-heme Fe(IV)-oxo synthetic
complexes. In addition, the LC-MS analyses and x-ray
crystallographic results strongly suggested that a step-wise
mechanism is operative in AsqJ catalyzed epoxidation, where
the Fe(IV)-oxo intermediate reacts with the olefin group of the
substrates by first attacking the C10 (benzylic) position of the
substrate. DFT calculations and MD simulations provide further
support for the experimental observations.
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through the stepwise mechanism.34,35 In addition, the
40–43
Compound I related model complexes
iron(IV)-oxo synthetic mimics
and the non-heme
44–46
generally exhibit strong
electrophilic nature during epoxidation. Specifically, the studies
conducted by using para-substituted styrenes revealed that the
electron-donating groups (EDGs) could significantly increase
the rate constant of the OAT reaction (by monitoring the decay
rate constant of Compound I or iron(IV)-oxo species), while the
electron-withdrawing groups (EWGs) exhibited the opposite
effect, and the rate constant could be modulated by 2 order of
magnitudes. However, Newcomb and coworkers showed that
the OAT reaction rate constant was largely independent of para
substitutions.47 More strikingly, a V-shaped Hammett plot was
observed
when
a
Mn(V)-oxo
complex,
[MnV(O)(TBP8Cz)(CN)]-, was examined, indicating a change
of mechanism when going from EDGs to EWGs where the
proposed high-valent metal-oxo species character shifts from
electrophilic to weak nucleophilic, respectively.48
Scheme 2. Proposed Reaction Mechanisms of Epoxidation
via Oxygen Atom Transfer (OAT).
Scheme 1. Examples of Epoxidation Catalyzed by Non-
Heme Iron Enzymes via (A) Dehydrogenation and (B)
Oxygen Atom Insertion.
Results and Discussion.
Synthesis and Characterization of 2-R (R = OMe, H, F, CF3)
The synthesis of substrate analogs (2-R, R = OMe, H, F, and
CF3) was carried out by using the published procedure with
different para-substituted benzaldehyde as the starting
material.49 The 1H NMR data of 2-OMe and 2-H in CDCl3 are
1
consistent with the literature reported values. The H and 13C
NMR data of 2-F and 2-CF3 are reported in the Supporting
Information with spectra shown in Figures S1 and S2.
1
Importantly, the H chemical shift at C10 of 2-R (~6.9 – 7.0
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ppm) exhibits downfield shift in going from 2-OMe to 2-CF3
2-CF3 (Figure S4). Thus taken together, both Mössbauer and
EPR results confirm that all the substrate and analogs bind to
AsqJ tightly.
1
2
3
(Table S1) and is consistent with the expected para-substituent
effect on the olefin moiety of 2-R in going from EDGs to
EWGs.
4
Binding Affinity of 2-R to AsqJ.
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To study AsqJ catalyzed epoxidation reaction mechanism, we
first establish that the binding affinity of the analogs (2-H, 2-F,
and 2-CF3) to AsqJ is not perturbed due to the para-substitution.
The data indicate that the substrate and the analogs bind to AsqJ
with similar affinity came from Mössbauer spectral analysis.
With addition of only one equivalent (relative to the Fe2+-loaded
enzyme) of 2-OMe or the analogs (2-H, 2-F, and 2-CF3), the
Mössbauer spectrum (Figure S3) was completely converted
from a broad quadrupole doublet (δ = 1.25 mm/s, |ΔEQ| = 2.26
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mm/s, linewidth 0.51 mm/s), representing the AsqJ⋅Fe2+⋅2OG
complex, to a sharp quadrupole doublet (δ = 1.25 mm/s, |ΔEQ|
= 2.54 mm/s, linewidth, 0.30 mm/s). In addition, a Voigt line
shape (a convolution of 50% Gaussian and 50% Lorentzian) had
to be used to achieve satisfactory simulation for the
AsqJ⋅Fe2+⋅2OG complex, as compared to a Lorentzian line
shape used for the AsqJ⋅Fe2+⋅2OG⋅substrate quaternary
complex, indicating substantial distribution of parameters in the
former sample. This observation indicates that the binding of
the substrate perturbs the electronic structure of the Fe(II)
center, and the AsqJ⋅Fe2+⋅2OG⋅substrate quaternary complex
was fully formed with the addition of stoichiometric amount of
substrate. To further validate this result, Mn2+ loaded AsqJ was
used to carry out substrate titration on 2-CF3. The ability of
Mn2+ ion binding to the metal binding site of Fe/2OG enzymes
has been reported for many crystal structures on Fe/2OG
enzymes where Mn2+ was used as an alternative metal ion to
reconstitute the enzymes. Indeed, when Mn2+ was incubated
with apo AsqJ and 2OG (0.5 mM Mn2+, 0.75 mM AsqJ, and 5
mM 2OG), the resulted EPR signal was drastically different
from the EPR signal of free Mn2+ (0.5 mM) in buffer solution
(Figure 1). This type of spectroscopic change has been
documented in the literature,50–53 and attributed to the change of
the coordination environment of Mn2+ from a symmetric
octahedral ligand field in the aqueous solution (with a close to
zero axial zero field splitting parameter, D ~ 0) to a distorted
octahedral environment in the protein metal binding site (with
increased D values). Consistent with our experimental
observation, the spectral simulations revealed that the AsqJ
bound Mn2+ signal (in the presence of 2OG) originated from a
single species with |D| ~ 0.08 cm-1, E/D ~ 0.20, and |A(Mn)| =
244 MHz. When an excess amount of 2-CF3 (2 eq. related to
Mn2+ concentration) was introduced into the AsqJ-Mn2+-2OG
complex, the Mn2+ EPR signal exhibited a subtle but clear
change, suggesting that the presence of substrate further
perturbs the metal site and indicates the formation of the
Figure 1. X-band EPR signals from samples containing Mn2+ in
buffer solution (top, grey), AsqJ⋅Mn2+⋅2OG ternary complex
(middle, red), and AsqJ⋅Mn2+⋅2OG⋅2-CF3 quaternary complex
(bottom, blue). The corresponding spectral simulations are shown
in black curves. The simulation parameters are for S = 5/2, I = 5/2.
For the ternary complex (middle), g = 2.03, D = 0.08 cm-1, E/D =
0.20, |AMn| = 243 MHz; for the quaternary complex (bottom), g =
2.02, D = 0.08 cm-1, E/D = 0.13, |AMn| = 254 MHz. Measurement
conditions: microwave frequency, 9.64 GHz; microwave power, 20
uW; modulation frequency, 100 kHz; modulation amplitude, 1 mT;
temperature, 12 K.
Kinetic Analysis of AsqJ Catalyzed Epoxidation Reaction.
To further probe the mechanism, kinetics study of the AsqJ
epoxidation reaction was carried out by rapidly mixing the
anaerobic AsqJ⋅Fe2+⋅2OG⋅substrate complex with oxygenated
buffer and recording time-dependent UV-Vis spectra using
stopped-flow absorption spectroscopy (SF-Abs). We took
advantage of the tight binding of the substrate analogs to AsqJ
to conduct experiments under single-turnover conditions (0.12
mM enzyme, 0.1 mM Fe2+, 0.1 mM substrate (2-OMe, 2-H, 2-
F, and 2-CF3), 4 mM α-KG and ~1.8 mM O2 before mixing) at
∘
5 C. When comparing SF-Abs derived kinetic traces, three
major features were identified (Figure 2 and S5). 1) A decrease
of the absorption band at 280-320 nm (for 2-OMe the band
extends to > 330 nm) mainly corresponds to the consumption of
the substrate. This assertion was derived from the difference of
the absorption features for the substrate and the product in the
near UV region.54 2) An initial decrease (up to ~ 0.4s) and
subsequent recovery of a band centered at ~470 nm that
corresponds to the Fe2+⋅2OG metal-to-ligand charge transfer
AsqJ⋅Mn2+⋅2OG⋅2-CF3
quaternary
complex.
