Visualizing the reaction cycle in an Iron(II)- and 2-(Oxo)-glutarate-dependent hydroxylase

Article abstract of DOI:10.1021/jacs.7b07374

Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cisrelated coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the L-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.

Full text of DOI:10.1021/jacs.7b07374

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Article
Visualizing the reaction cycle in an iron(II)-
and 2-(oxo)-glutarate-dependent hydroxylase
Andrew J. Mitchell, Noah P. Dunham, Ryan J. Martinie, Jonathan A. Bergman, Christopher J. Pollock, Kai Hu,
Benjamin D. Allen, Wei-chen Chang, Alexey Silakov, J. Martin Bollinger, Jr., Carsten Krebs, and Amie K. Boal
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07374 • Publication Date (Web): 20 Aug 2017
Downloaded from http://pubs.acs.org on August 21, 2017
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Visualizing the reaction cycle in an iron(II)- and 2-(oxo)-glutarate-
dependent hydroxylase
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Andrew J. Mitchell1‡, Noah P. Dunham1‡, Ryan J. Martinie2, Jonathan A. Bergman2, Christopher J.
Pollock2, Kai Hu1,3, Benjamin D. Allen1,3, Wei-chen Chang2†, Alexey Silakov2, J. Martin Bollinger
Jr.1,2*, Carsten Krebs1,2*, Amie K. Boal1,2*
1Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park PA 16802
2Department of Chemistry, The Pennsylvania State University, University Park PA 16802
3The Huck Institutes for the Life Sciences, The Pennsylvania State University, University Park PA 16802
ABSTRACT: Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are
often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orienta-
tion of the substrate C–H and ferryl Fe–O bonds, primarily by direction of the oxo group into one of two cis-related coor-
dination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the
ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental
validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo
position. Comparison of high-resolution x-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl
precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the L-arginine 3-hydroxylase, VioC, reveals coor-
dinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.
INTRODUCTION
industrially-relevant halogenation and cyclization reac-
tions.8 All members of the superfamily use their FeII co-
factor and a common metabolite, 2OG, to form an FeIV-
oxo (ferryl) intermediate;11 addition of O2 to the complete
preformed enzyme•substrate complex results in oxidation
of the iron and decarboxylation of 2OG to succinate. X-
ray crystal structures of the O2-reactive en-
zyme•FeII•2OG•substrate complexes, as well as inhibitor
and product-bound states, have been reported for many
Fe/2OG enzymes,12 and the ferryl intermediates have been
characterized by spectroscopic methods11, 13-18 in a smaller
number of cases. Importantly, neither the key FeIV=O
state nor any of its fleeting precursors have been structur-
ally characterized in any member of this enzyme super-
family.
Oxygen-mediated modification of biomolecules enables
a significant proportion of the structural diversity present
in natural products, molecules of unquestioned medicinal
and industrial importance.1-2 All biological catalysts that
rely on dioxygen as a source of oxidative power must ex-
ert tight spatial and temporal control over reactive O2-
derived adducts.3 X-ray crystallographic characterization
of such intermediates via reaction initiation in single crys-
tals has been important in understanding these mecha-
nisms of control in many classes of O2-reactive enzymes,
including the extradiol ring-cleaving dioxygenases,4 cop-
per amine oxidases,5 cytochrome P450s,6 and diiron hy-
droxylases.7 Crystallographic analysis of these reaction
pathways has also unmasked new intermediates and pro-
vided important structural context for states character-
ized by other methods.
