An Iron(IV)-Oxo Intermediate Initiating l -Arginine Oxidation but Not Ethylene Production by the 2-Oxoglutarate-Dependent Oxygenase, Ethylene-Forming Enzyme

Article abstract of DOI:10.1021/jacs.0c10923

Ethylene-forming enzyme (EFE) is an ambifunctional iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase. In its major (EF) reaction, it converts carbons 1, 2, and 5 of 2OG to CO2 and carbons 3 and 4 to ethylene, a four-electron oxidation drastically different from the simpler decarboxylation of 2OG to succinate mediated by all other Fe/2OG enzymes. EFE also catalyzes a minor reaction, in which the normal decarboxylation is coupled to oxidation of l-arginine (a required activator for the EF pathway), resulting in its conversion to l-glutamate semialdehyde and guanidine. Here we show that, consistent with precedent, the l-Arg-oxidation (RO) pathway proceeds via an iron(IV)-oxo (ferryl) intermediate. Use of 5,5-[2H2]-l-Arg slows decay of the ferryl complex by >16-fold, implying that RO is initiated by hydrogen-atom transfer (HAT) from C5. That this large substrate deuterium kinetic isotope effect has no impact on the EF:RO partition ratio implies that the same ferryl intermediate cannot be on the EF pathway; the pathways must diverge earlier. Consistent with this conclusion, the variant enzyme bearing the Asp191Glu ligand substitution accumulates μ4 times as much of the ferryl complex as the wild-type enzyme and exhibits a μ40-fold diminished EF:RO partition ratio. The selective detriment of this nearly conservative substitution to the EF pathway implies that it has unusually stringent stereoelectronic requirements. An active-site, like-charge guanidinium pair, which involves the l-Arg substrate/activator and is unique to EFE among four crystallographically characterized l-Arg-modifying Fe/2OG oxygenases, may serve to selectively stabilize the transition state leading to the unique EF branch.

Full text of DOI:10.1021/jacs.0c10923

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Article
An Iron(IV)−Oxo Intermediate Initiating L‑Arginine Oxidation but
Not Ethylene Production by the 2‑Oxoglutarate-Dependent
Oxygenase, Ethylene-Forming Enzyme
Rachelle A. Copeland, Katherine M. Davis, Tokufu Kent C. Shoda, Elizabeth J. Blaesi, Amie K. Boal,*
Carsten Krebs,* and J. Martin Bollinger, Jr.*
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ABSTRACT: Ethylene-forming enzyme (EFE) is an ambifunctional iron(II)- and
2-oxoglutarate-dependent (Fe/2OG) oxygenase. In its major (EF) reaction, it
converts carbons 1, 2, and 5 of 2OG to CO2 and carbons 3 and 4 to ethylene, a four-
electron oxidation drastically different from the simpler decarboxylation of 2OG to
succinate mediated by all other Fe/2OG enzymes. EFE also catalyzes a minor
reaction, in which the normal decarboxylation is coupled to oxidation of L-arginine
(a required activator for the EF pathway), resulting in its conversion to L-glutamate
semialdehyde and guanidine. Here we show that, consistent with precedent, the L-
Arg-oxidation (RO) pathway proceeds via an iron(IV)−oxo (ferryl) intermediate.
Use of 5,5-[2H2]-L-Arg slows decay of the ferryl complex by >16-fold, implying that
RO is initiated by hydrogen-atom transfer (HAT) from C5. That this large substrate
deuterium kinetic isotope effect has no impact on the EF:RO partition ratio implies
that the same ferryl intermediate cannot be on the EF pathway; the pathways must diverge earlier. Consistent with this conclusion,
the variant enzyme bearing the Asp191Glu ligand substitution accumulates ∼4 times as much of the ferryl complex as the wild-type
enzyme and exhibits a ∼40-fold diminished EF:RO partition ratio. The selective detriment of this nearly conservative substitution to
the EF pathway implies that it has unusually stringent stereoelectronic requirements. An active-site, like-charge guanidinium pair,
which involves the L-Arg substrate/activator and is unique to EFE among four crystallographically characterized L-Arg-modifying Fe/
2OG oxygenases, may serve to selectively stabilize the transition state leading to the unique EF branch.
and othersof which members of the versatile enzyme family
INTRODUCTION
■
are collectively capable.2,3,9,10 Most of these outcomes extract
Iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases
use a common cosubstrate (2OG) as the source of the two
electrons needed to balance the four-electron reduction of
two electrons from the prime substrate, thus resetting the
enzyme for another turnover.
Ethylene-forming enzyme (EFE) from pseudomonads and
fungi is a unique, ambifunctional Fe/2OG oxygenase that, in
its namesake, major reaction, diverges rather dramatically from
the chemical logic that characterizes the enzyme family.11,12 In
this reaction (Scheme 1, lower pathway), EFE confers all four
oxidizing equivalents of O2 to the cosubstrate, globally
fragmenting it to 3 equiv of CO2 (from carbons 1, 2, and 5)
and 1 equiv of ethylene (from carbons 3 and 4). The enzyme
also catalyzes a minor reaction (upper pathway) that does
conform to the family logic, in which oxidation of 2OG to
succinate is coupled to the oxidative fragmentation of L-
dioxygen with the two-electron oxidation of an Fe(II) cofactor
to an Fe(IV)−oxo (ferryl) intermediate.1−4 The cosubstrate
chelates the Fe(II) cofactor via its C1 carboxylate and C2
carbonyl oxygens, contributing to a square-pyramidal coordi-
nation sphere that is rounded out by (most often) three facially
disposed amino acid ligandstwo histidines and one
carboxylateprovided by the conserved β-sandwich protein
architecture that defines the enzyme family.5−7 Dioxygen adds
to the open axial site of this five-coordinate complex, and the
cosubstrate undergoes oxidative decarboxylation (C1−C2
bond cleavage) to produce succinate. In the process, one of
the two oxygen atoms from O2 is incorporated into the C2-
derived carboxylate, as the other oxygen remains bonded to
iron in the ferryl complex. The ferryl intermediate most often
abstracts a hydrogen (H•) from an aliphatic carbon of the
“prime” substrate (R−H) to form a fleeting Fe(III)−OH/R•
intermediate.8 Different fates of this intermediate lead to the
varied outcomeshydroxylation, halogenation, desaturation,
cyclization, ring expansion, stereoinversion, endoperoxidation,
Received: October 15, 2020
Published: February 1, 2021
https://dx.doi.org/10.1021/jacs.0c10923
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Scheme 1. Reactions Catalyzed by EFE
arginine to guanidine and L-glutamate semialdehyde (which
cyclizes in solution to pyrroline-5-carboxylate, P5C). Intrigu-
ingly, although it is transformed only in the minor reaction, L-
Arg is still strictly required for the major reaction.12,13 In
essence, the amino acid has distinct roles as prime substrate for
the ordinary, L-Arg-oxidizing (RO) reaction and essential
effector for the unique, ethylene-forming (EF) reaction.