Spectral
(MLCT) band. 3) An initial increase (peaking at around 0.03 s)
and subsequent decrease of a feature at 330-340 nm is also
observed. We propose this latter feature corresponds to the
formation and consumption of the Fe(IV)=O intermediate (see
below). We next modeled the SF-Abs traces at 310, 330 and 470
nm with a 3-step kinetic model (Scheme 3). A similar kinetic
model has been used to describe pre-steady state kinetics of
several Fe/2OG enzymes.55–58 Under the current experimental
conditions, the reaction likely proceeds through formation of
the Fe(IV)=O intermediate via addition of O2 to the
simulation showed that this new EPR signal originated from a
single species with |D| ~ 0.08 cm-1, E/D ~ 0.16, and |A(Mn)| =
255 MHz. In addition, when the AsqJ⋅Mn2+⋅2OG⋅2-CF3
complex was exposed to air for 20 min, no obvious change was
observed, indicating that the Mn2+ loaded quaternary complex
does not readily react with O2. Based on the two EPR signals
recorded under the conditions with and without substrate, we
carried out the substrate titration on 2-CF3. The titration
suggested that the dissociation constant (Kd) is ~ 0.005 mM for
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0.5
0.4
0.3
AsqJ⋅Fe2+⋅2OG⋅substrate quaternary complex with the rate
0.4
0.3
0.2
2-H
2-CF3
1
2
3
4
5
6
7
8
310 nm
310 nm
constant k1. The Fe(IV)=O intermediate subsequently oxidizes
the substrate to generate the AsqJ⋅Fe2+⋅succinate⋅product
complex with the rate constant of k2, which in turn releases
succinate and the product and regenerates the AsqJ⋅Fe2+⋅2OG
0.16
0.14
0.16
ternary complex in the presence of excess 2OG with the rate
constant of k3.
330 nm
470 nm
330 nm
470 nm
0.14
0.07
Scheme 3. Three-Step Kinetic Model Used for Analyzing
AsqJ Catalyzed Epoxidation.
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0.05
O2
AsqJ•Fe(II)•2OG•2-R
AsqJ•Fe(IV)=O•Succinate•2-R
0.06
k1
2OG Product + Succinate
AsqJ•Fe(II)•Succinate•3-R AsqJ•Fe(II)•2OG
k2
k3
The SF-Abs data and the kinetic simulations suggest that
the pre-steady state kinetics are very similar when 2-OMe, 2-H,
or 2-F are used as the substrate as shown in Figure 2 and Figure
S5 (the simulation parameters are shown in Table 1). The O2
addition rate constant (k1) is further verified by SF-Abs data
measured by using 2-H as the substrate and under varying O2
conditions (Figure S6). The formation and decay kinetics of the
putative Fe(IV)=O intermediate is reflected by the 330 nm
kinetic traces. The maximum accumulation of this intermediate
is occurred at ~ 0.03 – 0.04 s, which is consistent with the
previous published Mössbauer results.49 This observation
further validates the assignment that the 330 nm trace represents
mainly the Fe(IV)=O intermediate. However, for 2-CF3, the
enzyme kinetics is altered. This is clearly shown by the kinetic
trace at 470 nm, which mainly reflects the kinetics of the AsqJ
quaternary and ternary complexes (Figure 2). Compared with
the results obtained by using other analogs, both the decay and
the reformation rates of 470 nm absorption are faster and the
extent of absorption change in the reaction is only ~ 1/3 to that
with 2-H. Simulation using a three-step kinetic model at three
wavelengths (470nm, 330nm, and 310 nm) suggests that 2-CF3
has a faster formation rate constant (~ 2-3 times) and a slower
decay rate constant (~ 2-3 times) of the Fe(IV)=O intermediate
than those derived from the reaction with 2-H. The conversion
rate constant of the enzyme product complex to the enzyme
ternary complex is also faster (5-10 times) in the reaction with
2-CF3 (Table 1). These results indicate that the product
dissociation from the enzyme binding pocket might be more
rapid when 2-CF3 is used. In addition, an appreciable amount of
0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
Time (s)
Time (s)
Figure 2. SF-Abs derived kinetic traces of the time-dependent
absorption changes at various wavelengths in the AsqJ catalyzed
epoxidation by using 2-H (left panel) and 2-CF3 (right panel) as the
substrates. Experimental data are shown in black dots, the
corresponding kinetic simulations are shown in red curves, and the
corresponding rate constants are shown in Table 1. The kinetics of
individual species are shown at the bottom of both panels: purple,
the AsqJ•Fe2+•2OG•substrate complex; red, the Fe(IV)=O
intermediate; green, the AsqJ-Fe2+-succinate-product complex;
grey, the AsqJ•Fe2+•2OG complex. The results on the reactions
with 2-OMe and 2-F are shown in Figure S5.
Table 1. Simulated Kinetic Constants Based on the 3-Step
Kinetic Model.
Substrate
2-H
k1 (mM-1 s-1)
~14
k2 (s-1)
33
k3 (s-1)
0.1
2-F
~12
28
0.15
0.20
1.2
2-OMe
2-CF3
~12
42
~30
12
Freeze-Quench-Coupled Mössbauer Analysis for the AsqJ
Reaction with 2-CF3.
the AsqJ⋅Fe2+⋅2OG⋅2-CF3 complex may not (or very slowly)
react with O2 during the time period monitored by SF-Abs,
which provides a possible explanation to the small absorbance
changes at ~ 470nm in the reaction with 2-CF3.
To further validate the kinetic model derived from the SF-Abs
experiments, freeze-quench coupled Mössbauer analysis was
carried out for the AsqJ reaction with 2-CF3. The anaerobic
solution containing the AsqJ⋅Fe2+⋅2OG⋅2-CF3 complex (final
concentration: 0.75 mM AsqJ, 0.6 mM Fe2+, 6 mM 2OG, and
0.7 mM 2-CF3) was rapidly mixed with O2 saturated buffer
(final concentration: ~ 0.9 mM O2) and quenched at various
time points. The samples were subject to Mössbauer
measurements, and the resulted spectra are shown in Figure 3.
Four time-dependent iron species were observed: (1) a
quadrupole doublet representing the AsqJ quaternary complex
(δ = 1.25 mm/s |ΔEQ| = 2.54 mm/s, linewidth 0.33 mm/s), (2) a
second quadrupole doublet representing the Fe(IV)=O species
(δ = 0.31 mm/s |ΔEQ| = 0.68 mm/s, linewidth 0.38 mm/s), (3) a
third quadrupole doublet representing possibly the AsqJ
enzyme product complex (δ = 1.21 mm/s |ΔEQ| = 2.96 mm/s,
linewidth 0.32 mm/s), and (4) a fourth quadrupole doublet
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representing the AsqJ•Fe(II)•2OG ternary complex (δ = 1.25
mm/s |ΔEQ| = 2.26 mm/s, linewidth 0.55 mm/s with a Voigt line
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shape) Comparing with the previously published FQ Mössbauer
results in the reactions with 2-OMe and 2-H,49 the AsqJ
quaternary complex, the Fe(IV)=O species, and the AsqJ
enzyme product complex observed here show identical
Mössbauer parameters with those of the corresponding iron
species in the reactions with 2-OMe and 2-H. However, the time
dependent changes of these iron species are different. First, the
kinetics of the Fe(IV)=O species and the amount accumulated
(~ 20% of the total iron in the sample) are similar to the results
from the reaction with 2-OMe and 2-H (in comparison, the
maximum amount accumulated in the reactions with 2-OMe or
2-H was ~ 20-30%).49 However, a much smaller amount of the
AsqJ quaternary complex was consumed. For example, at the
time points (0.01, and 0.03 s) of the maximum accumulation of
the Fe(IV)=O species, there are still ~ 60% (relative to the total
iron in the sample) AsqJ quaternary complex left in the reaction
with 2-CF3, but for 2-OMe and 2-H, the quaternary complex
had diminished to ~ 30%.49 Even at a later time (1 s), the AsqJ
quaternary complex only reduces to ~ 50% in the 2-CF3
reaction. This reduced consumption of the quaternary complex
in the reaction with 2-CF3 is also correlated well with the
amount of the enzyme product complex (δ = 1.21 mm/s |ΔEQ| =
2.96 mm/s, linewidth 0.32 mm/s) observed by Mössbauer,
which is accumulated to a maximum of ~ 15% at 0.16 s. In
addition, the enzyme product complex is rapidly converted to
the AsqJ ternary complex (δ = 1.25 mm/s |ΔEQ| = 2.26 mm/s,
linewidth 0.55 mm/s) at 1 s in the 2-CF3 reaction, while in the
reactions with 2-OMe and 2-H, this enzyme product complex
has a much longer lifetime so that at 1 s 70%-80% of the iron
in the sample is still representing this species. Finally, ~ 10%
O2 insensitive Fe(II) species was observed, which has δ = 1.29
mm/s |ΔEQ| = 3.45 mm/s. It has been attributed to the inactive
portion of the enzyme. Thus far, combining the Mössbauer and
the SF-Abs results, we can conclude that regardless of the
nature of the para substitutions on the phenyl moiety of the
substrate, the decay kinetics of the Fe(IV)=O intermediate is not
strongly perturbed. Compare to strong electrophilicity
demonstrated by synthetic Fe(IV)=O complexes in the OAT
reactions, the OAT reactivity of the AsqJ Fe(IV)=O species
does not exhibit a similar trend, suggesting that the electron
richness of the olefin moiety is not the controlling factor in
promoting the OAT reactivity of the ferryl intermediate in AsqJ.