In the majority of cases, the ferryl oxidant initiates
transformation of the primary substrate by abstracting a
hydrogen atom (H•),13 yielding an intermediate with a
carbon-centered radical and FeIII-OH cofactor form. Dif-
ferent pathways emerging from this key branch-point
intermediate lead to the known divergent reaction out-
comes in the superfamily.8 The most common transfor-
mation is hydroxylation, accomplished by attack of the
substrate radical upon the coordinated oxygen ligand, a
step thought to have a low activation barrier. The product
is a new R-OH group and an FeII-succinate complex. Alt-
hough the precise fates of the initial substrate radical in
the alternative transformations are less well understood,
Fe(II)- and 2OG oxygenases constitute one of the pri-
mary enzymatic routes to O2-mediated functionalization
of natural products and are overrepresented in biosyn-
thetic pathways owing to their unique ability to selective-
ly functionalize otherwise chemically inert C-H bonds.8
More than 100,000 sequences in prokaryotes and eukary-
otes have been annotated as probable Fe/2OG oxygenases
on the basis of their conservation of an iron-binding mo-
tif, H-X-(D/E)-Xn-H (Fig. 1).9-10 Fe/2OG enzymes are func-
tionally diverse, catalyzing both simple –O atom inser-
tions and more complex transformations that include
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it is crucial to avoid facile coupling to the oxygen ligand. anoxically with d7-L-Arg were briefly exposed to O2 (for
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For some systems, distinct ligand geometries of the ferryl
state have been proposed18-19 as a mechanism to suppress
this step, but direct evidence for this notion is lacking.
less than 1 min) and flash frozen. Deuterium substitution
slows the H-atom transfer (HAT) step, which is rate-
limiting in solution.23-24 Initial efforts yielded many da-
tasets representative of the 3-S-OH-L-Arg/2OG complex
(Fig. 2B), suggesting that the reaction proceeds in crystal-
lo on this timescale, even with the heavy-isotope chal-
lenge. Despite evidence of L-Arg turnover, in no case was
the succinate coproduct also present; the electron density
was instead consistent with the larger cosubstrate 2OG
(present as a precipitant at ~1.0 M concentration), sugges-
tive of rapid diacid ligand exchange without dissociation
of the primary 3-OH-L-Arg product.
RESULTS AND DISCUSSION
VioC from Streptomyces vinaceus 1326 is an Fe/2OG ox-
ygenase that hydroxylates C3 of L-arginine on the biosyn-
thetic pathway to the tuberactin antibiotic viomycin,20
which was originally included in multi-drug cocktails tar-
geting Mycobacterium tuberculosis infections.21 In the
study elucidating its role in the viomycin pathway, multi-
ple structures of VioC complexes were solved to near-
atomic resolution by x-ray crystallography.22 However, the
x-ray datasets provide only limited insight into the reac-
tion mechanism because the complexes characterized are
not on the catalytic pathway. In one, 2OG was replaced by
tartrate; in a second, the 3-hydroxy L-Arg product was
bound along with succinate; and in a third, the iron cofac-
tor was absent (fig. S1). However, this initial study indi-
cated that VioC crystals routinely diffract to high resolu-
tion (< 1.8 Å) and might therefore be amenable to crystal-
lographic characterization of intermediates in the reac-
tion cycle. Reproduction of the previously reported crys-
tallization conditions but with O2 excluded and 2OG re-
placing tartrate or succinate as the precipitant yielded a
1.6 Å resolution structure of the complete O2-reactive re-
actant complex with highly reproducible crystallization
and x-ray diffraction (Fig. 2A) (Supporting Information).
