Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

Article abstract of DOI:10.1073/PNAS.2005327117

The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD⁺) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolved in crystallo reactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-? resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-? resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.

Full text of DOI:10.1073/PNAS.2005327117

Observing 3-hydroxyanthranilate-3,4-dioxygenase in
action through a crystalline lens
a,1
b,1
a
a
a,2
Yifan Wang , Kathy Fange Liu , Yu Yang , Ian Davis , and Aimin Liu

a
b
Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249; and Department of Biochemistry and Biophysics, Perelman
School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
Edited by John T. Groves, Princeton University, Princeton, NJ, and approved July 7, 2020 (received for review March 20, 2020)
The synthesis of quinolinic acid from tryptophan is a critical step in the
de novo biosynthesis of nicotinamide adenine dinucleotide (NAD ) in
shared in these two pathways (Scheme 1). HAO from all sources
+
presents in a homodimeric form, and each subunit consists of a
Cupin structural fold (12, 13). The catalytic machinery is orches-
trated by a nonheme iron ion coordinated by a 2-His-1-Glu facial
triad (14), which is located in the center of a Cupin domain. Some
bacterial HAOs contain a noncatalytic, rubredoxin-like site on the
protein surface for iron acquisition and storage (15). In the solu-
tion state, HAO is an efficient catalyst with a reported turnover
rate of 25 s− (12, 16). The fast turnover rate is partially due to
mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-
dioxygenase responsible for quinolinic acid production was studied by
performing time-resolved in crystallo reactions monitored by UV-vis
microspectroscopy, electron paramagnetic resonance (EPR) spectros-
copy, and X-ray crystallography. Seven catalytic intermediates were
kinetically and structurally resolved in the crystalline state, and each
accompanies protein conformational changes at the active site. Among
them, a monooxygenated, seven-membered lactone intermediate as a
monodentate ligand of the iron center at 1.59-Å resolution was cap-
tured, which presumably corresponds to a substrate-based radical spe-
cies observed by EPR using a slurry of small-sized single crystals. Other
structural snapshots determined at around 2.0-Å resolution include
monodentate and subsequently bidentate coordinated substrate,
superoxo, alkylperoxo, and two metal-bound enol tautomers of the
unstable dioxygenase product. These results reveal a detailed stepwise
O-atom transfer dioxygenase mechanism along with potential isomer-
ization activity that fine-tunes product profiling and affects the produc-
tion of quinolinic acid at a junction of the metabolic pathway.
1
substrate-induced loop dynamics that help to increase O con-
2
centration in the hydrophilic active site by making it more hy-
drophobic to accommodate the sequential binding of 3-HAA and
2
O , two substrates with disparate polarities, at the catalytic iron
center (16).
HAO has long been recognized as a potential drug target (17–22).
The tryptophan–kynurenine pathway produces a series of neuro-
logically active compounds, such as 3-HAA, 3-hydroxykynurenine,
kynurenic acid, and QUIN. The latter two are the only known en-
dogenous metabolites capable of modulating the activity of the
N-methyl-D-aspartate (NMDA) receptors, which are the predomi-
nant molecular devices responsible for controlling synaptic plasticity
and memory function (23–25). QUIN is also an agonist of NMDA
receptors. When the QUIN concentration exceeds basal levels,
neurons in the brain become overexcited, and apoptosis may follow,
thereby creating undesired biological consequences (18, 25, 26). The
metalloprotein mechanistic enzymology
| oxygen activation | kynurenine
|
pathway NAD biosynthesis
|
+
icotinamide adenine dinucleotide (NAD ) is an essential
+
N
and ubiquitous metabolite known for its role in transferring
kynurenine pathway also synthesizes a significant portion of NAD
+
electrons throughout metabolic networks. NAD is produced via
de novo biosynthetic pathways, or by salvage and recycling routes
Significance
(
1). All kingdoms of life use quinolinic acid (QUIN) as a uni-
+
versal precursor for the de novo biosynthesis of NAD . In
mammals, QUIN is synthesized from the tryptophan metabolite
+
NAD plays a critical role in redox-linked biological reactions as
a cofactor or substrate. The knowledge of its de novo biosyn-
thesis in mammals and certain bacteria is incomplete due to
missing information of a nonheme iron dioxygenase mecha-
nism and the conformation of its product that nonenzymati-
3
-hydroxyanthranilic acid (3-HAA) (Scheme 1). The production
of QUIN from 3-HAA involves O activation and aromatic ring
2
cleavage of the C3–C4 bond by 3-hydroxyanthranilate-3,4-diox-
ygenase (HAO). Dioxygenation of 3-HAA generates an unstable
acyclic product, 2-amino-3-carboxymuconate-6-semialdehyde
+
cally produces the universal NAD precursor, quinolinic acid. To
date, how the enzyme regulates the nonenzymatic product
remains unknown. This work fills the gap by combining single-
crystal spectroscopic and X-ray crystallographic studies. The
results provide a comprehensive view of the dioxygenase
mechanism by enabling step-by-step visualization of the cata-
lytic cycle and the protein dynamics during catalysis. Addi-
tionally, how the enzyme regulates the pathway product
distributions, including the nonenzymatic product of biologi-
cally significant compound, is proposed.