The small absorption change at ~ 470 nm in SF-Abs data from
the 2-CF3 reaction is most likely the consequence that only a
part of the AsqJ quaternary complex reacts rapidly with O2 as
shown by the Mössbauer data.
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Figure 3. Zero-field Mössbauer spectra of samples obtained by
freeze quenching the reaction of AsqJ with 2-CF3 at various time
points. Black vertical bars: experimental data; Black lines: overall
spectral simulation; the blue lines: the AsqJ⋅Fe2+⋅2OG⋅2-CF3
complex; the red areas: the Fe(IV)=O intermediate; the green areas:
the AsqJ product complex; the purple areas: the AsqJ⋅Fe2+⋅2OG
complex; the grey line: the inactive species. The simulation
parameters and the relative amount of various species observed in
different times are listed in the main text and in Table S2.
LC-MS Analysis of AsqJ Reactions with 2-R.
To further corroborate the spectroscopic results on AsqJ-
catalyzed epoxidation, enzyme activity assay was carried out
under multi-turnover conditions and the results were analyzed
by liquid chromatography coupled mass spectrometry (LC-
MS). As depicted in Figure 4, all analogs were converted to the
corresponding epoxides (3-OMe, 3-H, 3-F and 3-CF3)
respectively. Under the current experimental conditions, all
analogs were consumed at a similar level. If a cation species
centered at C10 is involved in the epoxidation, one anticipates
the electron withdrawing groups, e.g. F or CF3, will disfavor the
formation of such an intermediate, and likely result in decrease
of product formation. However, the observation of a similar
level product formation when replacing H with F and further by
-CF3 (2-H vs. 2-F and 2-CF3) is not consistent with such a
mechanism. In the literature, when a cation is involved in the
reaction, the electron withdrawing group, such as F or CF3,
usually diminishes the corresponding product formation
drastically.59,60 Since no obvious perturbation was observed
when different analogs were used, the LC-MS result is thus
consistent with a mechanism where the AsqJ catalyzed
epoxidation either initiates from the C10 position of the
substrate or it starts from C3 center, but does not involve a
cationic species at C10. In the latter scenario, the reaction likely
proceeds via homolytic cleavage of the C=C bond of the
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substrate to form the C3-O bond and the C10 radical which is with a di-oxygen species or two mutually exclusive, closely
not sensitive to the para-substituent on the phenyl ring of the
1
spaced solvent molecules with partial occupancy. By allowing
the occupancy of the proposed di-oxygen species to deviate
from unity, the occupancy and O1-O2 distance converged at 0.6
and 1.5 Å, respectively, after the refinement. From the reported
crystal structures on O2 activating mononuclear iron enzymes
in the literature,61–63 the Fe-O2 adducts generally exhibit an O-
O bond length of ~ 1.2 – 1.3 Å, and the Fe-(hydro)peroxo/Fe-
alkyperoxo species generally show an O-O bond length of ~ 1.4
– 1.5 Å. Thus, the O1-O2 distance of 1.5 Å seen in our structure
is close to those from the Fe-(hydro)peroxo/Fe-alkyperoxo
species. In the O2 activation mechanism of Fe/2OG enzymes,
two Fe-peroxy intermediates have been proposed.64–66 One is an
Fe(II)-peroxysuccinate intermediate that is formed after the
decarboxylation of 2OG while the O-O bond is not cleaved. The
other is a bicyclic Fe(IV)-peroxyhemiketal complex where the
peroxide bridges between the iron center and the C2 of 2OG.
Several DFT studies suggested that the former intermediate is
most likely involved in the O2 activation process based on
relative energetics of these intermediates.65,66 Recently, the
existence of the Fe(II)-peroxysuccinate intermediate has found
some further support from a crystal structure of VioC, a
Fe(II)/2OG dependent L-arginine hydroxylase, where the
electron density map at the iron center was best fitted with an
Fe(II)-peroxysuccinate moiety with O-O bond distance of 1.44
Å.67 In our case here, the existence of an intact 2OG molecule
is well supported by the electron density map, thus it is possible
that the extra electron density together with 2OG could
represent the Fe(IV)-peroxyhemiketal complex. However, a
close examination suggests it is unlikely because 1) the O2-
C1(2OG) and the O2-C2(2OG) distances are almost the same
(2.67 Å vs 2.66 Å), which rules out the possibility that a
covalent bond is formed between O2 and C2 of 2OG; and 2) the
Fe-O1 distance is 2.4 Å, which seems to be too long for an
Fe(IV) center. Thus, this structure may represent a 2OG-bound
Fe-(hydro)peroxo species. Alternatively, this extra piece of
electron density may represent two closely spaced partially
occupied solvent molecules. It should be noted that for all other
reported AsqJ crystal structures, one solvent molecule is always
present at the location occupied by O1.
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substrate.
It is also worth noting that although only ~ 80% of 3-CF3
was produced (relative to the amount of 3-H), it is possibly due
to substrate binding, rather than the substituent effect (see the
crystallographic section).
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Figure 4. LC-MS chromatograms of epoxide products formed
during AsqJ-catalyzed epoxidation when analogs (2-OMe, 2-H, 2-
F and 2-CF3) were used. The MS traces are monitored and recorded
using selected iron monitoring.
Crystallographic Characterizations of the AsqJ Catalyzed
Epoxidation.
To provide structural insights into the AsqJ-catalyzed
epoxidation, we have determined the crystal structures of AsqJ
at different stages of the catalytic cycle (Table S3). By using the
published procedure,54 we first obtained crystals with 1-H
(cyclopeptin) and acetate binding to the iron center. We then
replaced 1-H and acetate with 2-H and 2OG respectively by
post-crystallization soaking in the presence of O2. The resultant
electron density map with a resolution of 1.7 Å showed
unambiguously the presence of 2-H and 2OG (Figure. 5A),
indicating that our ligand-exchange strategy was successful. In
addition, an extra piece of electron density extending from the
iron center and trans to His134 was identified. It could be fitted
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Figure 5. Crystallographic snapshots of AsqJ-catalyzed epoxidation. (A) The AsqJFe2OG2-Hperoxide quinary complex (PDB code:
6K0E). (B) An intermediate structure show that the AsqJ-catalyzed epoxidation may start from the oxygenation of C10 (PDB code: 6JZM).
The red arrowhead points to the position of the oxygen atom. The magenta sphere (right panel) shows the modeled oxygen atom in an
idealized axial position. (C) The product-bound AsqJFe2OG3-Hperoxide quinary complex (PDB code: 6K0F). The 2Fo-Fc maps (grey
mesh) and unbiased Fo-Fc maps highlighting Fe and the extra piece of electron density (blue) are contoured at 1.0 σ and 3.4 σ, respectively.