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A few datasets, however, yielded a different and surpris-
ing result. In these structures, the electron density map is
consistent with the presence of the L-Arg substrate; no
electron density for the 3-OH group of the product is ap-
parent. New electron density bridging the iron cofactor
and the organic cosubstrate is inconsistent with the tar-
geted ferryl-succinate complex that accumulates in solu-
tion (Fig. 2C), nor could this density be modeled as an
FeII-succinate adduct or the photoreduced and protonat-
ed [FeII-O(H)x] form of the ferryl complex (fig. S2). At-
tempts to do so consistently yielded a large and elongated
peak of positive Fo-Fc difference electron density between
the metal and cosubstrate that is not fully accounted for
by these models (fig. S2). By contrast, a model of a com-
putationally-predicted precursor,25-26 in which the C1 car-
boxylate of 2OG has departed and an O2-derived peroxide
adduct bridges the metal and the succinate coproduct
with the O-O bond still intact (Fig. 2C), fits remarkably
well into the observed electron density. This complex is
one of two postulated ferryl precursors that have yet to be
detected (Fig. 1) by any experimental method. The other
option is a bicyclic FeIV-peroxyhemiketal complex27 with
the C1 carboxylate intact (Fig. 1) but this structure is clear-
ly too large to fit into the observed density. Refinement of
a model of the FeII-peroxysuccinate intermediate against
the electron density from these datasets gave Fe-O-O
bond distances and angles that agree with computational
predictions (fig. S3).
The observation that multiple sidechain motions asso-
ciated with conversion of the FeII•2OG•L-Arg reactant
complex to the FeII•succinate•3-S-OH-L-Arg product
complex (vide infra) have already occurred in the putative
intermediate state lends further credence to its assign-
ment as an on-pathway FeII-peroxysuccinate complex.
Prior to O2 exposure, the FeII center in VioC exhibits a
distorted 5-coordinate geometry in which the C1 carbox-
ylate of 2OG is shifted ~35° out of the equatorial plane
defined by His168, Glu170, and the 2OG ligand (figs. S4-S6).
C3 of L-Arg is positioned 4.8 Å from the cofactor, a dis-
tance suitable for HAT, and clear electron density is visi-
ble for all protein side chains in the vicinity of the active
site with the notable exceptions of Arg334 and Gln137 (fig.
S4). These side chains are best modeled as rotated away
from the active site to avoid clash with C1 of 2OG, but
Figure 1. Proposed mechanism of Fe/2OG hydroxylases.
With the goal of accumulating the H• abstracting ferryl
intermediate, crystals of the reactant complex prepared
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Figure 2. Crystal structures of VioC with L-Arg and 2OG (A), 3-OH-L-Arg and 2OG (B), L-Arg and peroxysuccinate (C), 3-OH-L-
Arg and succinate (D), L-Arg and succinate (E), and the photoreduced vanadyl complex (F). A 2Fo-Fc composite electron density
map is shown in black mesh and contoured to 1.5 σ. Selected amino acids and active site components are shown in stick format.
Iron (orange) and vanadium (teal) ions are shown as space-filling models.
torts significantly to enable coordination of the hydroxyl
group, and the guanidinium moiety rotates, accommo-
occupancy refinement (~33% for the terminal heteroa-
dated by accompanying protein side chain shifts within
toms) suggests that the amino acids are partially disor-
the L-Arg binding pocket. The -NH3+ group of L-Arg
dered in the reactant complex.
moves away from the Glu170 ligand to interact with Gln137,
To obtain the product complex via reaction in single
which can now be modeled unequivocally because it be-
crystals, we exposed VioC co-crystallized with 2OG to O2
comes well-ordered upon conversion of substrate to
for a longer period of time (~4 hours) in crystallization
product. Furthermore, the Glu170 carboxylate rotates to-
solution containing > 1.0 M succinate as the precipitant.
ward the sidechain of Arg334, which also becomes ordered
Electron density maps obtained from these crystals reveal
as it moves into the space vacated by C1 of 2OG to anchor
a new oxygen substituent at C3 of L-Arg and succinate
an H-bonding network involving the L-Arg carboxylate
bound in the co-substrate site (Fig. 2D). The coordination
and succinate O1 (Fig. 3). The motions of these residues
of the new oxygen of the 3-S-OH-L-Arg product to the FeII
are enabled by the additional space in the active site
cofactor trans to His316 suggests that it is not protonated.
