Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of α-Heteroatom Assistance

Article abstract of DOI:10.1021/jacs.8b01933

Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H?) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H?-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (²H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H? first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors ¹H or ²H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors ²H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 ?) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.

Full text of DOI:10.1021/jacs.8b01933

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Article
Two Distinct Mechanisms for C–C Desaturation
by Iron(II)- and 2-(Oxo)glutarate-Dependent
Oxygenases: Importance of #-Heteroatom Assistance
Noah P. Dunham, Wei-chen Chang, Andrew J. Mitchell, Ryan J. Martinie, Bo Zhang, Jonathan A. Bergman,
Lauren J. Rajakovich, Bo Wang, Alexey Silakov, Carsten Krebs, Amie K. Boal, and J. Martin Bollinger, Jr.
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01933 • Publication Date (Web): 30 Apr 2018
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Two Distinct Mechanisms for C–C Desaturation by Iron(II)ꢀ
and 2ꢀ(Oxo)glutarateꢀDependent Oxygenases: Importance of
αꢀHeteroatom Assistance
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┴
┴
Noah P. Dunham1, , Wei-chen Chang2,3, , Andrew J. Mitchell1,4, Ryan J. Martinie2, Bo Zhang2,
Jonathan A. Bergman1, Lauren J. Rajakovich1,5, Bo Wang2, Alexey Silakov2,*, Carsten Krebs1,2,*,
Amie K. Boal1,2,*, and J. Martin Bollinger Jr.1,2,*
Departments of 1Biochemistry and Molecular Biology and 2Chemistry, The Pennsylvania State University, Uni-
versity Park, PA 16802
ABSTRACT: Hydroxylation of aliphatic carbons by non-heme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-
atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated
oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either
suppress rebound or further process hydroxylated intermediates. For olefin-installing C–C desaturations, it has been
proposed that a second HAT to the Fe(III)–OH complex from the carbon α to the radical preempts rebound. Deuterium
(2H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two
related L-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this
key test, implicating different mechanisms. NapI, the L-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic
pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent
1
2
whether C4 harbors H or H. By contrast, an unexpected 3,4-desaturation of L-homoarginine (L-hArg) by VioC, the L-
Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when
2
C3 (the second hydrogen donor) harbors H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in
the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is re-
quired. The close proximity (≤ 3.5 Å) of both L-hArg carbons to the oxygen ligand in an x-ray crystal structure of VioC
harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results sug-
gest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hy-
droxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG de-
saturases.
INTRODUCTION
step, commonly referred to as oxygen rebound,13 regen-
erates the Fe(II) state of the cofactor for subsequent turn-
over. It is thought that rebound has a low activation bar-
rier,14 consistent with the failure of the Fe(III)-
OH/substrate-radical state to accumulate in reactions of
Fe/2OG hydroxylases.6,8 Recent work has suggested,
however, that this step can be suppressed to allow for
different fates of the intermediate.1,15 For example, in
stereoinversion of the bridgehead carbon (C5) of (3S,5S)-
carbapenam-3-carboxylate by carbapenem synthase
(CarC), HAT from C5 to the ferryl complex is followed
by a butterfly-like flexion of the bicyclic substrate, which
inverts the radical, moves it away from the Fe(III)-
coordinated oxygen to disfavor rebound, and approxi-
Iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG)
oxygenases hydroxylate, halogenate, epimerize, cyclize,
and desaturate unactivated aliphatic carbon centers.1-3
The pathways to these diverse outcomes are thought to
share common initial steps, which include addition of O
to the non-heme Fe(II) cofactor, cleavage of 2OG to CO
2
2
and succinate, formation of an Fe(IV)-oxo (ferryl) com-
plex, and hydrogen atom transfer (HAT) from the sub-
strate to the ferryl oxo.4-12 The resultant state, with
Fe(III)–OH cofactor and substrate radical, is the likely
branch point to the different outcomes.1 In the hydrox-
ylation pathway, the carbon radical attacks the oxygen
ligand to form a new C–O bond. This radical-coupling
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mates its opposite face to a tyrosine residue that donates
Scheme 1. Hypothetical Pathways for C–C Desatura-
tions Catalyzed by (A) Heme Cytochrome P450 (B)
Non-heme Diiron, and (C) Fe/2OG Enzymes.
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H• to complete the epimerization.12,16-18 In the Fe/2OG
aliphatic halogenases, a chloro or bromo ligand cis to the
oxygen couples to the carbon radical in preference to
oxygen rebound.9,19,20 The halogenase SyrB2 directs this
outcome by positioning the scissile C–H bond farther
from the oxygen and closer to the halogen, trading profi-
ciency in the HAT step for selectivity in the radical-
coupling step.20,21 Whereas the required positioning
might, in principle, have arisen by evolutionary reloca-
tion of the substrate-binding site within the conserved
protein scaffold, recent analysis has suggested that the
halogenases instead alter the configuration of the ferryl
complex to achieve the required substrate-cofactor dis-
position.22-24 For both the carrier-protein-dependent
SyrB2 and the recently discovered carrier-protein-
independent halogenase, WelO5 (the first small-
molecule halogenase identified), it has been proposed
that the oxo of the ferryl locates trans to the proximal
histidine ligand (found in the HXD coordination motif),
essentially replacing the departing C1 carboxylate oxy-
gen of 2OG.22,24 This hypothetical ferryl configuration has
been termed offline, to distinguish it from the inline con-
figuration with the oxo trans to the distal histidine. In
both halogenases, the offline position of the oxo group
should result in a perpendicular approach of the scissile
C–H bond to the Fe=O unit. Frontier-orbital analysis of
the SyrB2 reaction suggested that this disposition can
rationalize both the unusually slow HAT step and the
preference for C–Cl/Br over C–O coupling in the subse-
quent step.22,25,26 Enforcement of offline ferryl complexes
could, in principle, be a general strategy to suppress re-
bound in other Fe/2OG enzymes that catalyze reactions
other than hydroxylation.27
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understood. Such transformations require, formally, re-
moval of hydrogen atoms from two adjacent aliphatic
carbons, perhaps requiring two successive H•-accepting
intermediates. The modi operandi of other O -activating
2
iron enzymes, including those in the cytochrome P45028
and non-heme di-iron classes,29 appear well suited to this
requirement (Scheme 1). In the former case, the Fe(IV)-
oxo/porphyrin-radical intermediate known as com-
pound I could serve as the first H• acceptor,30 leaving
the Fe(IV)-OH complex, compound II (a strong oxidant
in its own right),31 to remove the adjacent hydrogen
(Scheme 1A). In the non-heme-diiron case, an Fe (IV)
2
intermediate similar to Q from the soluble methane
monooxygenase catalytic cycle32,33 could be the first HAT
acceptor, leaving an Fe
2
(III/IV) complex, analogous to
The mechanisms of known olefin-installing desatu-
the tyrosine-oxidizing cluster X in the β subunit of class
rations catalyzed by Fe/2OG enzymes remain poorly
Scheme 2. Naphthyridinomycin biosynthesis and the activities of NapI (top) and VioC (bottom).