(
ACMS) (2), which either metabolizes to acetoacetate through a
series of enzymatic reactions, or nonenzymatically (3) auto-
cyclizes to produce QUIN with the release of a water molecule
+
(Fig. 1A). The biosynthesis of NAD from QUIN is accomplished
firstly through a condensation reaction with phosphoribosyl py-
rophosphate (PRPP), creating nicotinic acid mononucleotide and
subsequently converting to nicotinic acid dinucleotide. Next,
amidation of nicotinic acid dinucleotide results in the synthesis of
nicotinamide mononucleotide, which is further converted to
+
NAD (SI Appendix, Fig. S1).
Author contributions: Y.W., K.F.L., and A.L. designed research; Y.W., K.F.L., Y.Y., and I.D.
performed research; Y.W., K.F.L., Y.Y., I.D., and A.L. analyzed data; and Y.W., K.F.L., I.D.,
and A.L. wrote the paper.
Unlike eukaryotes, prokaryotes primarily synthesize QUIN
from dihydroxyacetone phosphate and L-aspartate (4, 5). The
L-tryptophan–kynurenine pathway, however, is also found in a few
bacteria (6, 7), and vice versa, some eukaryotes are known to use
the prokaryotic pathway (8–10). Moreover, it is also known that
the majority of the kynurenine pathway has been utilized by some
prokaryotes to live on the toxic industrial waste, 2-nitrobenzoic
acid (2-NBA) as the sole source of carbon, nitrogen, and energy
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
1
Y.W. and K.F.L. contributed equally to this work.
2
To whom correspondence may be addressed. Email: feradical@utsa.edu.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2005327117/-/DCSupplemental.
(
11). The kynurenine pathway and the 2-NBA biodegradation
pathway unite at 3-HAA, where HAO is the first common enzyme
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Scheme 1. The L-tryptophan kynurenine pathway and the bacterial 2-nitrobenzoic acid (2-NBA) biodegradation pathway converge at the aromatic me-
tabolite 3-hydroxyanthranilic acid (3-HAA). The nonheme iron-dependent enzyme, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAO), sits at the critical
+
junction that directs most of the metabolic flux to the enzyme-controlled ketogenic pathway, which branches off for de novo biosynthesis of NAD via
quinolinic acid (QUIN). The precise conformation of the ring-cleaved product of the HAO reaction, 2-amino-3-carboxymuconic semialdehyde (ACMS), is critical
for determining the partitioning of the metabolite flux at the pathway junction for enzymatic and nonenzymatic routes.
in critical tissues such as the brain and heart (23). Despite its bio-
logical significance and the length of time it has been known, the
regulatory mechanisms of QUIN production through the kynurenine
pathway have remained elusive.
It has been demonstrated that embedding an enzyme in a
crystalline lattice could dramatically alter reaction kinetics, in-
cluding extradiol dioxygenases and other enzymes related to
HAO in the same metabolic pathway (29–33). Here, we first
tested the catalytic competency of single crystals of HAO using a
ACMS is the proposed enzymatic product of HAO, which
occupies a central position in both pathways (Scheme 1). How-
ever, this transient metabolite has not been characterized.