made available by departure of CO2. The observations
The presence of the alkoxide is somewhat surprising, giv-
indicate that the coordination mode of 2OG and the posi-
en the high pKa of the hydroxyl group (~ 17). The observa-
tions of Arg334 and Gln137 are markedly different in the
tion may, however, be consistent with proposals of initial
reactant and product states.
rebound of the deprotonated oxo unit28 and could have
important implications for bifunctional Fe/2OG enzymes
Arg/succinate VioC complexes also corroborate this mod-
that first hydroxylate their substrates and then effect dis-
X-ray structures of the 3-OH-L-Arg/2OG and L-
el for second sphere side chain motions associated with
2OG decarboxylation in progression to intermediate
tinct transformations of the alcohol (or alkoxide) inter-
mediates.8
states (Figs. 2B and 2E). In these structures, Arg334 re-
The product complex shows that, upon cleavage of the
mains ordered and in the inward-facing position, an-
chored by interaction with the product 3-OH group
and/or O2 of 2OG/succinate. The 2OG coordination
mode in the 3-OH-L-Arg/2OG complex is distinct from
C1-C2 bond of 2OG, the resulting succinate ligand relaxes
into the equatorial plane perpendicular to the Fe-His168
axis, yielding a more idealized octahedral coordination
geometry. The side chain of the 3-OH-L-Arg product con-
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that of the reactant complex: the C1 carboxylate is signifi- hyperfine couplings to nearby nuclei using hyperfine
cantly displaced to accommodate Arg334, a binding mode
that is more reminiscent of the structures with succinate
bound and is probably driven by coordination of the
product C3 oxygen (fig. S5A).
sublevel correlation (HYSCORE) spectroscopy. Field-
dependent H-HYSCORE spectra collected in the pres-
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ence of 3,3-d2-arginine can be simulated (Fig. 4A) to fur-
nish the disposition of the target hydrogen atom relative
to the vanadyl (fig. S8). Features of the spectra arising
from deuterium (fig. S9) can be simulated by a single axial
hyperfine coupling tensor with magnitude A = 0.33 ± 0.04
MHz (Euler angles [0, 37, 217] ± 10°) and quadrupole cou-
pling [-0.06, -0.06, 0.12] ± 0.01 MHz (Euler angles [0, 70,
30] ± 10°). The hyperfine coupling can be interpreted ac-
cording to a point-dipole model, indicating the presence
of a deuterium nucleus 3.3 ± 0.2 Å from the vanadium
nucleus, at an angle of 37 ± 10° relative to the V-oxo bond
(Fig. 4B).
In the structure of the posited L-Arg/peroxysuccinate
complex, Arg334 and Gln137 are both well-ordered and
found in their product-state conformations (fig. S4). This
observation provides support for the assignment of the
structure as a new intermediate and not simply a mixture
of reactant and product states. In the latter scenario, in-
creased disorder of the Arg334 and Gln137 side chains, mani-
fested as weaker electron density, would be anticipated.
We additionally ruled out the possibility of a mixture by
modeling and refinement of 2OG and the 3-OH-L-Arg
product into the observed electron density in the inter-
mediate structure (figs. S2, S6), yielding negative differ-
ence density for the 3-OH group and weak density for the
C1 2OG carboxylate. These observations are consistent
with a state that accumulates after loss of C1 as CO2 but
prior to substrate hydroxylation. Early movement of
Arg334 and Gln137 may also explain the surprising accumu-
lation of the peroxysuccinate complex in VioC crystals.
Full ordering of the side chains and/or formation of H-
bonds and other n0n-covalent interactions with oxygen-
derived ligand may be important for progression to the
ferryl-succinate complex and these steps could be dispro-
portionately impeded in the crystal lattice. Our crystallo-
graphic observation of the peroxysuccinate intermediate
implies mechanistic relevance of this state,29-30 here as a
transient on-pathway intermediate to the substrate-
targeting ferryl. The structure bears resemblance to a
metastable acylperoxo-FeIII intermediate that forms upon
incubation of biomimetic mononuclear FeII complexes
with peracetic acid.31-33 In those systems, the acylperoxide
ligand undergoes reversible O-O bond cleavage to yield a
C-H oxidizing FeV(O) species. It is also interesting to con-
sider the possibility that the FeII-peroxysuccinate complex
could undergo alternative (non-ferryl-generating) trans-
formations in other Fe/2OG enzymes, which would serve
to significantly expand the catalytic diversity in the super-
family.