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Ia ribonucleotide reductase,34 to cleave the adjacent C–H
bond (Scheme 1B). These pathways can rationalize
VioC can desaturate L-homoarginine (L-hArg, the L-Arg
analog with an additional –CH – unit in its side chain)
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known desaturations at even completely unactivated
aliphatic C–C bonds by heme-iron and non-heme di-iron
oxidases in, for example, steroid and fatty acid metabo-
lism, respectively.28,29
between carbons 3 and 4 in competition with hydroxyla-
tion of either site (Scheme 2, bottom). As just the second
demonstrated example (of which we are aware) of de-
saturation by an Fe/2OG oxygenase of a C–C unit lack-
ing an activating α-heteroatom, this reaction presented a
compelling case study to compare to the NapI reaction,
which conforms to the α-heteroatom rule. We show here
that the reactions actually proceed by different mecha-
nisms. In the efficient, native 4,5-desaturation of L-Arg
by NapI, anchimeric assistance (of either dehydration of
a hydroxylated intermediate or electron transfer from
the C5 radical) by N6 obviates the second HAT step in
favor of removal of the C4 hydrogen as a proton. By con-
trast, with no such sequential, polar pathway for remov-
al of the second hydrogen available in the absence of an
α-heteroatom, the VioC reaction follows the oft-
proposed, sequential-HAT pathway, but with marked
The catalytic manifold of Fe/2OG oxygenases seems
less well suited for such a sequential-HAT mechanism
(Scheme 1C): the Fe(III)-OH complex produced by HAT
to the ferryl intermediate is expected to have modest
oxidative potency. However, because the radical center
weakens bonds to the adjacent (α) carbon, this mecha-
nism has repeatedly been advanced in the literature.35-37
Two considerations motivate direct experimental scruti-
ny of the hypothesis. Firstly, in the absence of a very
rapid change in the substrate-cofactor disposition be-
tween the first and second HAT steps, the imperative to
suppress rebound could present a geometric conun-
drum. Perpendicular approach of the target C–H bond
to the ferryl could, as proposed for the halogenases,22,24
disfavor subsequent C–O coupling, but the need for the
adjacent carbon to approach the Fe(III)-OH species for
the second HAT step would raise the question of why
this carbon would not efficiently donate H• to the ferryl
in the first place. Secondly, the vast majority of sub-
strates for these enzymes have a lone-pair-harboring
heteroatom (O or N) adjacent to one of the carbon atoms
between which the olefin is installed.1 To our
knowledge, only PrhA and BcmF among the known
Fe/2OG desaturases are thought to transform a com-
pletely unactivated C–C unit,38,39 although an earlier ex-
ample of mixed desaturation and hydroxylation of a
non-native substrate by the Fe/2OG hydrox-
ylase/oxacyclase/desaturase, clavaminate synthase, pro-
vides a notable second exception to this α-heteroatom
rule.35 In the reactions conforming to the rule, the non-
bonding electrons on the heteroatoms could enable al-
ternative pathways via iminium or oxonium intermedi-
ates, from which the second hydrogen could be removed
as a proton by a basic amino acid.
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Figure 1. Kinetics of the ferryl complex in NapI monitored
by its absorbance at 320 nm. An anoxic solution of NapI (1.2
mM), Fe(II) (1.1 mM), 2OG (6.0 mM) and 6.0 mM of either L-
Arg (black), 4,4-d2-L-Arg (red), or 5,5-d2-L-Arg (blue) was
mixed at 5 °C with an equal volume of air-saturated buffer
(giving ~ 0.19 mM final [O2]). Regression fits to the data are
shown as solid lines. The equation for fitting assumes ab-
sorbance from two consecutive, irreversible reactions, with
isosbestic reactant and product and more intensely absorb-
ing intermediate.
As a model system to address these mechanistic
questions experimentally, we identified the L-arginine
4,5-desaturase, NapI, from the naphthyridinomycin bio-
synthetic pathway (Scheme 2, top).40 In this selection, we
viewed known L-Arg hydroxylases that are structurally
similar to NapI (e.g., the 3-hydroxylase, VioC, from the
viomycin pathway41,42 and the 3,4-dihydroxylase, OrfP,
from the streptolidine pathway43) as important assets,
because we anticipated that a comparative structural
and mechanistic analysis might identify the crucial de-
terminants of the divergent outcomes, an overarching
goal of studies on this enzyme family.1 In the course of
the mechanistic analysis, we fortuitously discovered that
diminution in selectivity. The results highlight a poten-
tially general limitation of Fe/2OG enzymes for C–C de-
saturation reactions: effective suppression of rebound
may be impossible for reactions that can proceed only by
close approximation of adjacent C–H bonds to the cofac-
tor, as is required for sequential HAT steps.
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RESULTS AND DISCUSSION
1
Kinetic Evidence for HAT from C5 of L-Arg to the
Ferryl Intermediate in NapI. In the NapI desaturation
reaction, the adjacent carbons from which hydrogens
must be removed (C4 and C5) are distinguished by their
β and α dispositions to the heteroatom, N6. To identify
the site from which H• is abstracted by the presumptive
ferryl intermediate, site-specifically deuterium-labeled
Scheme 3. Possible pathways for C–C desaturation by
2
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4
5
6
7
8
an Fe/2OG oxygenase. (A) Sequential HAT (blue), (B)
Intermediate Hydroxylation (red), and (C) Electron
Transfer (green).