Presently, the exact conformation of ACMS produced by the
HAO-mediated enzymatic reaction remains unknown due to its
short lifetime (2) and a large number of possible conformers.
ACMS has 32 potential conformers based on the combinations
of distinct tautomers, isomers, and rotamers (SI Appendix, Fig.
S2). On the basis of a synthetic modeling study, ACMS is
expected to be a 2Z,4E keto tautomer in solution, which un-
dergoes facile isomerization about the C2–C3 and C4–C5 double
bonds via transient formation of its enol tautomer to form QUIN
by an electrocyclization (3). Although HAO has been studied for
decades, the lack of solution-state intermediates renders the
catalytic mechanism challenging to study. How HAO balances
the catalytic activity of its downstream biomachinery and non-
enzymatic QUIN production remains an unanswered question.
To identify its catalytic intermediates and interrogate the
mechanism of HAO, as well as understand how HAO contrib-
utes to the regulation of product partitioning at the ACMS
junction, we describe an extensive effort to use the effects of
protein in the crystalline state to slow the reaction of HAO-
mediated catalysis, which becomes critical to understand the
mechanism used for fine-tuning production of QUIN.
benchtop spectrophotometer. Single crystals suspended in an O -
2
saturated crystallization mother liquor with cryoprotectant in a
cuvette containing 3-HAA were able to degrade the substrate.
We spectroscopically followed the conversion of 3-HAA to the
intermediate product ACMS, which then nonenzymatically
decayed to QUIN (Fig. 1C). After reacting for 70 min, all in-
cubated crystals were found intact by microscopy. Due to the
missing knowledge of the anticipated slower diffusion rate in the
crystalline lattice for both substrate and product, a detailed ki-
netic analysis is not warranted for the reaction in crystallo under
this condition.
Next, we conducted a time-dependent, single-crystal UV-vis
microspectroscopic experiment to detect whether any interme-
diate would accumulate during the catalytic reaction. Snapshots
of crystals flash cooled to 77 K at different time points of reac-
tion and examined at 100 K confirmed their catalytic competency
(Fig. 1D). Substrate-bound HAO crystals exhibited a λmax at 327 nm.
Crystals reacted for 2, 3, 5, 7, and 9 min or longer exhibited
optical features distinct from those of the enzyme–substrate
complex, suggesting specific intermediates populating at these
time points. The considerably slower reaction speed in crystals
provides a promising platform to investigate reaction interme-
diates and the enzyme-bound product throughout the HAO-
mediated reaction. This set of experiments indicates that the
catalytic rate constant is over 10,000-fold slower than that in
solution (25 s− ) (12, 16).
Results
1
HAO Is Catalytically Active in the Crystalline State with Significantly
Altered Kinetics. Stopped-flow UV-vis spectroscopy was used to
monitor the HAO reaction in the presence of excess O pro-
A Free-Radical Intermediate Populated and Characterized by
2
duced by the oxygen-evolving enzyme chlorite dismutase (27).
The production of ACMS was followed at 360 nm, which has an
Single-Crystal Electron Paramagnetic Resonance Spectroscopy.
Next, intermediates were probed for the presence of a radical
state in pre–steady-state conditions (Fig. 2A). A slurry of ap-
proximately 1,000 small-sized HAO single crystals was prepared
in crystallization mother liquor with cryoprotectant where O2
extinction coefficient (47,500 M− ·cm ) over one order of
magnitude greater than that of 3-HAA at 320 nm (2). The time
course of ACMS formation could be described by fitting with a
1
−1
double-exponential function with apparent rate constants of was limited. It is worth mentioning that the term “single crystal”
1
3
8.87 and 5.07 s− (Fig. 1B). We noticed no spectral features
refers to a monocrystalline state to differentiate from the
amorphous or polycrystalline state of crystals. HAO microcrys-
tals alone had no spectroscopic features at the g = 2 region at 70
K. When substrate (5 mM) was introduced aerobically at room
temperature, and freeze-quenched in liquid ethane at different
times, a g = 2.005 signal with a linewidth of 1.15 mT was ob-
served after incubation with 3-HAA for 30 s. The observed
linewidth is presumably generated from a conjugated system
(34–36) and is inconsistent with a superoxo anion radical (37).