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Figure 3. An H-bonding network involving Arg334 orients the
oxygen-derived ligand in the FeII-peroxysuccinate complex
(A) and vanadyl-bound state of VioC (C). An octahedral base
geometry in each state (B, D) leaves a second site open trans
to His168. Selected bonding interactions are indicated with
distances listed in angstroms (Å) and amino acid side chains
and substrate molecules are shown in stick format. Hydrogen
bonding interactions and weak coordination bonds are illus-
trated as black dashed lines. Metal ions and oxygen adducts
are shown as space-filling models.
To understand the macromolecular context of the key
H• abstracting intermediate, we implemented a structural
mimic of the ferryl state, in which the transition metal
component was exchanged for vanadium.34 High-valent
Fe centers in enzymes are notoriously fleeting and highly
reactive,24 but early transition metal oxycations are
known to be significantly more stable.34-35 The vanadyl ion
(VIV-oxo) has been shown to occupy divalent cation bind-
ing sites in proteins, and, in a recent study, to mimic the
H•-abstracting ferryl intermediate (FeIV=O) in Fe/2OG
enzymes.36 Vanadium(IV) is a d1 ion with a ground-state
total electron spin quantum number (S) of 1/2, and the
VioC•vanadyl•succinate•L-Arg complex therefore exhibits
an electron paramagnetic resonance (EPR) spectrum with
octet splitting due to hyperfine coupling with the 51V nu-
cleus (100% natural abundance, I = 7/2; fig. S7). Moreover,
structural information can be extracted by examining
To obtain global structural information for this ferryl-
mimicking complex, a 1.55-Å resolution structure was
solved from crystals obtained after incubation of VioC
with vanadyl ion, L-Arg, and succinate. Clear electron
density is observed for all components of the active site
(Fig. 2F), and the high degree of order is both notable and
consistent with significant stabilization of the ferryl in-
termediate by VioC. The V-oxo bond distance refines to
1.87 Å, noticeably longer than would be expected from the
values of 1.58-1.63 Å reported for inorganic models.37 This
deviation indicates that, although the crystals formed and
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Figure 4. (A) Field-dependent HYSCORE spectra (middle) of vanadyl-VioC collected in the presence of 3,3-d2-L-Arg, col-
lected at 282.0 (i), 297.5 (ii), and 342.5 (iii) mT and corresponding simulations (bottom). Orientation selectivities that
were probed by each magnetic field position (top) are color-coded using an RGB color scheme (red - fully excited; blue,
not excited). Spectra were collected with microwave frequency 9.46 GHz and temperature 35 K. (B) Comparison of crys-
tallographic (black) and HYSCORE (red) models of the C-H ꢀ V-oxo angle and distance.
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were frozen with VIV-oxo at the cofactor site, exposure to
x-rays during data collection resulted in photoreduction
of the vanadium to an oxidation state ƙ +III. Overlay of
the first coordination sphere with geometry-optimized
models of the VioC and TauD ferryl complexes,38 obtained
by density functional theory calculations, shows that the
remaining coordinating atoms can be superimposed al-
most exactly (fig. S10). The VioC DFT model was validated
by calculation of the ferryl Mössbauer parameters, which
resulted in excellent agreement with those obtained ex-
perimentally on a sample enriched in the intermediate
complex by rapid-freeze quench methods (fig. S11) [0.29
(exp) and 0.24 (calc) mm/s for the isomer shift and ±0.96
(exp) and -0.95 (calc) mm/s for ∆EQ]. In sum, the analysis
indicates that elongation of the metal-oxo bond, which is
unrestrained by covalent bonds to the protein or sub-
strate/product, is likely to be the only major structural
perturbation resulting from x-ray irradiation at 100 K.