9
substrates, 4,4-d
2
-L-Arg and 5,5-d -L-Arg, were chemical-
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ly synthesized (see Supporting Information) (Fig. S1) and
used in single-turnover experiments monitoring for-
mation and decay of the intermediate. Previous studies
on multiple Fe/2OG hydroxylases, halogenases, and the
epimerase/desaturase CarC established (1) that the con-
served ferryl intermediate has an ultraviolet absorption
feature at 320 nm and a Mössbauer quadrupole doublet
with low isomer shift (~ 0.3 mm/s) that allow it to be
monitored in time, and (2) that substitution of the ab-
stracted hydrogen with deuterium markedly extends the
lifetime of the intermediate, owing to the large, normal
deuterium kinetic isotope effect (2H-KIE) on the HAT
step.4,8,9,11,12,44,45 In accordance with these precedents, fol-
lowing rapid mixing of an anoxic solution of the NapI-
•Fe(II)•2OG•L-Arg complex with air-saturated buffer,
ꢀA320 rises and falls (Fig. 1, black) in temporal correlation
with the Mössbauer quadrupole doublet features diag-
nostic of the ferryl intermediate (Fig. S5A and C).45 In
desaturation. It has only modest intensity in reactions
the reaction with 5,5-d -L-Arg, the maximum absorbance
2
with the unlabeled L-Arg (Fig. 2C, black trace) but is con-
is greater and the decay phase markedly delayed (Fig. 1,
siderably (4–5-fold) more intense upon use of the 5,5-d -
2
2
blue), reflecting a large H-KIE on decay of the ferryl
L-Arg isotopolog (blue traces), confirming that the large
2H-KIE can partially redirect the ferryl complex to an-
other site and alter the outcome to stable hydroxylation.
complex (Fig. S5B). By contrast, deuterium substitution
at C4 has only a modest effect on the kinetics of the fer-
ryl complex (Fig. 1, red). Stabilization of the intermediate
by deuteria on C5 but not C4 establishes that the ferryl
complex in NapI abstracts H• from C5 to initiate the 4,5-
desaturation.
This complex of peaks is suppressed for the 4,4-d -L-Arg
2
isotopolog (red traces), implying that the hydroxylation
side reaction occurs primarily at C4.
Failure of C4 Deuteriation to Enhance C5 Hydrox-
ylation Disfavoring Sequential-HAT Mechanism. Ap-
plied to NapI, the most widely cited mechanism for C–C
desaturations would invoke a second HAT,35 from C4 to
the Fe(III)-OH complex, to convert the ferryl-generated
C5 radical directly to the 4,5-dehydro-L-Arg product
(Scheme 3, pathway A). The precedent of the halogenas-
es20 suggests that rebound to C5 might compete to some
extent with the second HAT step. Competing rebound
would generate an unstable hemiaminal-like species,
which, if not directed to eliminate water on the normal
desaturation pathway (Scheme 3, pathway B), would be
expected to break down preferentially by elimination of
Product Yields with Deuterium-labeled Substrates
Confirming Initial HAT from C5. Use of liquid chroma-
tography and mass spectrometry (LC-MS) to compare
the yields of the 4,5-dehydro-L-Arg product from the
three substrate isotopologs provided additional confir-
mation of C5 targeting by the ferryl complex. At equiva-
lent, limiting concentrations of 2OG, the L-Arg and 4,4-
d2-L-Arg substrates give indistinguishable yields, where-
as the C5-labeled substrate gives ~ 50% less desaturation
(Figs. 2A and S6). As shown in previous work on TauD,
SyrB2, and CarC, diminished yield of the primary prod-
uct can reflect unproductive decay of the ferryl com-
plex,12,46 its redirection to an adjacent site,20 or a combina-
tion of the two effects, caused by the large 2H-KIE on the
initiating HAT step.4 Indeed, a complex of peaks at m/z =
+16 relative to the substrate reflects stable hydroxylation
occurring in competition with the predominant 4,5-
guanidine (pK of 12.5 for guanidinium versus 15.7 for
a
water). Guanidinium (m/z = 60, green trace in Fig. 2B) is
indeed produced in the reaction of the unlabeled sub-
strate (black trace). The yield of the breakdown product is
diminished by the presence of deuterium at C5 (blue
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trace) to the same extent as the yield of the primary 4,5-
Inactivity of 6-Deaza L-Arg for Desaturation Sug-
1
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4
5
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desaturation product, implying that both products form
subsequent to HAT from C5. Under the assumption that
the sequential-HAT mechanism (pathway A) is operant,
guanidinium production would imply that the second
HAT from C4 is preempted by rebound to C5 in a small
fraction of events. In this scenario, because the second
gesting Assistance of Polar C4-H Cleavage by N6. The
two other most likely pathways for conversion of the C5
radical to 4,5-dehydro-L-Arg would involve rebound to
C5 followed by dehydration and C4 deprotonation
(Scheme 3, pathway B) or electron transfer from the rad-
ical to the Fe(III)-OH cofactor followed by C4 deprotona-
tion (pathway C). Both of these pathways include an
iminium-like intermediate involving the non-bonding
electrons on N6, and neither is expected to be feasible in
the absence of the α-heteroatom. To test this prediction,
the 6-deaza analog of L-Arg, known as indospicine, was
evaluated as a substrate for NapI. The analog is indeed a
substrate but undergoes stable hydroxylation, as
demonstrated by the prominent peaks at m/z +16 or +18
relative to the substrate in LC-MS chromatograms of the
2
HAT step should also exhibit a sizable H-KIE, oxygen
rebound to C5 should compete more effectively in the
9
reaction of the 4,4-d -L-Arg isotopolog, leading to in-
2
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creased guanidinium production and decreased desatu-
ration. This expectation is not borne out, as neither yield
is significantly altered by the presence of deuterium at
C4 (Fig. 2A, B; compare red and black traces). The failure
of this prediction of the sequential-HAT mechanism im-
plies that rebound to C5 does not directly compete with
HAT from C4 and, thus, that the reaction proceeds by a
different mechanism.
reactions carried out under air or 18O
, respectively (Fig.