To date, experimental investigations of EFE have been
limited to structural characterization of its substrate-bound
states, examination of the kinetics and ratios of products
(ethylene, pyrroline-5-carboxylate, and succinate) in multiple-
turnover assays of wild-type (wt) and variant enzymes, and
evaluation of substrate analogues for the ability to support one
or both reactions.12−18 By all accounts, wt EFE exhibits an
EF:RO partition ratio of ∼2−3:1 when turning over its native
substrates. Substitutions to active-site-proximal residues have,
in general, proven deleterious to both reactions, but a number
of those examined compromise the EF pathway more
profoundly than the RO pathway. By contrast, none has
been shown to increase the EF:RO partition ratio relative to
that of the wt enzyme. Experiments with L-Arg analogues have
suggested a remarkable specificity for the allosteric activator/
prime substrate. All analogues that have been tested are,
according to these reports, poorer activators of the EF
pathway.13,16 From the limited available mechanistic data, it
has not yet been possible to answer the central mechanistic
question posed by the unusual EF outcome: at which point,
and by what novel step, does this pathway diverge from the
more canonical RO pathway?
The conformity of the RO pathway to the characteristic Fe/
2OG-oxygenase logic suggests a mechanism involving ferryl-
mediated abstraction of H• from C5 of L-Arg to initiate either
(i) hydroxylation followed by C5−N6 fragmentation12,13 or
(ii) C5−N6 desaturation followed by hydrolysis.19 Two recent
X-ray crystal structures confirmed the required proximity of C5
to the metal center [N-oxalylglycine-coordinated Fe(II) or
2OG-coordinated Mn(II)], but no direct evidence for the
targeting of C5 by a ferryl complex has been reported.16,17 It is
less obvious that the major EF pathway should proceed
through a ferryl intermediate; mechanisms with and without
such a complex can be proposed. Indeed, two of the
aforementioned experimental studies posited different ferryl
complexesin one case, a succinate-coordinated ferryl
initiating ethylene production16 and, in the second case, an
oxalate-coordinated ferryl forming concomitantly with ethyl-
ene17whereas a recent computational study proposed an EF
pathway not proceeding through a ferryl intermediate.20
In the reactions of other Fe/2OG oxygenases, ferryl
intermediates have been demonstrated on the basis of three
hallmarks: (i) their increased UV absorption (typically
monitored at ∼320 nm) relative to the enzyme•Fe(II)
•2OG•substrate and enzyme•Fe(II)•succinate•product com-
̈
plexes; (ii) their characteristic Mossbauer isomer shifts (δ ∼
0.3 mm/s) and quadrupole splitting (ΔEQ ∼ −1 mm/s)
parameters;21,22 and (iii) the large substrate deuterium kinetic
isotope effects (D-KIEs) on their decay by H•/D• transfer,
which results in increased intensity and longevity of these
spectral signatures.3,8,23 The weak absorption feature of the
reactant complexes at 500−530 nm (ε ∼ 200 M−1 cm−1)
arising from a metal-to-ligand [Fe(II)-to-2OG] charge-transfer
(MLCT) transition5has also proven useful in transient-
kinetic monitoring of the reactions. The ferryl complexes are
approximately isosbestic in this region, but the enzyme•Fe(II)
•succinate•product complexes (referred to in the original
work on TauD as a “second intermediate”21) are transparent,
with the result that this optical feature provides an additional
way to monitor either conversion of the reactant complex to
the product complex in cases of a nonaccumulating ferryl
intermediate or decay of the ferryl intermediate to the
product(s) complex when the high-valent species accumulates
substantially.24,25
In this study, we used stopped-flow absorption (SF-Abs) and
̈
̈
freeze-quench Mossbauer (FQ-Moss) spectroscopies along
with substrate deuterium labeling, quantitative product
analysis, and site-directed mutagenesis to test for a ferryl
complex in either or both pathways of the EFE reaction, and
we solved the structure of the authentic EFE•Fe(II)•2OG•L-
Arg reactant complex. The kinetic and spectroscopic results
not only confirm the expectation that the RO reaction
proceeds via the canonical ferryl intermediate but also reveal
that the detected complex forms after the branchpoint of the
two pathways, meaning that it is not on the EF pathway. The
ability of a nearly conservative Asp → Glu ligand substitution
to almost completely derail the EF pathway while barely
affecting the RO pathway is consistent with unusually stringent
stereoelectronic requirements for the major reaction, and
structural features of the EFE reactant complex that distinguish
it from those of several other L-Arg-modifying Fe/2OG
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oxygenases characterized previouslymost notably, the
flipped orientation of L-Arg16 and resulting like-charge
guanidinium pair involving its side-chain and an active-site
Arg residuesuggest how the transition state leading to the
unique outcome might be selectively stabilized.