The bound ligands (cofactors and substrates) and peroxide are shown in stick form. The Fe is shown as orange sphere. The carbon atoms of
ligands and selected amino acid side chains are colored green. The remaining atoms are colored according to the CPK scheme
When the same post-crystallization ligand-exchange was
performed with a further addition of 0.5 mM ascorbate in the
substitute mother liquor solution in the presence of O2,
interestingly, difference electron density peaks (a resolution of
1.88 Å) appeared around the active center, suggesting that
chemical reaction may have taken place in the crystal under this
condition (Figure 5B). Reinterpretation of the electron density
maps of the active site indicated that 2OG and 1-H had been
converted to succinate and 2-H, respectively. Unexpectedly, a
continuous electron density was observed between the Fe and
the C10 of 2-H, which can be best fitted with an oxygen atom
that is covalently linked to the Fe and the C10 of 2-H with the
refined bond lengths of Fe-O and O-C10 are 2.1 and 2.0 Å,
respectively (Figure 5B). The Fe center of this structure is
penta-coordinated with a distorted trigonal bipyramidal
geometry. Thus this Fe-alkoxide structure may represent an
intermediate in AsqJ catalyzed epoxidation and implicates that
the epoxidation of 2-H is initiated by the oxygenation of C10,
which is fully consistent with the results obtained by SF-Abs,
Mössbauer and LC-MS. A similar Fe-alkoxide species has
recently been identified in a crystal structure of L-arginine-3-
hydroxylase, VioC,67 and has also been proposed in
taurine/2OG dioxygenase, TauD, based on resonance Raman
data.68 Although the catalytic role of such an Fe-alkoxide
species in aliphatic hydroxylation reaction, such as VioC
catalyzed L-arginine hydroxylation, is still unclear, a similar
species in the epoxidation reaction has long been proposed in
experimental and computational studies on both heme
dependent enzymes,30–36,39 and heme and non-heme biomimetic
model complexes.37,38,45,69–74 Yet, it is the first time that such a
species is experimentally identified in an enzymatic
epoxidation reaction. In addition, the Fe-alkoxide species has
also been proposed as the key intermediate for aromatic
hydroxylation catalyzed by non-heme iron enzymes.75–77
Recently, an Fe-alkoxide species has been implicated in the
reaction of a Rieske-type aromatic hydroxylase, Salicylate-5-
Hydroxylase (S5H), based on the observed hyperfine
broadening of the EPR resonance at g = 4.3 through a 17O2
experiment.78 Furthermore structure information for this type of
species involved in aromatic hydroxylation has been reported
on a non-heme iron model complex supported by a pyridine-
based ligand that is involved in intramolecular aromatic
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hydroxylation.79,80 The existence of a similar Fe-alkoxide
species in all these reaction types suggests strong similarities in
their reaction mechanisms.
green and yellow, the remaining atoms are colored according to the
CPK scheme.
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Consistent with the occurrence of in-crystal epoxidation,
A further examination of this Fe-alkoxide structure
revealed that C10 becomes sp3-hybridized and exhibits
tetrahedral geometry. In contrast, C3 was found to adopt
trigonal planar geometry in this epoxidation intermediate
structure (Figure 6A), suggesting that C3 has remained in the
sp2-hybridized state following the attack on C10. To further
verify the observed bonding geometry, we deliberately modeled
C3 as a tetrahedral carbon, and the hypothetical structural
model was subjected to additional refinement cycles by Phenix.
While planar C3 is fully enclosed within the electron density
map, the tetrahedral C3 model was not justifiable due to a poor
fit to both omitted and refined electron density maps (Figure
6B-D). Moreover, the refined bond length of C3-C10 is 1.47 Å
(Figure 6B), which is shorter than the expected value (1.54 Å)
for a C-C single bond. Taken together, these findings support
the interpretation that C3 is likely in a sp2-hybridized state,
which may imply a carbocation species at C3 (but see more
discussion in the DFT section).
the electron density maps (a resolution of 1.63 Å) derived from
a 1-H-bound crystal exposed to excess amount of 2OG, 1.0 mM
ascorbate, and O2 revealed the presence of 3-H, and 2OG in the
active site (Figure 5C). This structure represents a product-
bound, AsqJFe2OG3-H complex. The presence of 2OG
instead of succinate in the active site indicates that AsqJ likely
binds 2OG with higher affinity than that of succinate.
Collectively, these structures derived from our crystallographic
studies provide new insights for the epoxidation steps of AsqJ
and are fully consistent with other experimental data presented
in the previous sections.
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Figure 7. Crystal structures of AsqJ complexed with the 2-CF3
substrate analog. (A) and (B) Electron density maps and structures
of the bound analog observed in space group P212121 (PDB code:
6KD9) and C2221 (PDB code: 6K3O), respectively. The 2Fo-Fc
electron density maps (contoured at 1.0 σ) are shown in grey mesh.
(C) Superimposition of the bound 2-CF3 analog. The conformations
observed in space group P212121 and C2221, are in green and cyan,
respectively. The orange-shaded region highlights the difference in
ring-puckering seen in 2-CF3. (D) Structural comparison between
AsqJ-bound 2-H (magenta) and 2-CF3 (green) in the orthorhombic
crystal form (space group P212121) revealed the movement of 2-
CF3 with respect to Fe, H134 and P132.
Furthermore, we determined the 2-CF3 bound structures by
cocrystalization of 2-CF3 with iron bound AsqJ protein. The
crystal structures of the complex were determined in different
space groups C2221 and P212121 at 2.68 and 2.45 Å resolution
(Table S4) with one and two AsqJ molecules per asymmetric
unit, respectively. For both crystal forms, the electron density
maps covering the benzyl moiety of 2-CF3 become bulkier,
consistent with the presence of CF3 substituent (Figure 7A and
7B). Superimposition analysis showed that, while the spatial
positions of active site residues are largely invariant between
these two analog-bound structures, the plane defined by N1, C2
carbonyl (C2=O), and C3 of 2-CF3 exhibits a conformational
change (Figure 7C). This finding indicates a greater flexibility
Figure. 6. A sp2-hybridized C3 of the subtrate following the initial
attack on C10 by the ferryl during epoxidation. (A) A wall-eye
stereo diagram showing two orthogonal views of the observed
epoxidation intermediate structure superimposed on the 2Fo-Fc
electron density. (B, C) C3 of the epoxidation intermediate was
modeled as sp2 (B, green) or sp3 (C, yellow) for structural
refinement. (D) 2Fo-Fc electron density superimposed on the two
refined structural models of the epoxidation intermediate with C3
being constrained to either adopt a planar (sp2, green) or tetrahedral
(sp3, yellow) geometry. The red arrowhead indicates the position of
C3. The 2Fo-Fc electron density maps (contoured at 1.0 σ) are
shown in grey mesh. The Fe is shown as an orange sphere. The
bound ligands are in stick form with carbon atoms colored either
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in the binding of 2-CF3 to AsqJ than that of other substrates, and seen in the crystal structures (the offline mode) to the online
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that the heptameric ring of 2-CF3 may undergo a ring-puckering
change in the AsqJ active site. Moreover, compared to the 2-H-
bound structure (Figure 7D), the trifluoromethylbenzene
moiety seen in the P212121 structure displays a lateral movement
with respect to the Fe and Fe-coordinating H134 (Figure 7D),
likely caused by the steric repulsion between P132 and
trifluoromethyl group of the analog. Due to the positional and
ring-puckering change, the Fe-C3 distance seen in the P212121
structure (5.3 Å) is longer than that of the 2-H-bound structure
(5.0 Å) (Figure 7D). We suspect the substrate binding mode
seen in the P212121 structure is not optimal for catalysis, which
may be a possible reason to explain why this analog is less
reactive toward epoxidation.
mode results in little change in either the isomer shift or the
quadrupole splitting. However, the 6-coordinate complexes
generally have a larger |ΔEQ| than 5-coordinate complexes by
approximately 0.4 mm/s. This difference is generally observed
in all the DFT models tested in this study, showing a good
correlation between the coordination number (four-, five- and
six-coordinate) and the calculated quadrupole splittings (the
quadrupole splitting increases with increasing coordination
number, Figure S8). Thus, the Mössbauer parameters for the
AsqJ quaternary complex are in best agreement with a 6-
coordinate iron center. In addition, although in all the published
crystal structures of AsqJ in the presence of 2OG and the
structures included in this study, 2OG always shows an offline
binding mode, based on the current DFT results the Mössbauer
parameters (δ and ΔEQ) cannot be used to readily distinguish
the 2OG binding configuration to the iron center (offline vs.
online). Yet, our current calculations suggest that the 2OG
online mode in a 6-coordinated iron center is energetically more
stable than that of the offline mode by 4.5 kcal/mol (including
only the electronic energy). For a 5-coordinate iron center
(without the additional water molecule), the structures with two
different 2OG binding modes are essentially isoenergetic, with
the offline mode structure as the more stable structure by only
~1 kcal/mol. Therefore, two 2OG binding modes could both be
present in aqueous solution in the AsqJ quaternary complex.