2
3, black and red traces). Notably, no peak at m/z = –2 rela-
tive to indospicine develops (blue trace), confirming that
Figure 2. Single-ion LC-MS chromatograms monitoring (A) desaturation (m/z = 173) (B) guanidine production (as a
consequence of C5 hydroxylation), and (C) stable hydroxylation (m/z = 191) of substrates, L-Arg (black), 4,4-d2-L-Arg
(red), and 5,5-d2-L-Arg (blue), by NapI. The green trace in B is the chromatogram of a 13C-guanidine standard. The
lighter red and blue traces in C are traces for the m+15 ions (where m is the m/z value of that substrate) resulting from
hydroxylation initiated by abstraction of deuterium. Peak areas relative to the normalized (independently for each
panel) areas of the traces for unlabeled L-Arg are given to the left of each trace, with standard deviations from three
trials in parentheses. Details of the analytical procedure are provided in the Supporting Information.
a, n.d. = not detected
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Figure 3. Positive-mode single-ion LC-MS chromatograms
showing that NapI can hydroxylate but not desaturate the
6-deaza analogue of L-Arg, L-indospicine. The front three
traces are from a control lacking the co-substrate, 2OG.
The middle traces are from analysis of a reaction carried
out under atmospheric O2. The rear traces are from a reac-
tion under 18O2. Coloring coding of the traces is: black, m/z
= 174 (indospicine); green, m/z = 190 (H16O-indospicine);
gray, m/z = 192 (H18O-indospicine); blue, m/z = 172 (dehy-
droindospicine; not observed).
Figure 4. Structural analysis of NapI by x-ray crystallog-
raphy. A, Active site model from a structure of L-
Arg•FeII•NapI rationalizing initial HAT from C5. The
enzyme exhibits a single conformation of L-Arg with con-
tinuous 2Fo-Fc electron density (black mesh) for the sub-
strate when the map is contoured at 1.0 σ. B, Relative dis-
position of substrate and cofactor showing unusual prox-
imity of C5 of L-Arg to the iron cofactor (orange sphere).
Selected ligands and second-sphere side chains are shown
in ball-and-stick format and colored by atom type. A pair
of acetate ions (OAc; shown in purple) from the crystalli-
zation solution occupy the 2OG binding site.
N6 of L-Arg plays an essential role in the native 4,5-
desaturation. These observations further validate the
conclusion that the sequential-HAT mechanism (path-
way A) is not operant for NapI.
the stably hydroxylated product, thus weighing against
the intermediacy of its hemiaminal cognate in pathway
B and in favor of pathway C.
Kinetics of Reaction with 6-Deaza L-Arg Favoring
Electron Transfer over Intermediate Hydroxylation.
The distinction between pathways B and C is whether 5-
hydroxy-L-Arg is (in B) or is not (in C) a precursor to the
iminium intermediate that undergoes deprotonation of
C4 to form 4,5-dehydro-L-Arg. Kinetic analysis of the
reaction with L-indospicine suggests that pathway C is
more likely. The hydroxylated product of the 6-deaza
analog, 5-hydroxy-L-indospicine, would be expected to
mimic the unstable hemiaminal-like intermediate of
pathway B, but it would be foreign to pathway C. As
unstable intermediates are generally held tightly within
enzyme active sites, slow dissociation of the 3,4-dehydro
product would be expected for the case that pathway B
is operant, whereas rapid dissociation might be antici-
pated for a mechanism not involving a hydroxylated
intermediate, such as pathway C. Previous work on
TauD showed that product dissociation is ordered: suc-
cinate dissociation is the last and slowest step and is
prevented by occupancy of the taurine site (by either
substrate or product).6 Redevelopment of the 520-nm
absorption band of the reactant complex in single-
turnover reactions thus occurs only after release of both
products. In single-turnover, stopped-flow experiments
on NapI, the absorption feature of the reactant complex
actually re-develops more rapidly in the L-indospicine
reaction than in the reaction with the native substrate
(Fig. S7). This result suggests that NapI readily releases
X-ray Crystal Structure of NapI Rationalizing HAT
from C5 to the Cofactor. NapI shares ~ 50% sequence
identity with the structurally characterized
L-Arg 3-
hydroxylase, VioC, from the viomycin biosynthetic
pathway. The two proteins are thus expected to have
similar structures. The demonstration that the NapI fer-
ryl intermediate abstracts H• from C5 (by contrast to
abstraction from C3 in VioC) raises the intriguing ques-
tion of how the shared substrate is differently positioned
within the conserved protein architecture to permit dif-
ferent sites to be targeted for distinct outcomes. To an-
swer this question, we solved an x-ray crystal structure
of NapI in complex with Fe(II) and L-Arg in the absence
of O
2
(Fig. 4A, B). A superposition of the 2.1-Å-
resolution NapI structure with the published structure of
VioC (PDB accession code 6ALM) shows that the two
proteins share an identical topology (rmsd of 1.13 Å over
314 Cα atoms), apart from a small difference in the fold
of a dynamic lid loop (Fig. 5A).
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Figure 5. Comparison of the structures of VioC (PDB: 6ALM)44 and NapI. A, side-by-side comparison of the overall fold and
conformation of the lid loop (inset). B and C, Comparison of the L-Arg side chain interactions in VioC and NapI, respectively,
showing that their different sets of H-bonding partners position the common substrate uniquely within the active site. Select-
ed side chains, backbone, and substrate molecules are shown in ball-and-stick format. Water molecules (red) and FeII cofactor
(orange) are shown as spheres.
Two acetate ions from the crystallization solution
nection involves an alternate conformation of the sub-
strate sidechain and different interacting atoms. Interest-
ingly, NapI employs H-bonds between the terminal
guanidine nitrogens and a pair of symmetrically dis-
posed, bound water molecules to position C5 closest to
the Fe(II) cofactor, just 3.9 Å away. C4 and C3 are more
distant, at 4.9 Å and 4.7 Å, respectively (Fig. 4B). By con-
trast, in VioC, a single bound water molecule and an
additional Asp residue (270) on the same side of the ac-
tive site hydrogen bond with the terminal guanidine
nitrogens, and these contacts are associated with a ~ 90°
occupy the 2OG binding site in the NapI structure; at-
tempts to obtain high-quality crystals with the co-
substrate bound were unsuccessful. However, the bind-
ing orientation of L-Arg, which is readily discernible in
electron density maps of the complex (black mesh in Fig.