RESULTS
■
Activity and Partition Ratio of Our Preparations of
EFE. To verify the integrity of our preparations of Pseudomonas
savastanoi pv phaseolicola EFE, we quantified the products of
multiple-turnover reactions by liquid chromatography with
mass spectrometric detection (LC-MS) and gas chromatog-
raphy with flame-ionization detection (GC-FID). We detected
pyrroline-5-carboxylate (P5C), a product of the RO pathway,
by first reducing it to proline with sodium cyanoborohydride
and then appending an Fmoc protecting group prior to LC-MS
analysis. Our results corroborate previous reports12,13 that,
with unlabeled L-Arg as substrate, ∼70% of the 2OG consumed
by wild-type EFE yields ethylene, while the remaining ∼30% is
converted to succinate (Figure 1). The rates ([product] time−1
[EFE]−1) of succinate and ethylene production that we
measured (∼15 and ∼40 min−1, respectively) are also
consistent with published reports.13,14,26
Figure 2. Kinetic analysis of the wt and D191E EFE reactions with
protium- and deuterium-bearing L-Arg substrates by stopped-flow
absorption spectroscopy. The change in absorbance (A) at 318 nm
and (B) at 510 nm was monitored after mixing (at 5 °C) of an anoxic
solution containing 0.7 mM wt EFE (black) or D191E EFE (red), 0.6
mM Fe(II), 8 mM 2OG, and 8 mM L-Arg (filled circles) or d7-L-Arg
(open circles) in 40 mM sodium HEPES (pH 7.5) buffer with an
equal volume of the same buffer containing ∼0.48 mM O2. The solid
lines are the results of global fitting of the data to a kinetic model in
which two rapidly equilibrating reactant complexes react with O2, as
described in the Supporting Information and shown in Scheme S1.
Use of deuterated L-Arg also resulted in a modest decrease in
the downward deflection of the ΔA510-vs-time trace (Figure
2B, open black circles), but importantly, the temporal delay
and diminution of the amplitude of this trace were not as
profound as one would have expected in view of the markedly
greater accumulation of the presumptive ferryl complex, which,
as noted, absorbs significantly in the visible regime in other Fe/
2OG enzymes. This observation is explained by the facts that
(i) only approximately one-third of the reaction flux (the RO
pathway) is sensitive to the presence of deuterium in L-Arg,
and (ii) the other two-thirds (the EF pathway) converts the
reactant complex to a transparent product complex without
substantial accumulation of an intervening, 510 nm-absorbing
state (as explained more below). These observations are
consistent with a modest but detectable accumulation of the
ferryl intermediate with the protium-containing substrate and
enhanced accumulation with d7-L-Arg as a result of a large,
normal D-KIE on HAT to the high-valent complex.
To confirm the expectation that the H• donor to the
presumptive ferryl complex is C5, we also examined the
reaction with site-specifically C5-deuterated L-Arg (5,5-[2H2]-
L-Arg or d2-L-Arg). As anticipated, the increased accumulation
and slower decay seen with the d7-L-Arg substrate were also
observed with the specifically labeled substrate (Figure S3).
The fact that the kinetics of decay with d2-L-Arg and d7-L-Arg
were essentially identical implies that the ferryl complex cannot
abstract H• from a different site with an appreciable rate
constant, a conclusion supported by our inability to detect any
other L-Arg-derived product in the reaction with d2-L-Arg.
Figure 1. Yields of succinate, Fmoc-proline (P5C), and ethylene after
incubation of 10 μM wt or D191E EFE with 40 μM (NH4)2Fe(SO4)2,
200 μM 2OG, and 500 μM of unlabeled L-arginine (L-Arg),
perdeuterated L-arginine (d7-L-Arg), or 5,5-d2-L-arginine (d2-L-Arg)
in air-saturated 40 mM sodium HEPES (pH 7.5) buffer at 21 °C for
∼10 min. 2OG was completely consumed during the reaction. P5C
was derivatized to Fmoc-proline prior to quantification. Ethylene
analysis was performed by GC-FID; succinate and Fmoc-proline were
quantified by LC-MS. Error bars correspond to standard errors of the
mean values for two experiments. Detailed procedures are provided in
the Supporting Information.
Evidence for an L-Arg-Oxidizing Ferryl Intermediate
in the EFE Reaction. To test for accumulation of a ferryl
intermediate in the EFE reaction, we mixed the anoxic
EFE•Fe(II)•2OG•L-Arg complex with O2-containing buffer at
5 °C in a stopped-flow apparatus. Weak UV absorption
developed within ∼0.07 s and decayed by more than 90%
within 1 s (Figure 2A, solid black circles). The loss of
absorbance at 510 nm in this same time regime (Figure 2B,
solid black circles) signified conversion of the reactant state to
the transparent Fe(II)-containing product state(s) via a barely
accumulating (presumptive) ferryl complex. Use of L-Arg
bearing deuterium at C5 (here, 2,3,3,4,4,5,5-[2H7]-L-Arg or d7-
L-Arg) resulted in 4−5-fold increases in both the upward
deflection in the ΔA318-vs-time trace and the time for its return
back to the initial absorbance (Figure 2A, open black circles).
̈
We next confirmed by freeze-quench Mossbauer spectros-
copy that the UV-absorbing intermediate is, in fact, a ferryl
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complex. The spectrum of the frozen EFE•57Fe(II)•2OG•d7-
L-Arg reactant solution acquired at 4.2 K without an applied
magnetic field (Figure 3A, i, solid lines) consists of a
quadrupole doublet with isomer shift (δ) and quadrupole
splitting (ΔEQ) parameters characteristic of N/O-coordinated,
high-spin Fe(II) ions (δ ∼ 1.1 mm/s and ΔEQ ∼ 2.6 mm/s).27
This reactant solution was mixed at 5 °C with an equal volume
of buffer containing ∼1.8 mM O2, and the resulting solution
was frozen after 0.34 s, the reaction time at which the UV-
absorbing complex was seen to accumulate maximally (Figure
S4). The 4.2 K/zero-field spectrum of this sample (Figure 3A,
ii, solid lines) exhibits a broadened doublet for the Fe(II)
species (indicated by black arrows) and a new peak at ∼0.8
mm/s (indicated by the red arrow). The latter feature is at the
position typical for the high-energy line associated with the
high-spin ferryl complexes detected in other Fe/2OG
enzymes.8 Both sets of features are less sharp in the spectra
acquired at the same temperature (4.2 K) with a 53 mT
magnetic field oriented parallel to the γ-beam (Figure 3A, i and
ii, vertical bars), as has also been seen in studies of other Fe/
2OG enzymes. Subtraction of the zero-field spectrum of the
frozen reactant solution from the zero-field spectrum of the
0.34-s reaction sample resulted in a difference spectrum that
can be simulated as a superposition of three quadrupole
doublets (Figure 3A, iii, vertical bars and black solid line). Of
the two downward-pointing quadrupole doublets (which arise
from the species that form in the 0.34 s incubation), ohas
parameters typical of a high-spin ferryl complex (δ = 0.26 mm/
s, ΔEQ = 0.96 mm/s, Γ = 0.25 mm/s, red line) and contributes
̈
11% of the total intensity. The 4.2 K/53 mT Mossbauer
spectrum of a sample reacted with O2 for 5 sby which time
the 318 nm-absorbing intermediate was observed to decay in
SF-Abs experimentslacks the line at 0.8 mm/s, demonstrat-
ing decay of the ferryl complex and the transient UV
absorption on the same time scale (Figure 3A, iv). Together,
̈
the SF-Abs and FQ-Moss results establish the accumulation of
a ferryl intermediate that can initiate L-Arg oxidation by
abstracting H• from C5.