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Density Functional Theory Calculations of the AsqJ
Quaternary Complex and the Enzyme Product Complex.
DFT calculations were performed to provide structural models
for the different experimentally observed iron species, in
particular to help interpret the information about the iron sites
and their coordination environment provided by Mössbauer
81–85
spectra. Previous works on model complexes
have
demonstrated that quantum chemical calculations can estimate
the Mössbauer parameters with good agreement to experiments.
Recently, Lai and coworkers have also calculated parameters
for the possible structures of the Fe(IV)-oxo intermediate of
AsqJ.86 We performed DFT calculations using possible
structures of the mononuclear iron active sites of AsqJ
quaternary and product complexes. Of particular interest is the
quadrupole splitting, ΔEQ, which reflects the charge distribution
around the iron nucleus, especially the Fe valence electrons and
the first coordination sphere. It provides signatures for different
Fe species even if they have the same (formal) valence and spin
states. For high-spin Fe(II) species with O/N ligands, |ΔEQ| is
typically in the range of 1.6-3.2 mm/s.87
Models of the AsqJ mononuclear Fe active site were
constructed based on a crystal structure reported in our previous
work (PDB ID 5Y7R).54 The model contains an octahedral, 6-
coordinate Fe, with the facial triad, the bidentate 2OG (keto
group trans to Asp136, and carboxylate group trans to His211),
and a water as the ligands, as well as the substrate 2-H in close
vicinity but not directly bonding to the Fe ion (Figure S7). The
“core” of the active site is modified according to different
possible configurations. The geometries were optimized with
the coordinates of certain anchoring atoms on the periphery of
the model fixed at their crystal structure positions, as detailed
in the SI. Based on the optimized structures, the Mössbauer
parameters δ and ΔEQ were further calculated using the method
described in the SI, which was also validated against several
high-spin Fe(II) model complexes with known experimental
|ΔEQ| (Table S5).
For the AsqJ⋅Fe2+⋅2OG⋅2-H complex (δ = 1.25 mm/s, |ΔEQ|
= 2.54 mm/s, Figure S3), we considered the following
possibilities: the 2OG could be chelating the iron with the
carboxylate trans to His211 as in the crystal structure (the
offline mode), or its C2-C3 bond could be rotated so that the
carboxylate is trans to His134 (the online mode); the 6th ligand
position could be occupied by a water, or unoccupied leaving a
5-coordinate species. The models and the calculated Mössbauer
parameters are shown in Scheme 4. The calculated isomer shifts
are similar across all models and consistent with experimental
value, although the 5-coordinate species generally have lower
isomer shifts. Rotating the 2OG from the binding configuration
Scheme 4. Schematic drawing of possible Fe center geometry of
the AsqJ⋅Fe2+⋅2OG⋅2-H complex and their calculated Mössbauer
parameters.
The intermediate Fe(II) species (δ = 1.21 mm/s, |ΔEQ| =
2.96 mm/s, See Figure 3 and our previous work49) appearing
after the decay of the Fe(IV)=O intermediate is most likely a
product complex with a bound succinate. To explore the
structural models for such a product complex, we considered
the following possibilities (Scheme 5): (i) a 4-coordinate
complex without iron-bound water molecules; (ii) a 5-
coordinate geometry with an iron bound water molecule either
in the plane or out of the plane defined by the oxygens from
succinate and Asp136 and nitrogen from His134; (iii) a 6-
coordinate species with two water molecules, one of which
interposes between the iron and the epoxide oxygen; (iv) an
alternative 6-coordinate species with the epoxide oxygen
directly coordinating to the iron. In addition, we considered the
possibility of succinate carboxylate being a bidentate ligand to
the iron center in the product complex, in light of various crystal
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structures as such included in 88–90. However, geometry
optimization attempts starting with bidentate binding invariably
reverted back to a monodentate binding for succinate, likely
dictated by a hydrogen bond interaction with Gln131, consistent
with the arguments by Solomon and coworkers.91
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For the first two models listed above, the optimized
structures exhibit Fe-O(epoxide) distances of 3.53, 3.46, and
4.03 Å respectively, and only the 6-coordinate species with two
iron-bound waters reproduces the experimentally observed
quadrupole splitting and isomer shift well (Scheme 5). For 5-
and 4-coordinate species, the predicted quadruple splittings are
generally much smaller than the experimentally determined
one. It is also worth noting that the experimental and
computational quadrupole splittings presented here for
potentially 6-coordinate iron centers are also consistent with the
observed |ΔEQ| for two 6-coordinate high-spin Fe(II) complexes
supported by 2-pyrazinecarboxylate ligand (3.04 and 3.16
mm/s).92,93 The iron coordination environment of these
complexes closely mimics that of the succinate-bound product
complex, with two aromatic N ligands with neutral formal
charge cis to each other, and two negatively charged
carboxylate ligands trans to each other on an axis perpendicular
to the N-Fe-N plane.
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Scheme 5. Possible Fe site structures of the AsqJ⋅Fe2+⋅succinate⋅3-
H complex and the calculated Mössbauer parameters. For Fe-O
distance scan in model (iv), see Table S6.
We further investigated the possible electronic structure of
the Fe-alkoxide species observed in one of the AsqJ crystal
structures. Using the crystal structure of the Fe-alkoxide species
(PDB code 6JZM) as the initial structure, the structural
optimization cannot stabilize the Fe-alkoxide species, rather it
causes the structure to relax into a fully formed epoxide with
the C3-O and C10-O distances of 1.42 and 1.43 Å (Scheme 5,
model (i’)). Interestingly, the optimized structure also features
a 5-coordinate iron center with an Fe-O(epoxide) distance of
2.26 Å, suggesting that the formed epoxide is still bound to the
iron center. In addition, in this structure the H-bond between
Asn70 and the epoxide product is broken, with a distance of
5.70 Å between the C1 carbonyl oxygen of the 3-H and the
nitrogen of Asn70, as compared to 3.10 Å in the model (i)
discussed above. This optimized structure is essentially an
alternative configuration of the product complex model with a
4-coordinate iron center (Scheme 5, model (i)). In that 4-
coordinate iron center structure, the O(epoxide) does not
coordinate to the iron center while the H-bond between Asn70
and the product is maintained. The electronic energy difference
between these two optimized structures (model (i) vs. model
(i’)) is only ~2 kcal/mol with the model (i) structure being a
more stable structure. This further suggests that the modulation
of the H-bond between Asn70 and the epoxide product may be
important in determining the interaction between the iron center
and the epoxide. Nonetheless, the quadrupole splitting
predicted from this 5-coordinate Fe-epoxide structure (|ΔEQ| =
2.48 mm/s) is still smaller than that of the experimental value
for the product complex ((|ΔEQ| ~ 3 mm/s).
For the last model (model (iv), Scheme 5), geometry
optimization did not result in the complex with directly
coordinating epoxide oxygen in a 6-coordinate iron center.