4A and Fig. S8), and the network of hydrogen-bonding
contacts that anchor it in the active site (Fig. 5B-C) are
unlikely to be affected by the absence of 2OG. Compari-
son of the VioC and NapI active site models (Figs. 5B-C
and S9) shows that, despite their nearly identical folds,
the two enzymes orient their common substrate using
completely distinct contacts to the L-Arg amine, carbox-
ylate, and guanidinium groups. Only a salt bridge be-
tween the substrate side chain and a second-sphere Asp
(D245, NapI numbering) is conserved, but even this con-
rotation of the L-Arg guanidine that serves to move C5
away from the cofactor and instead position C3 proximal
to the Fe(II).
Active-site Metrics by HYSCORE Spectroscopy Ra-
tionalizing HAT from both C5 and C4. Analysis of hy-
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perfine couplings between substrate hydrons and EPR-
active (S = 3/2) iron-nitrosyl ({FeNO}7) cofactor adducts
(Fig. 4B), offering the possibility of cation-π stabiliza-
1
2
3
4
5
6
7
8
tion51 of the full positive charge that would develop on
N6 in pathways B and C of Scheme 3. Given the likeli-
hood that the guanidine sidechain would already be
protonated, formation of the iminium-like intermediate
in pathway B or C could also involve coupled proton
transfer from one of the terminal side-chain nitrogens to
the salt-bridged Asp245 or an active-site water (Fig. 5C)
to obviate formation of a di-cation intermediate. Both
routes, B and C, would be completed by removal of a
proton from C4, and the structural and HYSCORE data
reveal two excellent candidates for the acceptor, the car-
boxylate side chain of D200 (which resides on a lid loop
that closes over the substrate) and the Fe(II)-OH center
itself, to act as the general base for this step. In the latter
scenario, rotation of C4 to a position closer to the metal
center (as suggested above) would have to occur. An
additional possibility, suggested by the unusually short
3.9-Å distance between the H•-donating carbon and the
Fe(II) cofactor (which, to our knowledge, is the shortest
yet observed for any Fe/2OG oxygenase reactant com-
plex3), is that the cofactor could function as a Lewis acid
to promote elimination of hydroxide from C5 in path-
way B of Scheme 3.
previously afforded active-site structural information for
O
-activating non-heme-iron enzymes,47 including the
2
Fe/2OG hydroxylase TauD48-50 and halogenase SyrB2.21
Orientation-selective HYSCORE spectra of the {FeNO}7
form of NapI in complex with either 5,5-d
2
-L-Arg or 4,4-
d -L
2
-Arg reveal deuterons 3.3 ± 0.1 and 3.6 ± 0.2 Å from
the iron, respectively (Fig. 6). For both substrates, the
analysis yields angles between the Fe-2H and Fe-N(O)
vectors of 55 ± 5° (Figs. S10 and S11). The HYSCORE
data are thus also consistent with HAT to the ferryl
complex primarily from C5. However, comparison of the
metrics obtained by the two structural methods suggests
that oxidation of the iron cofactor from +II in the crystal
structure to (formally) +III in the complex probed by
HYSCORE results in a leveling of the Fe–C5 and Fe–C4
distances. The implied movement of C4 toward the co-
factor (Fig. 6), should it occur also upon conversion of
the reactant complex to the ferryl intermediate, would
explain the capacity of C4 to serve as H• donor, espe-
cially when HAT from C5 is slowed by the presence of
deuterium.
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Structural Features of NapI Potentially Promoting
Polar C4-H Cleavage. The position of C5 in the substrate
binding pocket, directly over the H146 ligand contribut-
ed by the HXD motif, is different from the positions of
H• donors in other structurally characterized en-
zyme•Fe(II)•2OG•substrate complexes (Fig. S12). The
binding mode allows for close interaction (3.1 Å) be-
tween the adjacent N6 and the imidazole ring of H146
Discovery of Desaturation of
L-homoarginine (L-
hArg) by the -Arg 3-Hydroxylase, VioC. Given the
L
demonstrated importance of N6 in the 4,5-desaturation
of L-Arg catalyzed by NapI, the discovery that the struc-
turally similar (~50% identical) L-Arg 3-hydroxylase,
VioC, from the viomycin biosynthetic pathway in Strep-
tomyces vinaceus (Sv)41,52 can desaturate a C–C unit of L-
Figure 6. Analysis of substrate positioning in NapI by HYSCORE spectroscopy on its EPR-active, S = 3/2 iron-nitrosyl
{FeNO}7 complex. A, Comparison of HYSCORE spectra (left) and simulations (right), collected at 330 mT on samples of Na-
pI•{FeNO}7•2OG•5,5-d2-L-Arg (top) and NapI•{FeNO}7•2OG•4,4-d2-L-Arg (bottom). Spectra were collected with a micro-
wave frequency of 9.77 GHz at a temperature of 4.0 K. Experimental conditions and simulation parameters are given in the
legends to Figures S10 and S11. B, Comparison of distances obtained by x-ray crystallography (black) and HYSCORE spec-
troscopy (red), and structural models based on these distances. The transparent C3-C5 segment is based on the HYSCORE
results. Hydrogens are shown as purple spheres.
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hArg lacking an α-heteroatom (Scheme 2, bottom) pre- Characterization of the 3,4-Desaturated and 3/4-
1
2
3
4
5
6
7
8
sented an opportunity to probe experimentally how the
requirement for anchimeric assistance is obviated. The
discovery was made in an initial application of the LC-
MS method used for NapI to the reaction of Sv VioC,
Hydroxylated Products from VioC/L-hArg Reaction. To
determine the structure of the ꢀm/z = -2 product, the
VioC/L-hArg reaction was carried out on a large scale,
and the product was chromatographically purified and
characterized by NMR spectroscopy (as summarized in
the Experimental Procedures). Spectra from a suite of
correlation experiments identify it as the Fmoc deriva-
tive of trans-3,4-dehydro-L-homoarginine (Fig S15-S19).