Evidence That the Ferryl Intermediate Is Specific to
the RO Pathway. The modest (11%) accumulation of the
ferryl intermediate arises from two factors. First, despite the
retarding effect of the large D-KIE, the decay of the
intermediate in the d7-L-Arg reaction is still not fully kinetically
resolved from its formation, and this competition suppresses
accumulation. Second, intermediacy of the ferryl complex in
only the minor, RO pathway diminishes the quantity that can
accumulate to only ∼0.3 times (the fraction proceeding down
the RO branch) the limit imposed by the former effect. This
more mechanistically informative origin of ferryl suppression is
established by the quantitative, global simulations of the
stopped-flow data (Figure 2, Table 1, and the Supporting
Information), but it can also be understood qualitatively by
comparing the deflections in the ΔA510 and ΔA318-vs-time
traces from the reactions with the unlabeled and d7-L-Arg
substrates (Figure 2). From anoxic titrations of the enzyme
with Fe(II) in the presence of the other substrates (Figure S1),
Figure 3. Analysis of the reactions of (A) wt and (B) D191 EFE by
̈
freeze-quench Mossbauer spectroscopy. Experimental spectra (i, ii,
and iv) were collected at 4.2 K with either no applied magnetic field
(solid lines) or a field of 53 mT applied parallel to the γ-beam
(vertical bars). In preparation of the freeze-quenched samples (spectra
ii and iv), an anoxic solution containing 1.8 mM EFE (wt or D191E),
1.4 mM 57Fe(II), 5 mM 2OG, and 5 mM d7-L-Arg in 40 mM sodium
HEPES buffer (pH 7.5) containing 5% glycerol was mixed at 5 °C
with an equal volume of O2-saturated (∼1.8 mM O2) buffer, and the
reaction was allowed to proceed for the indicated time before being
terminated by rapid freezing in the cryosolvent (2-methylbutane at
∼−150 °C). For samples of the reactant complexes (spectra i), the
same anoxic protein reactant solution was manually frozen (wt, panel
A) or diluted with an equal volume of O2-free buffer before being
manually frozen (D191E, panel B). Spectra iii (vertical bars) result
from subtraction of the experimental zero-field spectra (ii − i) to
illustrate the changes that occur at early time. These difference spectra
can be accounted for as summations (black lines) of either three (wt,
panel A) or two (D191E, panel B) quadrupole-doublet components
(colored lines) corresponding to features that form (downward) and
decay (upward) over short reaction times. The colored theoretical
spectra are associated with the ferryl intermediate (red), the Fe(II)-
containing reactant complex (blue), and the Fe(II)-containing
product complex of the EF pathway (green in panel A only). The
parameters used to generate these theoretical spectra are given in the
text and Table S8.
we determined a molar absorption coefficient at 510 nm (ε510
of 110
)
10 M−1 cm−1 for the EFE•Fe(II)•2OG•L-Arg
complex. This value is somewhat greater than that reported by
Martinez and Hausinger (∼80 M−1 cm−1),26 but problems
with determination of enzyme concentration13,26 in that study
allay our concern over the discrepancy. With the protium-
bearing substrate, the ferryl complex accumulates minimally,
and the ΔA510-vs-time trace (Figure 2B, solid black circles)
reflects (primarily) a cycle of net conversion of the absorbing
reactant complex to the transparent product complex(es)
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competition between decay and regeneration of the absorbing
reactant complex. At the approximately equal concentrations of
EFE•Fe(II)•2OG•L-Arg and O2, the comparable rates of the
two steps, combined with the second-order nature of the first
step, suppress accumulation of the product complexes (i.e., net
loss of the reactant) to ∼40% the concentration of the limiting
reactant. The ferryl complexes that have been characterized in
other Fe/2OG enzymes all have molar absorption coefficients
(ε318) of ∼1500 M−1 cm−1, and our global kinetic model (see
the Supporting Information) is consistent with an equivalent
ε318 for the EFE ferryl complex. The upward deflection of the
ΔA318 vs time trace from the d7-L-Arg reaction of 0.05 at 0.55 s
thus implies accumulation of a maximum of only 0.033 mM of
the ferryl intermediate (0.11 equiv), consistent with the
Table 1. Rate Constants for Formation and Decay of the RO
Ferryl Complex Obtained by Fitting the ΔA318 vs Time Data
Shown in Figure 2 to an A + B → C → D Model Using
a
KinTek Explorer
substrate
kformation, net (103 M−1 s−1
)
kdecay (s−1
)
kH/kD
wt
wt
D191E
D191E
L-Arg
54
54
31
31
16
1
42
1.6
d7-L-Arg
L-Arg
16
26
d7-L-Arg
a
Initial reactant and O2 concentrations were reduced by a factor that
reflects flux through the RO pathway (i.e., divided by 3.5 for wt and
1.05 for D191E). ε318 and ε510 values for the reactant complexes were
∼40 and 90 M−1 cm−1, respectively, and ε318 and ε510 for the ferryl
complexes were ∼1450 and 190 M−1 cm−1, respectively.