Rather, the structure relaxed into the 5-coordinate geometry of
model (ii) above. This is possibly due to a hydrogen bond
between the cyclopenin (3-H) C1 carbonyl and the acetamide
group of Asn70 (See Figure S7), whose Cα is fixed in the DFT
models. A close Fe-O (epoxide) distance would break this H-
bond. We next brought the epoxide O close to the iron in a 6-
coordinate geometry by freezing the Fe-O distance, then
optimized a series of structures with fixed Fe-O distances
ranging from 2.0 to 2.7 Å (which would break this H-bond) and
calculated the Mössbauer parameters as well. The results are
shown in Scheme 5 and Table S6. Again, the |ΔEQ| follows a
decreasing trend with decreasing coordination number. The Fe-
O distance scan results are also consistent with this trend, as the
|ΔEQ| decreases with increasing Fe-O distance, essentially
transitioning from 6- to 5-coordination. When the Fe-O distance
is in the range of typical Fe(II)-O single bond (loosely defined
to be 2.0 - 2.2 Å), |ΔEQ| is in the range of 2.98 - 3.34 mm/s,
which is close to the experimentally observed value. However,
as described above, the 6-coordinate complex with two iron-
bound waters is also calculated to have a similar |ΔEQ| = 3.2
mm/s. Therefore, although the exact iron coordination
environment cannot be exclusively established for the Fe(II)
product complex based on these DFT calculations, it is clear
that the Mössbauer parameters of the Fe(II) product complex
support a 6-coordinate iron center with most likely 2 histidine
nitrogen and 4 oxygen ligands. It should be noted that the Asn70
residue is located on a flexible loop near the surface of the
protein (Figure S7).Therefore, it is possible that protein
dynamics during the enzymatic reaction could modulate the H-
bond between Asn70 and the substrate to allow the substrate to
come close to the iron center during the epoxidation step,
making it possible for an iron-bound epoxide to exist
transiently.
Since the structural optimization by DFT cannot stabilize
the Fe-alkoxide species, we performed a more constrained
geometry optimization by only allowing the iron atom, the three
protein ligands and the succinate (but not the alkoxide) to move.
This optimization resulted in a structure with a shorter Fe-O
bond length of 1.78 Å, compared to 2.1 Å in the crystal
structure. Significant spin populations were found mostly on Fe
(+4.02) and C3 (-0.57), while the population on O is only +0.13
(Figure S7). Calculation of Mössbauer parameters yielded δ =
0.54 mm/s and ΔEQ = 1.42 mm/s. The result signifies that the
electronic structure of the iron center is essentially a high-spin
Fe(III) (S = 5/2) antiferromagnetically coupled to a C3-radical.
The spin density is visualized (contoured at 0.01 au-3) in Figure
S9. <S2> was calculated to be 6.76, which were deviated from
a <S2> = 6 for an isolated S = 2 iron center, but were consistent
with a broken-symmetry solution having an S = 5/2 Fe(III)
center antiferromagnetically coupled with a substrate radical.
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In summary, DFT calculations suggest that the AsqJ
1
quaternary complex with 2-H binding as well as the product
complex (with the presence of the epoxide) most likely feature
a 6-coordinate iron center based on the DFT predicted
quadrupole splittings and isomer shifts on various models. The
epoxide may directly coordinate to the iron center during the
initial epoxide ring formation, and its dissociation from the iron
center may rely on the hydrogen bond interaction between the
epoxide product and the protein residues, most likely Asn70, as
well as extra water molecules close to the iron center. The Fe-
alkoxide species could not be stabilized during geometry
optimization, suggesting that this species could only be
captured in crystal due to the restraining crystal environment.
In addition, its electron structure may be best described as a
high-spin Fe(III) center antiferromagnetically coupled with an
S = ½ radical localized at the C3 position of the substrate. This
is a species that has long been proposed to be a potential
intermediate in epoxidation reaction catalyzed by both heme
and non-heme iron enzymes.33-38,86,94,95 Lastly, the offline and
the online 2OG binding configurations to the iron center in the
AsqJ quaternary complex cannot be readily distinguished by
Mössbauer parameters and may coexist in aqueous solution
based on close electronic energies of the two binding
configurations. This is also predicted by a recent QM/MM study
of an Fe/2OG-dependent histone demethylase.96
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Molecular Dynamics Simulations on the Relevance of the Two
2OG Binding Configurations to the Initial O2 Reactivity
towards the AsqJ Fe(II) center.
Two 2OG-binding configurations to the iron center have been
observed in many published crystal structures of Fe/2OG
enzymes.97 Although the C2-carbonyl oxygen of 2OG
invariantly trans to the carboxylate of the 2-His-1-carboxylate
facial triad, the position of the C1 carboxylate of 2OG adopts
two orientations. In one orientation, the C1 carboxylate group
is located trans to the proximal (closer to the N-terminus of the
protein) histidine of the 2-His-1-carboxylate facial triad (the
online configuration). In the other orientation, the C1
carboxylate group is located trans to the distal histidine (the
offline configuration). For AsqJ, all the available crystal
structures show an offline configuration for 2OG. As a result,
the potential O2 binding site, which is normally occupied by a
solvent molecule in the crystal structures, is pointed away from
the substrate. Then the subsequently formed Fe(IV)=O
intermediate followed by the O2 addition and the oxidative
decarboxylation of 2OG would result in an offline mode ferryl
intermediate where the Fe(IV)=O unit points away from the
substrate, thus creating a problem for the subsequent chemical
reaction between the Fe(IV)=O intermediate with the enzyme
substrate. In our previous study on AsqJ catalyzed epoxidation
reaction, a possible oxo-hydroxo/water tautomerism of the
Fe(IV)=O intermediate has been proposed. Based on QM/MM
calculations, Lai and coworkers suggested that this tautomerism
could be the key mechanism to effectively rotate the Fe(IV)=O
unit and bring the oxo close to the olefin moiety of 2-H for the
subsequent steps of the reaction.86 The different orientations of
the Fe(IV)=O moiety of the ferryl intermediate (online vs.
offline) in the Fe/2OG dependent halogenase, SyrB2, have also
been proposed to explain why SyrB2 favors halogenation than
hydroxylation. Here, we investigate another possibility.
Namely, which 2OG configuration (the online mode vs. the
offline mode) is thermodynamically preferred for the initial O2
addition to the iron center of AsqJ.
Figure 8. Free energy difference (ΔΔG) of O2 addition to the Fe(II)
center of AsqJ in two different 2OG binding configurations derived
from MD simulations. (a) Cumulative ΔΔG for four system
configurations averaged over 0.5ns MD simulation intervals by
using the the 2OG offline configuration without substrate as the
reference state. (b) Cumulative distribution function of water
molecules around iron-bound water or O2 in presence or absence of
a substrate in two 2OG binding configurations. (c) Root-mean-
squared deviation (RMSD) of the protein backbond and the
substrate 2-H derived from MD simulations with either water or O2
bound to the iron and the 2OG in the online configuration. The
simulations were performed with no positional restraints on either
the substrate 2-H or the protein.
For non-heme O2 activating mononuclear iron enzymes, it
is generally accepted that the initial O2 addition to the Fe(II)
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center is largely facilitated by the generation of a five- Fe(IV)=O moiety pointing towards the substrate to carry out
1
coordinated iron center via the removal of the solvent
molecule.98,99 Therefore, to answer the aforementioned
question, we used thermodynamic integration (TI) to calculated
free energy change in the process of replacing a coordinated
water with an O2 at the iron site of AsqJ in the two 2OG
configurations (online vs. offline). The TI simulations were
performed repeatedly in presence and in absence of the enzyme
substrate, 2-H. To fully account for the solvation effect and the
dynamic nature of the enzyme, we used molecular dynamics
(MD) simulations for these TI calculations (Scheme S2). The
initial protein structures for both the online and offline modes
of the 2OG binding were prepared from the crystal structure
(See the SI for the details). The MD-equilibrated protein
remained stable in the simulations in both the 2OG online and
the 2OG offline modes. Simulated structures maintained
conformations of the protein secondary structures and of the
active site pocket, which can be overlapped with the initial
crystal structure (Figure S10, S11). For calculating the free
energy changes, the coordinated water in the initial structure is
gradually transformed to an O2 for both the online and the off-
line mode structures in the presence or the absence of 2-H (total
four configurations were modeled, see the SI for the simulation
details). The Gibbs free energy of the transformation was
calculated by using the multistate Bennett acceptance ratio
(MBAR) algorithm. Figure 8a shows free energies of
transformation of a bound water to an O2 computed for four
system configurations cumulatively averaged over 0.5ns MD
intervals to demonstrate the statistical convergence of the
simulated results. Computed ΔΔG are reported with respect to
the free energy of the water to O2 substitution in the offline 2OG
configuration with no substrate in the protein active site.
rapid chemical transformations.