Evidence summarized below confirms that the two hy-
droxylated species are 3S-hydroxy-L-homoarginine (3-
OH) and 4-hydroxy-L-homoarginine (4-OH), implying
that all three primary products form subsequent to HAT
from either C3 or C4 to the ferryl intermediate.
Fe(II), 2OG, L-hArg and O . Two major peaks, with ꢀm/z
2
values of +16 and –2 relative to L-hArg, develop in this
reaction; they reflect hydroxylation and desaturation,
respectively, of the substrate analog (Fig. S13A). With
varying, limiting quantities of 2OG, the intensities of
both chromatographic peaks vary proportionally to
[2OG] (Fig. S14), confirming that the species are authen-
tic enzymatic products. Derivatization of the reaction
products with the fluorenylmethyloxycarbonyl (Fmoc)
protecting group and use of reverse-phase HPLC affords
better chromatographic resolution of the products and
reveals the presence of two hydroxylated species, for a
total of three primary products (Fig. 7A, black trace).
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Mechanistic Diagnosis of Partition Among 3,4-
Desaturation and 3/4-Hydroxylation Outcomes. Chemi-
cal synthesis of 2,3,3-d
3
-L-hArg, 4,4-d
2
-L-hArg, and per-d -
9
L-hArg isotopologs (see Supporting Information, Fig. S2)
afforded the tools to dissect the VioC/L-hArg desatura-
tion reaction in the same manner as for NapI. Stopped-
flow absorption experiments analogous to those on NapI
were uninformative: although the ferryl intermediate
was previously shown to accumulate substantially in the
reaction of VioC with its native substrate (L-Arg),44 the
Use of 18O
as a substrate results in a further +2 shift in
2
ꢀm/z (to +18) for the two hydroxylated products, show-
ing that they form by the canonical O-atom-insertion
(HAT-rebound) mechanism. Not surprisingly, use of 18
O
2
does not change the ꢀm/z (–2) for the presumptively de-
saturated product. (Fig. S13B).
Figure 7. LC-MS analysis of the transformations of L-hArg deuterium isotopologs by VioC, and interpretation of the results in
terms of mechanistic pathways, 2H-KIE (kH/kD) values, and impacts on partition ratios. A, All-protium L-hArg. B, C3-labeled L-
Arg (red), C4-labeled L-Arg (blue), and uniformly labeled L-Arg (purple). The red values are partition ratios for (i) the initial
HAT to the ferryl complex from C3 or C4 (upper in A), (ii) the fate of the C3 radical, rebound or second HAT (middle in A),
and (iii) overall products (lower in A and B). The red numbers in parentheses are the standard deviations of the values from
2
three independent trials. The H-KIE values that rationalize the impacts of deuterium substitution on the partition ratios are
shown in gray and black (in A). The arrows and dashes (in B) illustrate the qualitative impacts of the 2H-KIEs on the partitions
between HAT from C3 or C4 to the ferryl complex (gray), between the two pathways for decay of the C3 radical (black), and
among the three possible products (gray and black). Details of the analytical procedure are provided in the Supporting Infor-
mation. The values resulting from product detection by A260 (from the Fmoc group) are compared in Table S3.
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Figure 8. X-ray crystal structures of VioC complexes revealing changes in the conformation of the substrate/product side
chain through the reaction cycle. A, Reactant complex with Fe(II) (orange), 2OG (yellow), and L-hArg (pink). B, Complex
with vanadium [cyan; (hydr)oxo shown in red], succinate (purple), and L-hArg (pink). C, Complex after exposure of crystals
of the reactant complex to O2 showing electron density consistent with formation of (3S)-OH-L-hArg (green) (color coding
of other ligands as in A). In all structures, a 2Fo-Fc electron density map for selected active site components is shown in
black mesh and contoured to 1.5 σ.
complex with the non-native L-hArg substrate reacts
with O too slowly to support measureable ferryl accu-
shifted to Δm/z = +15 (–2 for loss of 2H and +17 for gain of
–OH) in the reaction with 2,3,3-d -L-hArg, showing that
2
3
mulation, even with uniform deuterium substitution to
minimize its decay rate. Nevertheless, LC-MS analysis of
the reactions with the series of isotopologs afforded sig-
nificant mechanistic insight, revealing (1) that HAT from
either labeled carbon to the ferryl complex is disfavored
relative to HAT from the adjacent carbon lacking deuter-
ium, thus changing the initial partition between C3 and
C4 targeting, and (2) that the partition between C4 hy-
droxylation and 3,4-desaturation is shifted in favor of
the former outcome by the presence of deuterium at C3.
The measured product distributions across the series of
L-hArg isotopologs could be rationalized by the two
simple assumptions embodied in Fig. 7a. First, HAT to
the ferryl intermediate from C3 (right pathway) commits
the reaction to hydroxylation of this site, whereas HAT
from C4 initiates either C4 hydroxylation (center) or 3,4-
it is associated with the 3-hydroxylated product (3-OH).
Conversely, the peak eluting at ~ 5 min was diminished
and shifted to Δm/z = +15 in the reaction with 4,4-d -L-
2
hArg, thus associating it with the 4-hydroxylated prod-
uct (4-OH).
Effects of Deuterium at C3 and/or C4 on Partition
Ratios: Evidence for Sequential HAT Steps. This as-
signment of the chromatographic peaks enabled deter-
mination of the relative yields of the three major prod-
ucts with each of the four L-hArg isotopologs (specifical-
ly labeled at either site, globally labeled, and unlabeled).