̈
quantity detected by freeze-quench Mossbauer spectroscopy.
The time scales for decay of the ferryl complex in the d7-L-Arg
reaction and regeneration of the reactant complex in the
reaction with unlabeled substrate differ by no more than a
factor of 1.5, and so a kinetic origin of the ∼3-fold shortfall in
ferryl accumulation relative to reactant decay can be ruled out.
followed by regeneration of the reactant. The downward
deflection of 0.011 in this trace implies a net loss of 0.10 mM
of the reactant complex (0.3 iron equiv) at ∼0.85 s, the
minimum of the trace. This shortfall from the maximum
imposed by the [O2] of 0.24 mM is accounted for by kinetic
Scheme 2. Potential Mechanisms of Ethylene Formation by EFE
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The explanation is that the ferryl intermediate accumulates
only in the minor, RO pathway.
least 16 on decay of the intermediate with either the d7- or the
d2-L-Arg substrate. This D-KIE is simply too large for there to
be more than a few percent of flux (ferryl decay) through
another step that competes with HAT from C5 to the
intermediate complex (at least for the case of the protium-
bearing L-Arg substrate). The failure of these predictions of
Scheme 2 rules out this latest possible EF/RO branchpoint,
with the ferryl complex as a common intermediate.
̈
The Mossbauer spectra from the freeze-quench experiment
provide additional evidence for this conclusion. In addition to
the downward features for the ferryl complex that develops
(11% of total intensity), the difference spectrum showing the
changes that occur in the first 0.34 s of the reaction (Figure 3A,
iii, vertical bars) also exhibits upward features associated with
loss of the Fe(II)-containing reactant complex (δ = 1.10 mm/s,
ΔEQ = 2.47 mm/s, line width Γ = 0.24 mm/s, blue line)
corresponding to 39% of the total intensity. The quantitative
mismatch in these contributions is accounted for by the
signature of a new Fe(II) species (δ = 1.16 mm/s, ΔEQ = 3.10
mm/s, Γ = 0.54 mm/s, 32% of total intensity, green line) that
develops along with the ferryl complex. The combined analysis
Demonstration of a Selectively Compromised EF
Pathway and Enhanced Ferryl Accumulation in the
D191E Ligand Variant. The conclusion that the detected
ferryl complex is specific to the RO pathway raised the
question of whether perturbations impacting the EF:RO
partition ratio (e.g., amino acid substitutions or substrate
modifications) might also impact the quantity of ferryl complex
to accumulate. We were encouraged to address this question
by previous studies, in which single substitutions near the
cofactor were reported to alter the partition ratio, compromis-
ing ethylene production to a greater extent than L-Arg
oxidation.17 However, none of these variant proteins have
(to the best of our knowledge) been examined in the transient
state for detriments to O2 activation leading to intermediate
formation, which, in past work, has generally been seen for
such active-site-proximal substitutions.28,29 Seeking a minimal
structural perturbation to alter the partition ratio without
causing such unwanted, global kinetic effects, we substituted
the facial-triad ligand, Asp191, with Glu. LC-MS/GC-FID
assays showed that the D191E variant degrades L-Arg and 2OG
to P5C (again detected after reduction to proline and Fmoc
derivatization) and succinate nearly stoichiometrically, with
only ∼5% of the cosubstrate being fragmented to ethylene
(Figure 1). As with the wt enzyme, mixing of the anoxic EFE-
D191E•Fe(II)•2OG•L-Arg complex with O2 at 5 °C resulted
in development of transient absorption at 318 nm in tens of
milliseconds and subsequent decay over seconds (Figure 2A,
solid red circles), and use of d7-L-Arg in place of the protium-
bearing substrate increased the amplitude and decay time by a
factor of ∼4 (open red circles). Importantly, the deflections in
the ΔA318-vs-time traces were, for both substrates, ∼3.5 times
as large as for wt EFE under the same reaction conditions,
suggesting that the variant protein accumulates more of the
ferryl complex.
̈
of the SF-Abs and FQ-Mossbauer data reveals that only
approximately one-third of the reactant complex that decays in
this short reaction time yields the ferryl intermediate, while
approximately two-thirds yields a new Fe(II)-containing
species, which is presumably the product complex of the EF
pathway.
Implications of the L-Arg C5 D-KIE on Ferryl Decay
and Absence of Its Effect on the EF:RO Partition Ratio.
Further analysis lends even greater weight to the conclusion
that the detected ferryl complex is specific to the RO pathway
and not a branchpoint between the pathways. In a scenario
involving partitioning of a common ferryl intermediate
between HAT from C5 of L-Arg and succinate fragmentation
(e.g., by proton-coupled electron transfer or PCET) to
ethylene and 2 (additional) equiv of CO2 (Scheme 2, orange
vs purple or blue dotted arrows), a large D-KIE on the C5-
HAT step should impact the partition ratio, making the EF
pathway even more dominant. We verified that, to the
contrary, the presence of deuterium does not measurably
impact the EF:RO partition ratio (Figure 1). The observed
partition ratio (FEF/FRO, where F is “fraction”) would reflect
the relative magnitudes of the rate constants for the two
competing steps, kHAT and kPCET, and the observed rate
constant for decay of the ferryl complex (kobs) would be the
sum of the rate constants for the two steps (eqs 1−4).
FEF = kPCET/(kPCET + kHAT
)
(1)
(2)
(3)
(4)
F
RO = kHAT/(kPCET + kHAT
)
̈
We confirmed this implication by freeze-quench Mossbauer
spectroscopy. Figure 3B shows the difference spectrum (iii,
vertical bars) obtained by subtracting the 4.2 K/zero-field
spectrum of the frozen D191E-EFE•57Fe(II)•2OG•d7-L-Arg
reactant complex (i, solid line) from the corresponding
spectrum of a sample frozen after this complex was exposed
to O2 for 0.14 s (ii, solid line). A developing quadrupole
doublet from the high-spin ferryl complex appears as
downward features contributing 43% of the total intensity
(red line). The upward features arising from the decaying
reactant complex have essentially the same relative intensity
FEF/FRO = kPCET/kHAT
kobs = kHAT + kPCET
In this case, with succinate oxidation being faster than L-
arginine oxidation by a factor of ∼2.5 (kPCET = 2.5kHAT), the
intrinsic D-KIE on the HAT step (kHAT/kDAT) would be only
minimally “expressed” in the observed D-KIE on decay of the
ferryl complex (kobs,H/kobs,D) as a consequence of the
insensitivity of the other step (PCET) to deuterium
substitution in L-Arg. Thus, even an extremely large intrinsic
D-KIE on the HAT step giving a very small value of kDAT
would yield an observed effect of only ≤1.4 (eqs 5 and 6).