Therefore, we conclude that the photoreduced vanadyl-
VioC structure is a valid and stable structural mimic of
the ferryl state, as previously proposed.34
the HAT step (if it were the actual ferryl complex), as an-
ticipated from the stereochemistry of the observed prod-
uct and consistent with the C3-H…VIV-oxo distance and
angle measurements obtained in the HYSCORE analysis
(Fig. 4B). The metal ion can be viewed as six-coordinate
(Fig. 3D) but is severely distorted from idealized octahe-
dral symmetry toward a more nearly square-pyramidal
geometry. Five of the metal-ligand distances are normal
for such complexes (1.9-2.3 Å), and there is clear continu-
ous electron density along the inter-nuclear axes of these
bonds (Fig. 2F). By contrast, the sixth site appears open,
because O2 of succinate is positioned > 2.3 Å from the
vanadium, and there is a distinct break in the electron
density along the bond axis. As in the peroxysuccinate
structure, the succinate coproduct and Arg334/Gln137 side
chains in the vanadium complex are in their product con-
formations. This arrangement locates the terminal nitro-
gen atoms of Arg334 within 3.1 Å of both the O2 atom of
succinate and the vanadyl (derived) oxygen. The proximi-
ty of a competing H-bond donor to the coproduct oppo-
site the vanadium ion could be responsible for the weaker
coordination by succinate O2. Although the marked dis-
tortion from idealized octahedral symmetry may result, at
least in part, from photoreduction of the vanadyl com-
plex, it nevertheless highlights the potential vacancy of a
symmetry-equivalent coordination site, cis to the ob-
served oxo position and trans to the other His ligand
(His168), that H-atom abstracting ligand could potentially
occupy. The structures of the vanadyl and peroxysuccin-
ate intermediates suggest a key role for outer sphere resi-
due Arg334 in stabilization of Fe-O adducts in certain co-
ordination positions. Direct interaction between the O-O
unit of the FeII-peroxysuccinate complex and the side
In the structure of the vanadium VioC complex with
substrate, the V-O vector undergoes a minor shift of ~25°
from its location in the peroxysuccinate complex such
that it is now ~180° away from His316 (Fig 3B, 3D). Of the
two atoms ultimately derived from dioxygen, the O2 atom
of succinate undergoes a much more significant confor-
mational change, rotating 33° and moving ~2 Å after O-O
bond cleavage. These observations suggest that such mo-
tions may represent the largest activation barrier in the
crystal lattice. The vanadium oxo ligand is positioned ap-
propriately to accept the C3 pro-S hydrogen of L-Arg in
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chain of Arg334 in its newly ordered position suggests a
CONCLUSIONS
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1
mechanistic role for the second-sphere residue, perhaps
in favoring the different charge distribution of the nas-
cent peroxide adduct in order to promote decarboxylation
and C2–O-bond formation. In Fe/2OG halogenases, se-
cond sphere H-bonding to the oxo ligand has been impli-
cated in stabilization of alternative ferryl geometries, in-
ferred from computational modeling of the active site.18-19
The VioC structure with the vanadium mimic offers the
opportunity to fully map all of the H-bonding interactions
in the active site immediately prior to the HAT step (figs.
S12-S14), including the bonds between active site side
chains and the oxo ligand. Surprisingly, this analysis re-
veals that Arg334 does not H-bond directly to the vanadi-
um oxygen ligand. It is not clear if H-bond stabilization of
the oxo is necessary in hydroxylation reactions but, in
VioC, the -ammonium group of the L-Arg substrate is
instead optimally oriented for this role. The likely H-
bonding partners for the Arg334 side chain are the succin-
ate O2 atom and the L-Arg carboxylate, although the lat-
ter interaction indirectly stabilize the oxo via H-bonding
by control of substrate position. In VioC, Arg334 therefore
promotes an inline oxo configuration via a combination of
electrostatic interactions, steric block of ligand dynamics,
and maintenance of an extended active site H-bonding
network.