Two independent methods of detection, mass spectrom-
etry and UV absorption (at 260-nm, from the amine-
appended Fmoc), gave mutually consistent results (Ta-
ble S3). The anticipated redirecting effect of the large 2H-
KIE on the initial HAT to the ferryl was confirmed by
comparison of the chromatograms for the reactions of
2
desaturation (left). Second, H-KIEs of ~ 10 on the HATs
to the ferryl complex (from either C3 or C4) and the re-
moval of the second hydrogen (exclusively from C3)
impact the fates of the branch-point intermediates (the
ferryl and Fe(III)-OH/substrate-radical states) to dictate
the product distribution.
the unlabeled, 4,4-d
2
, and 2,3,3-d -L-hArg isotopologs
3
(Fig. 7). The partition ratio for HAT to the ferryl from
carbons 3 and 4 (C3:C4) was diminished from ~ 1 (55:45)
with the unlabeled substrate (panel A) to ~ 0.1 (8:92)
with 2,3,3-d -L-hArg (panel B, left), whereas it was in-
3
LC-MS Analysis of Reactions with Deuterium-
creased to ~ 10 (94:6) with 4,4-d -L-hArg (panel B, center).
2
2
labeled
L
-hArg Substrates to Assign Products. The first
The effect of the H-KIE on the second hydrogen remov-
step in the analysis was to associate the two ꢀm/z = +16
chromatographic peaks with the appropriate hydrox-
ylated products. The distinct impacts of C3 versus C4
deuterium substitution on the yields and ꢀm/z values of
the two hydroxylation products enabled this assign-
ment. The peak eluting at ~ 6 min was diminished and
al could be resolved by including the results for per-d -L-
9
2
hArg, because the very similar H-KIEs for HAT to the
ferryl from carbons 3 and 4 resulted in a C3:C4 partition
ratio identical to that for the unlabeled substrate. The ~
10-fold increase in the 4-hydroxylation:3,4-desaturation
partition ratio, from ~ 0.7 (18:27) with the unlabeled sub-
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-L-hArg, thus reflected ex-
strate to ~ 7 (43:6) with per-d
9
1
2
3
4
2
clusively the H-KIE on removal of the second hydrogen
from C3. Importantly, the 4-hydroxylation:3,4-
desaturation partition ratio of ~ 7 (82:12) for 2,3,3-d
3
-L-
hArg was almost identical to that for per-d -L-hArg, thus
9
5
establishing a consistent effect of C3 2H substitution,
regardless of the value of the initial C3:C4 partition ratio
for HAT to the ferryl complex. The existence and magni-
tude of this effect, not observed for the anchimerically-
assisted desaturation mediated by NapI, are most con-
sistent with the oft-proposed sequential-HAT mecha-
nism (Scheme 3, pathway A).35
6
7
8
9
Figure 9. Views of the active site models for the VioC reactant
and vanadium complexes showing substrate side chain con-
formational changes potentially associated with ferryl for-
mation. A, Reactant complex (from Figure 8A), in which C3 is
proximal (4.0 Å) to the metal center but C4 is too far removed
(5.1 Å) and poorly oriented for HAT to the cofactor. B, Vana-
dium complex showing C3 and C4 comparably well poised
(3.1 Å and 3.4 Å, respectively) for HAT, consistent with the
ability of both carbons to be targeted by the ferryl intermedi-
ate.
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Structure of VioC•Fe(II)•2OG•L-hArg Complex
Rationalizing HAT to Ferryl from C3. To underpin
mechanistic interpretation of the product distribution
and impact of specific deuterium labeling of the sub-
strate thereupon, we solved the structure of the
VioC•Fe(II)•2OG•L-hArg complex to 1.7 Å resolution.
Continuous composite and difference electron density is
visible for the iron cofactor, its ligands, and L-hArg (Fig.
8A and Fig. S20A). The extended (4-guanidinobutyl)
side chain is readily accommodated in the active site,
and the analog forms the same hydrogen-bonding inter-
plex is consistent with HAT from C3 to either the ferryl
complex (to initiate 3-hydroxylation) or the Fe(III)–OH
intermediate (to complete 3,4-desaturation), the position
of C4 seems inconsistent with HAT from this carbon to
the ferryl complex (to initiate either 4-hydroxylation or
3,4-desaturation). C4 is 5.1 Å from the iron with its hy-
drogens directed away from the Fe(II) center, not suita-
bly poised for abstraction by either of the oxidized in-
termediates. The structure thus implies that the confor-
mation of the substrate side-chain must change signifi-
cantly during the reaction to permit C4 to donate H•, as
deduced above for the NapI/L-Arg case. To obtain evi-
dence for such substrate dynamics, we determined the
structure of VioC in complex with a mimic of the ferryl
center. Recent studies have shown that the stable V(IV)-
oxo (vanadyl) dication can bind in the active sites of
Fe/2OG oxygenases, including VioC.44,50 Specific reloca-
tions of active-site residues in the VioC vanadyl com-
plex, matching V–ligand and Fe–ligand distances deter-
mined by extended X-ray absorption fine structure
(EXAFS) spectroscopy, accuracy of ferryl Mössbauer
parameters calculated from the geometry of the vanadyl
complex, and a marked stabilization of the otherwise
dynamic interaction between the halogenase, SyrB2, and
its carrier-protein substrate, SyrB1, in both its ferryl and
vanadyl forms provided evidence for faithful “metallo-
mimicry” of the reactive states. A 1.70-Å-resolution
structure obtained by x-ray-diffraction analysis of crys-
tals of the VioC•VO•succinate•L-hArg complex (Fig.
8B) provides even stronger evidence. Although the re-
fined V–O distance of 1.86 Å is consistent with the previ-
ously reported photo-reduction of the vanadyl ion in the
x-ray beam, details of the structure suggest that this pro-
cess does not abrogate the ferryl-mimicking features of
the original (unreduced) complex.44 The observed inter-
actions of the protein with the substrate are nearly iden-
actions with second-sphere residues as the native L-Arg
substrate does in the previously published structure.44
As in the prior structure, hydrogen bonding of the sub-
strate α-amine to iron ligand E170 and interactions of
both D268 and D270 with the guanidine moiety combine
to position C3 closest to the Fe(II) center (4.0 Å), explain-
ing its preferential targeting by the ferryl complex (Fig.
9A and Fig. S21A).
Structure of Product Complex Revealing C3 Hy-
droxylation and its Stereochemistry. Exposure of the
crystals of the VioC•Fe(II)•2OG•L-hArg complex to O
2
and subsequent freezing allowed for solution of the
structure of a product complex at 1.95-Å resolution (Fig.