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Conclusions
In this study, the AsqJ-catalyzed epoxidation was studied using
the native substrate, 2-OMe, and several para-substituted
substrate analogs (2-H, 2-F, and 2-CF3). All compounds
triggered a rapid formation of the iron(IV)-oxo intermediate
under pre-steady state conditions. Importantly, the decay
kinetics of the ferryl species was largely insensitive to the para-
substituents on the phenyl moiety bearing electron withdrawing
or electron donating group (only 2-3 times of variation on the
rate constant are observed). Thus, the OAT reactivity of the
Fe(IV)-oxo intermediate in AsqJ does not depend on the
electron richness of the olefin moiety of the substrate. This
catalytic behavior is very different from non-heme Fe(IV)-oxo
model complexes. In all the reported model complexes, the
OAT reactivity for Fe(IV)-oxo species generally demonstrated
strong electrophilic nature so that the para substitution on the
phenyl moiety of the substrate (e.g. styrene) dramatically
modulates the life-time of such a species (up to 100 fold in
going from electron donating substituents to electron
withdrawing ones). The lack of such a correlation in the AsqJ-
catalyzed epoxidation also suggests that there is no significant
charge separation (e.g. cationic species) in the transition state of
the rate-determining step of the reaction. These experimental
observations were further supported by the crystallographic
data and the reaction product analysis obtained via LC-MS. The
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LC-MS analyses showed
a similar level of substrate
consumption and product formation when the enzymatic
reactions were carried out in the steady-state turnover
conditions regardless of the compound used. The observation of
an Fe-alkoxide species in one of the crystal structures provided
important support for a step-wise epoxidation mechanism
where an initial attack from the Fe(IV)-oxo intermediate to the
olefin moiety happens at the benzylic (C10) position of the
substrate. This observation is also consistent with the substrate-
bound crystal structures, which showed a shorter distance
between the Fe and the C10 position than that between the Fe
and the C3 position (4.4 Å vs. 5.0 Å).
The results suggest that the presence of 2-H greatly
facilitates the replacement of a bound water to an O2 only when
2OG is in the online mode (computed ΔΔG = -6.5 kcal/mol
relative to the 2OG-online and no substrate configuration). At
the same time, the 2OG offline mode is not affected
significantly (computed ΔΔGs are within 1 kcal/mol for
configurations with and without the substrate). Given that all
other factors in the structure are similar this result suggests that
the O2 addition rate should be much more rapid (>10000 times
Furthermore, the DFT calculations suggested that the Fe-
alkoxide species observed in the crystallographic data may be
best described as an Fe(III) center antiferromagnetically
coupled with a substrate radical located at the C3 position of the
substrate. However, this species was not observed in solution,
suggesting that a restraining crystal environment is necessary to
stabilize such an intermediate. In addition, the calculations also
suggested that AsqJ quaternary complex as well as the product
complex with the presence of the epoxide product most likely
feature a 6-coordinate iron center. The epoxide may directly
coordinate to the iron center during the initial epoxide ring
formation, and its dissociation from the iron center may rely on
the hydrogen bond interaction between the epoxide product and
the protein residues, most likely Asn70, as well as extra water
molecules close to the iron center. Finally, the MD simulations
supports a scenario where the initial O2 addition to the iron
center could happen in the online-binding mode of 2OG to the
iron center, not the offline mode seen in the crystal structure.
This is most likely due to the substrate binding, which provides
a hydrophobic environment to facilitate the O2 addition to the
iron center. Such a hydrophobic environment is more
effectively formed when 2OG binds to the iron center in the
online mode.
o
faster at 4 C) in the 2OG online mode structure than in the
offline mode once the substrate is present. This is likely because
the O2 binding pocket created by the substrate binding is more
hydrophobic in the online mode structure than in the offline
mode. Indeed, Figure 8b shows the cumulative distribution
function of water molecules around iron-bound water or O2 and
suggests that the substrate binding squeezes water more
strongly from the active site in the 2OG online configuration
that results in a stronger destabilization of iron-bound water
molecule. Figure 8c shows results of the 40 ns MD simulations
of AsqJ in the 2OG online mode with unrestrained 2-H, with
water or O2 bound to Fe. It shows that in the 2OG online mode
the substrate binding is stable when oxygen is bound to iron but
unstable when water is bound to iron. This may partially explain
the difficulty to experimentally observe the online OG
configuration of AsqJ/substrate complex in crystal structure.
In summary, although the 2OG-binding configuration in all
the available crystal structures of AsqJ is in the offline mode,
the rapid O2 addition to the Fe(II) center in solution could likely
occur in the online binding mode of 2OG. Thus the
subsequently formed ferryl intermediate will have the
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experiment. Time-resolved absorbance scans from 300 to 700 nm
In summary, all the experimental and computational results
strongly suggest a step-wise mechanism for AsqJ-catalyzed
epoxidation reaction. The initial oxygen addition from the
Fe(IV)-oxo species to the olefin moiety of the substrate happens
at the benzylic position, which is most likely followed by the
generation of a Fe(III)-alkoxide species with a carbon radical
located at the C3 position of the substrate. The subsequent C3-
O bond formation complete the epoxide ring formation.
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were collected with a diode-array detector. The resulting data were
processed using KaleidaGraph software and shown in Figure S5.
The kinetic analysis was carried out by using a 3-step kinetic model
depicted in Scheme 3, and the simulations were performed by
Kintek Explorer software.100,101 Details of sample preparation are
provided in the Supporting Information.
Freeze-quench Mössbauer Sample Preparation. Freeze-
quench experiments were performed using a KinTek quench-flow
instrument. An oxygen-saturated buffer solution (~1.8 mM O2 in a
50 mM Tris-HCl, pH 7.5) buffer solution prepared at ~ 4 °C was
rapidly mixed with an equal volume of an oxygen-free solution
containing AsqJ (1.5 mM), 57Fe(II) (1.25 mM), 2OG (12.5 mM),
and substrate (2-H or 2-CF3, 1.5 mM) to initiate the reaction at 5
°C. The resulting reaction was terminated by injection of the
solution into liquid ethane maintained at 90 K at various time
points. The resulting samples were first pumped to remove liquid
ethane. Then the dry frozen solution powder was packed into in-
house designed freeze quench Mössbauer sample cups cooled at
liquid nitrogen temperature to generate Mössbauer samples. The
reaction time of a freeze-quenched sample is the sum of the aging
time and the quench time. The aging time was the transit time for
the reaction mixture through the aging hose. The quench time
corresponded to the time required after injection into the
cryosolvent for the reaction mixture to be cooled sufficiently to
prevent further reaction and was estimated to be ~ 5 ms.
EXPERIMENTAL SECTION
9
General Procedures. All reagents were used directly as obtained
from the commercial sources. Analytical thin layer
chromatography (TLC) was carried out on pre-coated TLC
aluminum plate (silica gel, grade 60, F254, 0.25 mm layer
thickness) acquired from EMD Chemicals (Gibbstown, NJ). Flash
column chromatography was performed using silica gel (230-400
mesh, grade 60) obtained from Sorbent Technologies. NMR
spectra were recorded on a Bruker 500 MHz spectrometers.
Chemical shifts (in ppm) are referenced using solvent (CHCl3,
DMSO, water) peaks, with coupling constants reported in Hertz
(Hz). The relative molecular mass and purity of enzyme samples
were determined using SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). The protein molecular weight marker used was
purchased from Bio-Rad Laboratories (Precision Plus ProteinTM
All Blue Standards).
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Protein Overexpression and Purification. Plasmids (pET-28a)
carrying the Aspergillus nidulans AsqJ gene were synthesized by
the commercial GeneArt Gene Synthesis service (Invitrogen). The
plasmids were transformed into BL21 (DE3) E. coli cells (New
England Biolabs) and cultured in LB medium. Overexpression was
induced by Isopropyl β-D-1-thiogalactopyranoside (GeminiBio) at
27 °C for 4 hours before cells were harvested. The enzyme was
purified using a Ni-NTA agarose column, and excess salt and
protein-bound metal ions were removed by dialysis. Oxygen was
removed from the concentrated protein solution by degassing on a
Schlenk line and equilibrating in a glove box (MBraun). The
protein stock was aliquoted and stored at -80 °C. Details of the
experimental conditions are provided in the Supporting
Information.