The structures reported here provide a complete and
fully contextualized view of the reaction cycle in Fe/2OG-
dependent oxygenase VioC. The study reveals a previously
unobserved FeII-peroxysuccinate intermediate and an x-
ray structure of a stable vanadyl-substituted enzyme-
substrate complex, representative of the substrate-
targeting ferryl intermediate that accumulates in solution.
The vanadyl probe is a viable crystallographic tool that
may be applied more generally to other enzymes that de-
ploy high-valent iron species in order to map the struc-
tural changes that accompany intermediate formation
and control its reactivity. The structures of VioC at inter-
mediate states in the reaction illustrate how second-
sphere residues in the active site might control geometric
configuration of ligands at key points, providing new
routes to enzyme engineering and functional prediction
in the larger Fe/2OG enzyme superfamily.
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ASSOCIATED CONTENT
Supporting Information
Experimental section, SI figures S1-S17, and tables S1-S4.
The Supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
Analysis of the available Fe/2OG oxygenase structures
(Table S1) reveals that an Arg is often conserved near the
active site, consistent with a general role in directing the
location of the oxygen ligand. The residue can be mapped
to the last beta strand in the core fold and it is located 4-6
residues C-terminal to another strictly conserved Arg im-
plicated in ionic bonding with the C5 carboxylate of
2OG/succinate (fig. S15A). In most structures, the side
chain resides in the second sphere and is positioned simi-
larly to its counterpart in VioC. In a minority of cases, the
site is occupied instead by a hydrophobic amino acid (fig.
S15B). Strikingly, these enzymes often catalyze transfor-
mations other than hydroxylation. A recent structure of
an Fe/2OG chlorinase, WelO5, which harbors a Phe at the
site occupied by Arg334 in VioC, suggests use of a geomet-
rically distinct ferryl located in the equatorial plane to
achieve selective halogenation in this system.19 For this
reason, the coordination geometry observed in the VioC
vanadium complex, in which a site cis to the present loca-
tion of the oxo is accessible for its possible migration dur-
ing or after ferryl formation and controlled by interaction
of the ligand (or lack thereof) with outer-sphere residues,
is intriguing. Notably, the amino acid hydroxylase, SadA,
Supporting information (PDF)
AUTHOR INFORMATION
Corresponding Author
* Correspondence to: akb20@psu.edu; ckrebs@psu.edu;
jmb21@psu.edu.
Present Addresses
†North Carolina State University, Department of Chemistry,
Raleigh NC 27695.
Author Contributions
‡These authors contributed equally.
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health
(GM119707 to A.K.B. and GM113106 to J.M.B. and C.K.), a Na-
tional Science Foundation Graduate Research Fellowship
Program (Grant No. DGE1255832) to R.J.M, and an NIH Na-
tional Research Service Award to C.J.P. (GM113389). We
gratefully acknowledge the resources of the Advanced Pho-
ton 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 No. 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).
GM/CA@APS has been funded in whole or in part with Fed-
eral funds from the National Cancer Institute (ACB-12002)
and the National Institute of General Medical Sciences
(AGM-12006). The Eiger 16M detector was funded by an
which has Phe at the position equivalent to Arg334 in VioC,
39
can be modified to halogenate its substrate,
demon-
strating successful reprogramming of function via site-
directed mutagenesis in an Fe/2OG enzyme. Family-wide
bioinformatic analysis for Arg334 substitutions could be a
viable strategy to identification of enzyme platforms that
enable different outcomes, natively or following modifica-
tion.
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