8C). The composite and difference electron density clear-
ly reveal conversion of L-hArg to the C3-hydroxylated
product (3-OH) with S stereochemistry (Fig. S20C). The
apparent accumulation of only the 3-OH product in crys-
tallo could be a consequence of restraints on the active
site that disfavor the 4-hydroxylation and 3,4-
desaturation pathways (vide infra). Alternatively, the 3-
OH product could selectively accumulate, owing to its
slow dissociation, tight binding, or both. As in several
previously reported structures of product complexes of
Fe/2OG hydroxylases,44,53 the newly installed oxygen
coordinates the Fe(II) cofactor, an interaction that could
contribute to its predominance in the active site.
Structural Analysis of a Ferryl-Mimicking Vanadium
Complex Rationalizing Ambiguous C3/C4 Targeting
and Desaturation by Sequential HATs. Although the
structure of the VioC•Fe(II)•2OG•L-hArg reactant com-
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tical to those in the reactant complex, but, importantly, non-native 3-4-desaturation of deoxyproclavaminate by
1
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4
5
6
7
8
C4 is much closer to the metal center (Fig. 9B). Subtle
changes in the conformation of the L-hArg sidechain
relative to that in the reactant complex bring carbons 3
and 4 into nearly equal proximity to the vanadyl oxo (3.1
versus 3.4 Å, respectively). In this configuration, the two
carbons would be similarly well-poised to serve as HAT
donors, thus rationalizing the observed distribution of
products.
clavaminate synthase is reportedly more selective (90%
desaturation versus 10% hydroxylation) than the
VioC/L-hArg desaturation.35 Similarly, reactions tenta-
tively attributed to PrhA and BcmF in the paraherquonin
and bicyclomycin biosynthetic pathways, respectively,
would be examples of desaturations without the possi-
bility of heteroatom assistance;38,39 these native desatura-
tions are likely to be relatively efficient. Given that
Fe/2OG oxygenases appear to present an outstanding set
of targets for development of new biocatalytic activities
and that olefin insertion between unactivated carbons
would be an immensely useful synthetic capability, de-
fining the inherent limitations of the mechanistic motif
for this type of reactivity is a worthy goal.
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HYSCORE analysis of the vanadyl complex itself (not
photo-reduced) provides support for the conclusions
from the x-ray crystal structure. Hyperfine coupling of
the vanadyl center to deuterium is observed in the com-
plex with either 3,3-d
2
-L
-hArg or 4,4-d -L-hArg bound.
2
Analysis of the orientation-selective HYSCORE spectra
gives distances of 3.4 ± 0.2 Å and 3.8 ± 0.4 Å between the
vanadium ion and the C3 and C4 deuterons, respectively
(Figs. S22 and S23). The magnitude and rank order of
these V–2H distances, and the difference between them,
match well to those indicated by the crystallographic
structural model (3.6 Å for the pro-S C3-2H and 4.4 Å for
the pro-R C4-2H). These comparisons imply that, other
than the low-barrier elongation of the V–O and other
metal-ligand bonds, the cryogenic photo-reduction of
the vanadyl unit in the x-ray beam does not grossly alter
the chemically relevant dispositions of atoms in the ac-
tive site, in agreement with the conclusions from the
earlier study.44 The implications are: (i) that crucial dis-
positions of substrate atoms may change in these en-
zymes in progression through the catalytic cycle, (ii) that
reactant complexes may, therefore, fail to recapitulate
reactivity-determining features of the ferryl complexes,
and (iii) that the vanadyl complex may provide a better
basis for rationalizing and perhaps even predicting reac-
tion outcomes.
ASSOCIATED CONTENT
Experimental section, Figures S1-S23, and Tables S1-S3. This
material is available free of charge via the internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*email addresses: aus40@psu.edu; ckrebs@psu.edu;
akb20@psu.edu; jmb21@psu.edu
Present Addresses
3Department of Chemistry, North Carolina State University,
Raleigh, NC 27695
4Whitehead Institute for Biomedical Research, Cambridge,
MA 02142
5Department of Chemistry and Chemical Biology, Harvard
University, Cambridge, MA 02138
Author Contributions
┴N.P.D. and W.-c.C. contributed equally.
ACKNOWLEDGMENT
CONCLUSIONS
The selective, natural desaturation of L-Arg by NapI
and the relatively unselective, non-native desaturation of
L-hArg by VioC appear to proceed by different mecha-
nisms, as judged by the divergent impact of deuterium
substitution of the second hydrogen donor on the extent
of competing oxygen rebound to the substrate radical
formed by the first HAT. Facilitation of polar cleavage of
the second C–H bond by the α-heteroatom, present in
the NapI reaction but absent in the VioC/L-hArg desatu-
ration, is most likely responsible and can rationalize the
presence of α-heteroatoms in the majority of known,
natural Fe/2OG-desaturase substrates.1 Given the clear
evidence for divergent mechanisms, continuing study of
desaturations of substrates lacking a heteroatom to de-
fine the limitations of the Fe/2OG mechanistic manifold
seem warranted. For example, the previously reported
This work was supported by the National Institutes of
Health (GM119707 to A.K.B; GM113106 and GM118812 to
J.M.B. and C.K.) and the National Science Foundation
Graduate Research Fellowship Program (Grant No.
DGE1255832 to R.J.M.). We thank Professor Scott A.
Showalter for assistance with collection of NMR spectra.
We gratefully acknowledge the resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office
of Science User Facility operated for the DOE Office of Sci-
ence by Argonne National Laboratory under Contract No.
DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was
supported by the Michigan Economic Development Corpo-
ration and the Michigan Technology Tri-Corridor (Grant
085P1000817). GM/CA@APS has been funded in whole or in
part with Federal funds from the National Cancer Institute
(ACB-12002) and the National Institute of General Medical
Sciences (AGM-12006). The Eiger 16M detector was funded
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Martinie, R. J.; Livada, J.; Chang, W.-c.; Green, M. T.;
by an NIH-Office of Research Infrastructure Programs,
1
2
Krebs, C.; Bollinger, J. M., Jr.; Silakov, A. J Am Chem Soc 2015,
137, 6912.
High-End Instrumentation Grant (1S10OD012289-01A1).
3
4
5
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8
9
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