LC-MS experiments. High performance liquid chromatography
(HPLC) with detection by mass spectrometry (MS) was conducted
on an Agilent Technologies (Santa Clara, CA) 1260 infinity series
system coupled to an Agilent Technologies 6120 single quadruple
mass spectrometer. The associated Agilent MassHunter software
package was also used for data collection and analysis. Assay
mixtures were separated on an Agilent ZORBAX Extended-C18
column (4.6 x 50 mm, 1.8 μm particle size) with an isocratic system
of 40% of solvent A (25 mM aqueous ammonium formate, pH =
6.0) and 60% solvent B (methanol). Detection was performed using
electrospray ionization in positive mode (ESI+). The corresponding
products were extracted at the corresponding M+1 m/z values.
Reactions associated with Figure 4 was performed as follows: Each
reaction contained a mixture of AsqJ, Fe(II), 2OG, substrate in 0.1
M tris buffer (pH = 7.5) with the final concentrations of 0.11 mM
AsqJ, 0.1 mM Fe(II), 0.1 mM substrate, and 0.2 mM 2OG. Once
all reaction samples were prepared in the glove box, samples were
exposed to air by opening the reaction vials to allow oxygen
infusion. After 20 mins under ambient environment exposure,
proteins were removed using centrifugal filtering tubes using
Nanosep® centrifugal filters.
Mössbauer analysis. Mössbauer spectra were recorded with
home-built spectrometers using Janis Research SuperVaritemp
dewars, which allowed studies in the temperature range from 1.5 to
200 K, and applied magnetic fields up to 8.0 T. Mössbauer spectral
simulations were performed using the WMOSS software package
(SEE Co., Edina, MN). Isomer shifts are quoted relative to Fe metal
at 298 K. All Mössbauer figures were prepared using SpinCount
software.102
EPR analysis. All EPR samples were produced in a N2 glove box
with a sample volume of 250 uL and flash-frozen in liquid nitrogen
with 10% (v/v) glucerol added as a glassing agent. X-band EPR
spectra were recorded on a Bruker Elexsys spectrometer equipped
with an Oxford ESR 910 cryostat and a Bruker bimodal cavity for
generation of microwave fields parallel and transverse to the
applied magnetic field. The modulation amplitude and frequency
were 1mT and 100 kHz for all spectra. First-derivative spectra were
recorded at 1024point with an integration time of 150 millliseconds
per point. The microwave frequency was calibrated with a
frequency counter and the magnetic field was calibrated with a
calibrated Hall probe. The temperature was calibrated with a
carbon glass resistor (Lakeshore CGR-1-1000). EPR signals were
quantified by relative to a 1 mM Cu(II)EDTA standard in 10%
glycerol. The spectral simulations were performed by using the
SpinCount software.102
Protein Crystallization. Crystallization of Fe(II)-bound
complexes AsqJ•Fe•succinate•1-H and AsqJ•Fe•succinate•2-CF3
complexes was accomplished using the sitting-drop method as
reported in previous work and detailed in the Supporting
Information.54 Crystals of the peroxide-bound AsqJ•Fe•2OG•2-H
were prepared by soaking the AsqJ•Fe•acetate•1-H crystals with a
substitute mother liquor containing 25% PEG 1000, 100 mM
imidazole/HCl (pH 8.0),
5 mM 2OG and 1 mM of
dehydrocyclopeptin under aerobic condition for 24 h at 4°C.
Crystals of AsqJ•Fe•succinate•2-H (with the O-C10 bond
formation being observed) and AsqJ•Fe•2OG•3-H were obtained
by soaking the pre-grown crystals of AsqJ•Fe•acetate•1-H in a
substitute mother liquor containing 25% PEG 1000, 100 mM
imidazole/HCl (pH 8.0) and 5 mM 2OG supplemented with 0.5 and
1 mM of ascorbate, respectively, for 24 h at 4°C.
Stopped-Flow Absorption Spectroscopy.
Stopped-flow
absorption spectroscopy (SF-Abs) experiments were performed on
an Applied Photophysics SX20 stopped-flow spectrometer
operating in an MBraun Unilab glove box filled with nitrogen gas
and maintained at < 0.5ppm O2 level. Sample and reaction
chambers were kept at 5 oC for the entire duration of the
Crystal Structure Determination. The X-ray diffraction data of
the crystals were collected at the BL15A1 and TPS beamline 05A
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at NSRRC (National Synchrotron Radiation Research Center,
‡ These authors contributed equally.
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Taiwan). The data were indexed, integrated and scaled with the
HKL-2000 program suite,103 while phasing and model building
were accomplished with various components of the CCP4 suite,104
as detailed in the Supporting Information. The data collection and
refinement parameters are listed in Tables S3 and S4. Atomic
coordinates and structure factors have been deposited in the PDB
with accession codes 6K0E (AsqJFe2OG2-Hperoxide), 6JZM
(O-C10 bond formation), 6K0F (AsqJFe2OG3-Hperoxide), and
6KD9 and 6K3O (AsqJ•Fe•succinate•2-CF3 in space group P212121
and C2221, respectively).
Density Functional Theory Calculations. Models for DFT
calculations were built based on previously reported crystal
structure (PDB ID 5Y7R)54 with truncation to keep only residues
close to the active center. Various ligands are edited in and models
are subject to geometry optimization using the Gaussian 09
software, revision D01.105 The Mössbauer parameters of the
models were calculated using the ORCA software package, version
4.0.1.2.106 The calculated electron density was converted to isomer
shift using a set of calibration formula from Pápai et al.84 The
quadrupole splitting was calculated from the electric field gradient
tensor at the Fe nucleus. The method for quadrupole splitting was
also validated with a small set of model complexes, as detailed in
Table S5. Further details of models, geometry optimization, and
calculations were provided in the Supporting Information.
Molecular Dynamics Simulations. MD simulations were
carried out using the GROMACS software package.107 We
performed thermodynamic integration for the free energy changes
for the replacement of Fe-coordination water molecule by O2. The
calculation was accomplished using the MBAR (Multistate Bennett
Acceptance Ratio) method108 as implemented in gmx bar utility of
GROMACS, applying an alchemical transformations of the Fe
ligand O2 into H2O. 10 steps (11 λ points) have been used in the O2
to H2O transformation. Calculations have been carried out for 4
system configurations, namely with and without the bound 2-H
substrate for the online and offline orientations of 2OG in the
coordination complex. Each structure has been solvated and
equilibrated for 20 ns before production MD runs. The length of
production MDs was 5 ns for each of 11 intermediate (λ) states
(vide infra). Mixed topology files have been prepared where the
initial state is O2 coordination and the final state is H2O
coordination. Details of model building, force field parameters, and
restraints are provided in the Supporting Information.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by grants from the National Institutes of
Health (GM125924 to Y.G., W.-c. C, and M. K.), National Taiwan
University, and Ministry of Science and Technology grant 106-
2113-M-002-021-MY3, R.O.C., Taiwan. We thank Prof. Michael
P. Hendrich at CMU for the use of the FQ apparatus. The resource
for computational work is partly provided by Extreme Science and
Engineering Discovery Environment (XSEDE) and Pittsburgh
Supercomputing Center (allocation number TG-CHE180042).
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pp 139–154.
Aziridines and Epoxides in Organic Synthesis; Yudin, A. K., Ed.;
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS
Publications website.
Additional experimental and computational details and data,
including Schemes S1, Figures S1-S11, Tables S1 – S6 (PDF)
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Cartesian coordinates of all DFT structural models (Zip)
AUTHOR INFORMATION
Corresponding Author
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* ysguo@andrew.cmu.edu
* nlchan@ntu.edu.tw
* wchang6@ncsu.edu
* kurnikova@cmu.edu
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Author Contributions
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TOC Figure
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O
O
N
Me
HN
O
N
Me
O
OMe
HN
O
OMe
-OOC
O
-OOC
2OG
“offline”
O2
O
O
FeII
His134
His134
O
O
O
FeII
O
O
Asp136
Asp136
H2
H2
O
His211
His211
2OG
“online”
O
N
Me
HN
-OOC
OMe
CO2
O
O
His134
Asp136
O
O
N
FeII
Me
O
HN
-OOC
OMe
O
His211
O
His134
O
9
FeIV
O
O
Asp136
N
Me
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His211
HN
-OOC
OMe
O
O
His134
Asp136
O
Fe
His211
O
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