̈
(40%, blue line). Interestingly, the Mossbauer parameters of
the ferryl complexes in the variant (δ = 0.32 mm/s, ΔEQ = 0.88
mm/s, Γ = 0.27 mm/s) and wt (see above) enzymes are not
identical, and those of their reactant complexes differ to an
even greater extent (Table S8). As in the reaction of the wt
enzyme, the ferryl complex in the D191E variant was seen to
decay within 5 s, as indicated by the absence of a feature at
∼0.8 mm/s in the 4.2 K/53 mT spectrum of the sample frozen
at this reaction time (Figure 3B, iv).
k
obs,H/kobs,D = (kHAT + kPCET)/(kDAT + kPCET
)
(5)
kobs,H/kobs,D = (kHAT + 2.5kHAT)/2.5kHAT = 1.4
(6)
By contrast, the global simulations summarized in Table 1 and
the Supporting Information imply an observed D-KIE of at
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Figure 4. X-ray crystallographic models of the EFE active site with 2OG and L-Arg bound. (A) Comparison of the 1.83-Å-resolution wild-type
EFE•Fe•2OG•L-Arg structure (PDB accession code 6VP4) to the published 1.43-Å-resolution EFE•Mn•2OG•L-Arg structure (PDB accession
code 5V2Y) reveals no significant differences. (B) One of two conformations of the carboxylate side chain observed in the 1.97-Å-resolution D191E
EFE•Fe•2OG•L-Arg structure (conformation A) closely resembles that observed in the wild-type enzyme. In (C) and (D), 2Fo−Fc maps
contoured to 1.0σ are shown in black for wild-type and D191E EFE, respectively. Conformation A of the E191 side chain forms a single hydrogen
bond with the L-Arg substrate (E), whereas conformation B forms two hydrogen bonds (F).
̈
The Mossbauer spectra of Figure 3 confirm the implication
complex is associated with accumulation of both the ferryl
intermediate (11%) and a new Fe(II)-containing “product”
(32%). With the reaction of the D191E variant proceeding
almost homogeneously through the RO pathway, the ferryl-
stabilizing effect of deuterium at C5 of L-Arg results in a simple
net conversion of the reactant complex to the intermediate
without significant “loss” to the nonabsorbing Fe(II) product
statesover short reaction times. This simpler conversion has
a noteworthy manifestation also in the ΔA520 vs time traces
an initial rise phase as a result of the fact that the ferryl
complex, which in other Fe/2OG enzymes is nearly isosbestic
with its precursor, here has a greater molar absorptivity than
the (unusually hypochromic) EFE•Fe(II)•2OG•L-Arg reac-
tant complex. This fact would not have been obvious solely
from the data on wild-type EFE, because the net conversion of
the reactant complex to the ferryl complex is, in that case,
of the stopped-flow absorption data that 3−4 times as much of
the ferryl complex accumulates in the reaction of the D191E
variant as in the reaction of wt EFE. This difference does not
have a kinetic origin in formation and decay rates, which are
similar for the two reactions. Rather, it is explained by the
almost complete elimination of flux through the EF pathway
and the consequent ∼3.5-fold increase in flux through the RO
pathway. This explanation is further supported by the
̈
observations that, in the Mossbauer difference spectrum
associated with the reaction of the D191E variant protein,
the contribution from the developing ferryl complex
quantitatively matches that from the decaying reactant
complex (∼40%), and no new features for an Fe(II)-
containing product complex are seen. This behavior contrasts
with that of the wt EFE reaction, in which loss of the reactant
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Figure 5. Comparison of the active sites of EFE and three other L-Arg-modifying Fe/2OG oxygenases, NapI (PDB: 6DAW), OrfP (PDB: 4M27),
and VioC (PDB: 6ALM), showing a distinct L-Arg binding mode in EFE that features (i) a stacked guanidinium pair involving the substrate and
Arg171 and (ii) projection of C3 and C4 over His189 to shield them from HAT.
accompanied by ∼2.5 times as much flux directly to a
transparent Fe(II)-containing product complex. These obser-
vations on the variant protein corroborate that the detected
ferryl complex is specific to the RO pathway.
heme-iron enzymes. In many of these systems, the ferryl
complexes can be redirected to neighboring sites (leading to
different outcomes) when challenged by substrates bearing
deuterium only at the preferred H•-donor sites.19,30−34 The
different behavior of EFE can be explained, at least in part, by
the fact that the binding orientation of the amino acid is
essentially opposite (rotated by ∼180°) to that in these other
enzymes. Zhang et al. previously noted this flipped L-Arg
orientation in the EFE active site relative to its orientation in
the L-Arg 3-hydroxylase, VioC, from the viomycin biosynthetic
pathway,35,36 which processes L-Arg itself and clavaminate
synthase, CAS, which acts on a β-lactam derivative of L-
Arg.16,37,38 Figure 5 illustrates the intriguing fact that the
VioC/CAS-like orientation of the amino acid is, in fact,
conserved in two additional, structurally characterized L-Arg
modifying Fe/2OG enzymes: the 3,4-dihydroxylase, OrfP,
from the streptolidine biosynthetic pathway and the 4,5-
desaturase, NapI, from the naphthyridinomycin path-
way.19,39,40 Indeed, the flipped binding mode appears, at
present, to be unique to EFE. The comparison with NapI is
particularly germane because the 4,5-desaturase also targets C5
to initiate its reaction but can, unlike EFE, be redirected to C4
and perhaps even C3 by the presence of deuterium at the
preferred HAT site(s).19 In the NapI binding mode, the
guanidinium functional group of the side chain is poised over
His146 of the His2Asp “facial triad”, and the propyl tether
(C5−C3) to the α-carbon (C2) projects away. Consequently,
the propyl moiety projects over the cofactor in space that is not
occupied by protein ligands and is thus potentially available for
the H•-abstracting ferryl oxo ligand to take up residence.
Although it has been posited that, in general, the oxo ligand
can be located trans to either the more C-terminal His ligand
(the so-called inline position) or the more N-terminal His
ligand (offline),2 the vector of the C5−C3 chain potentially
exposes each of these carbons for HAT to a ferryl complex in
either configuration. By contrast, the EFE binding mode
centers C5 above the cofactor but projects C4−C2 toward the
His2Asp facial triad, making these carbons inaccessible for
hydrogen abstraction. Its unique L-Arg binding mode nicely
rationalizes the unusually strict regiochemistry of HAT to the
ferryl complex in EFE. Might it also hold clues to the
mechanism of the novel EF outcome?
Structural Basis for Effect of the D191E Substitution
on the EF:RO Partition Ratio. To address why the EF:RO
partition ratio is so markedly perturbed by the nearly
conservative ligand substitution, we solved crystal structures
of the wt and D191E variant proteins in complex with Fe(II),
2OG, and L-Arg. Crystals were grown under anoxic conditions
to prevent turnover, and structures were solved to resolutions
of 1.83 Å (wt) and 1.97 Å (D191E). Our structure (the first)
of the authentic reactant complex of wt EFE closely matches
both the A conformation in the structure of the EFE•Fe(II)
•N-oxalylglycine•L-Arg complex reported by Zhang et al.
(PDB accession code 5LUN, rmsd ∼ 0.31 Å over 329 Cα
atoms) and the structure of the EFE•Mn(II•2OG•L-Arg
complex presented by Martinez et al. (PDB accession code
5V2Y, rmsd ∼ 0.40 Å over 329 Cα atoms, Figure 4A).16,17
Comparison of our structural models of the wt and D191E
EFE•Fe(II)•2OG•L-Arg complexes reveals no global differ-
ences between the two proteins (root-mean-square deviation
of ≤0.12 Å over 328 Cα atoms). The longer tether of the
ligand in the D191E variant causes the only significant
deviation in the active site. For all monomers in the
asymmetric unit, the new glutamate is modeled best in two
different, approximately equally prevalent side chain con-
formations (Figure 4D), of which one is analogous to that of
the Asp ligand in the wild-type protein (Figure 4B). In the
other conformation, the carboxylate remains coordinated to
the cofactor and hydrogen bonded with the L-Arg substrate,
but the projection of both carboxylate oxygen atoms toward
the substrate enables the engagement in an additional
hydrogen bond with the guanidino group (Figure 4F). The
ligand substitution does not, however, detectably perturb the
position of either substrate. Moreover, neither of our data sets
suggests the presence of the alternative L-Arg binding mode
that Zhang et al. posited to enable the EF pathway.16
DISCUSSION
■
Regioselectivity of HAT by EFE in Comparison to
Other L-Arg-Oxidizing Fe/2OG Oxygenases. The inability
of deuterium-bearing isotopologues to generate alternative
products of L-Arg oxidation and the identical rates of ferryl
decay observed with perdeuterated L-Arg and 5,5-d2-L-Arg
demonstrate site specificity in the HAT step by EFE that
contrasts with the behavior of several other ferryl-utilizing non-
Implications for the Mechanism of Ethylene For-
mation. The failure of the large D-KIE on HAT from L-Arg to
alter the EF:RO partition ratio implies that the EFE•Fe(IV)-
oxo•succinate•L-Arg complex that initiates RO cannot be the
branchpoint of the two pathways, but it does not rule out the
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possibility that a different, fleeting (nonaccumulating)
succinate-ligated ferryl complexpossibly in an “offline”
configuration and originating from earlier pathway bifurca-
tioninitiates ethylene production. Indeed, Zhang et al.
proposed a ferryl-mediated Grob-like fragmentation of
succinate involving migration of electron density over seven
atomic centers (this type of concerted fragmentation more
typically involves only five centers), in which the Fe(IV) center
would function, uncharacteristically, as a nucleofuge by
accepting two electrons from the coordinated succinate
carboxylate (Scheme 2).16 An alternative means by which a
distinct, undetected ferryl complex could initiate ethylene
production would involve single-electron transfer (most likely
coupled to proton transfer) to the Fe(IV) center from the
succinate carboxylate (purple dotted arrow in Scheme 2)
followed by radical β-scission of the C1−C2 bond, a
mechanism akin to those of the electrolytic Kolbe reaction
and the oxidation of 4-hydroxyphenylacetate initiated by a thiyl
radical in hydroxyphenylacetate decarboxylase (HPAD).41
We consider it more likely that an unusual fragmentation of
an intermediate that would normally be a precursor to the
succinate-coordinated ferryl complex, such as the unstable
Fe(IV)-peroxyhemiketal complex or Fe(II) persuccinate
intermediate, preempts its formation in the EF pathway
(Scheme 2). In a mechanism envisaged by Martinez et al.,13,17
the unstable Fe(IV)-peroxyhemiketal complex would fragment
between C2 and C3 rather than between C1 and C2, thus
generating an oxalate-coordinated rather than a succinate-
coordinated ferryl complex. Oxidative fragmentation of the
oxalate to the C1- and C2-derived CO2 molecules would
proceed by inner-sphere ET to the ferryl complex, decarbox-
ylation of the oxalyl radical, and reduction of the Fe(III) center
by the formate radical, similar to the mechanism of the
manganese-dependent oxalate oxidase.42,43 In principle, the
unique C2−C3 fragmentation could occur by a polar or radical
mechanism (Scheme 2), but the heterolytic pathway would
require the strained bicyclic complexwhich is already
thought to be unstable44to adopt a conformation with
appropriate orbital alignment across the seven atomic centers
involved, and the homolytic pathway would generate an Fe(V)
complex, unprecedented for the Fe/2OG-oxygenase class.
Interestingly, in proposing the Fe(IV)-peroxyhemiketal com-
plex as the likely branchpoint and thereby implying that
pathway bifurcation precedes ferryl formation, Martinez et al.
also posited that encroachment of the Phe283 side chain
toward the open coordination site of the cofactor upon L-Arg
binding could promote ethylene production by impeding
rearrangement of an “offline” ferryl complex to an inline
configuration required for L-Arg hydroxylation. According to
this hypothesis, displacement of the active-site Phe caused by
the increased length/bulk of the Glu ligand in the D191E EFE
variant might, in principle, cause the change in partition ratio.
However, the position of the Phe283 side chain is essentially
unchanged by the ligand substitution (Figure S6). Given that
we do not discern an obvious structural rationale for the
marked shift in the EF:RO partition ratio, it is possible that the
basis is more dynamic than structural or, alternatively, arises
from structural changes too subtle to detect.
(Scheme 2). One possible mechanism of ethylene formation
from the Fe(II) persuccinate intermediatea seven-center
Grob-like fragmentation, wherein the nucleofugal oxygen is
bound to the iron centerwould seem likely to require
concomitant protonation of this oxygen to avoid formation of
an extremely basic Fe(II)−oxo complex. An extensive
hydrogen-bonding network, which includes the cosubstrate
along with Arg171, Glu84, and the L-Arg substrate, could
deliver the requisite proton(s). Alternatively, as has been
explored extensively in studies of heme enzymes and model
complexes,46 homolysis of the iron-coordinated peroxide could
compete with the canonical heterolysis, generating the
Fe(III)−(hydr)oxo/succin-1-yl state that would break down
as previously delineated. Given that neither of these two
alternative fragmentations of the Fe(II) peroxysuccinate
complex is known to occur in any other Fe/2OG enzyme,
unique electrostatic features within the EFE active site would
seem to be required to counteract the favored polarity of the
O−O cleavage step. In this respect, it seems possible that the
flipped L-Arg orientation could have greater import than
merely ensuring strict regiochemistry for the ferryl-mediated
HAT step in the minor RO pathway. In this orientation, the
substrate guanidinium stacks with the side chain guanidinium
of Arg171 (Figure 5). This counterintuitive pairing of like-
charged Arg side chains in peptides and proteins has received
considerable attention over the past decade and has been
explained computationally.47−50 Its uniqueness to EFE among
the L-Arg processing Fe/2OG oxygenases, the reported
sensitivity of the EF pathway to modifications to L-Arg and
substitutions of residues that interact with it, and the close
proximity of the guanidinium pair to the cofactor (especially to
the expected location of the coordinated peroxide of the
peroxysuccinate intermediate) all suggest that this feature of
the EFE•substrates complex might have a role in lowering the
barrier for a noncanonical fragmentation of the intermediate
that commits to ethylene production.
CONCLUSIONS
■
A ferryl complex accumulates in the reaction of EFE, but
quantitative analysis of time-dependent absorption and
̈
Mossbauer spectra implies that it forms in only approximately
one-third of the events. The 16-fold extension of its lifetime by
the presence of deuterium at C5 of L-Argwithout impact to
the EF:RO partition ratioproves that the ferryl complex
forms only along the RO pathway, beyond the reaction
branchpoint. Thus, the D191E ligand substitution, which
virtually abolishes the EF reaction, also promotes accumulation
of ∼4 times as much of the ferryl complex. The identity of the
branchpoint intermediate and mechanism of the step leading
to ethylene production remain to be established, but it is clear
from results presented here and elsewhere that the marginal,
selective stabilization of the transition state for this step by the
wt enzyme is quite fragile. The basis for the differential
robustness of the two pathways and whether an even greater
degree of relative stabilization of the ethylene-committing
transition state is possible are intriguing questions for the
future.
In our view, the crystallographically validated Fe(II)-
persuccinate complex,45 which canonically undergoes O−O
bond heterolysis to generate the succinate-coordinated ferryl
intermediate, is a more likely branchpoint between the RO and
EF pathways than the Fe(IV)-peroxyhemiketal complex
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c10923.
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■
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AUTHOR INFORMATION
Corresponding Authors
Amie K. Boal − Department of Chemistry and Department of
Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, Pennsylvania 16802, United
States; orcid.org/0000-0002-1234-8472;
■
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J. Martin Bollinger, Jr. − Department of Chemistry and
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Authors
Rachelle A. Copeland − Department of Chemistry, The
Pennsylvania State University, University Park, Pennsylvania
16802, United States; orcid.org/0000-0002-8052-7843
Katherine M. Davis − Department of Chemistry, The
Pennsylvania State University, University Park, Pennsylvania
16802, United States; orcid.org/0000-0002-0258-8907
Tokufu Kent C. Shoda − Department of Chemistry, The
Pennsylvania State University, University Park, Pennsylvania
16802, United States; orcid.org/0000-0002-0238-0356
Elizabeth J. Blaesi − Department of Chemistry, The
Pennsylvania State University, University Park, Pennsylvania
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c10923
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the Office of Basic Energy Science
within the Department of Energy Office of Science (BES
Award DE-SC0016255). This research used resources of the
Advanced Photon Source, a U.S. Department of Energy
(DOE) Office of Science User Facility operated for the DOE
Office of Science by Argonne National Laboratory under
Contract DE-AC02-06CH11357. Use of the LS-CAT Sector
21 was supported by the Michigan Economic Development
Corporation and the Michigan Technology Tri-Corridor
(Grant 085P1000817). Data were also collected at the
Advanced Light Source, a Department of Energy Office of
Science User Facility under Contract DE-AC02-05CH11231.
The Berkeley Center for Structural Biology is supported by the
Howard Hughes Medical Institute and the National Institutes
of Health, National Institute of General Medical Sciences,
Grant P30 GM124169. K.M.D. is grateful for support from the
National Institutes of Health, Pathway to Independence Award
(4R00GM129460). We thank Dr. Benjamin Allen for technical
assistance and Prof. Todd Sowers at the Pennsylvania State
Earth and Environmental Systems Institute for GC-FID
analysis.
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Zhang, B.; Bergman, J. A.; Rajakovich, L. J.; Wang, B.; Silakov, A.;
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