The sample was then thawed, aged for another 30 s, and rapidly
indicative of an intermediate in the difference spectra. One
possibility is that an intermediate is present, but it does not
exhibit a distinct absorbance at most wavelengths. Another
possibility is that no catalytic intermediates populate in the
millisecond or longer time window in the aqueous phase. The
two summed exponential fitting is most likely required due to
the dimeric nature of HAO with inequivalent iron centers, as
found in the first enzyme of the pathway, a homotetramer of a
heme-based dioxygenase (28).
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Fig. 1. Crystalline state of HAO alters the reaction rate and populates catalytic intermediates that are kinetically unresolved in the solution state. (A) The
chemical reaction catalyzed by HAO and the nonenzymatic cyclization. (B) Reaction of anaerobically premixed enzyme–substrate complex (0.10 mM) and O
5 mM, generated by chlorite dismutase) in solution monitored by stopped-flow photodiode array spectroscopy. The Inset shows the time course of ACMS
2
(
production monitored by the single-wavelength detector at 360 nm, fitted with the double-exponential function. (C) The catalytic activity of the HAO single
crystals was monitored at room temperature by a benchtop spectrophotometer. Seven crystals were incubated in crystallization mother liquor (pH 8.5) with
the cryoprotectant described in Experimental Procedures, 3-HAA (0.3 mM), and O
2
(1.3 mM). (D) Representative absorption spectra of time-resolved in
crystallo reaction obtained from single-crystal UV-vis microspectroscopy. Each spectrum was collected from one single crystal at 100 K.
refrozen in liquid ethane to acquire a second spectrum, resulting
in the same signal with less intensity. The g = 2.005 radical sig-
nals continued decreasing over 50 min with multiple cycles of
thawing and freezing. The protein crystal electron paramagnetic
resonance (EPR) experiments suggest that a substrate-based
radical intermediate accumulates under pre–steady-state condi-
tions using the crystallized enzyme as a catalyst. Notably, the rate
of the reaction in crystallo is affected by the dimension and shape
of a crystal (38, 39). The maximal intensity of radical species at
ACMS, while the peak at 8.4 min had the same retention time
and absorbance spectrum (λmax at 294, 335 nm) as the 3-HAA
standard (Fig. 2C). These absorbance maxima were observed
under acidic HPLC conditions, which are distinct from those
observed at pH 8.5. We further characterized the eluted fractions
using high-resolution mass spectrometry (Fig. 2D). The results
confirmed that the 2.1-min fraction was the newly formed QUIN
(m/z = 168.0292, mass accuracy of 0.60 ppm), and the 8.4-min
fraction was the unreacted substrate 3-HAA (m/z = 154.0499,
mass accuracy of 1.95 ppm). Based on the integrated peak area,
0.15 mM QUIN was formed during the in-tube reaction with the
single-crystal slurry, which corresponds to approximately one
catalytic turnover.
3
0 s in microcrystals corresponds to a later time point of the
reaction with the larger-sized crystals used in the X-ray diffrac-
tion experiments.
After completion of the in-tube EPR catalytic reaction with
HAO single crystals, the crystals and aqueous solution were
separated by gentle centrifugation. Those crystals were dissolved,
resulting in a 200-μL aqueous solution with a protein concen-
tration of 185 μM. The aqueous portion of EPR sample was
subjected to high-performance liquid chromatography (HPLC)
for product analysis. As shown in Fig. 2B, two significant peaks
were separated by chromatography. The peak at 2.1 min had the
same retention time and absorption spectrum (λmax at 272 nm) as
a QUIN standard, the nonenzymatically cyclized product of
Observing Substrate Binding to the Catalytic Iron Via X-Ray
Crystallography. The slower turnover rate in crystals provides an
opportunity to probe the reaction cycle by X-ray crystallography
to trap and characterize catalytic intermediates that are other-
wise not populated in the solution reaction. To this end, we
performed time-resolved reactions in crystallo, first in the ab-
sence (for substrate binding) and then in the presence of O . The
2
II
substrate-free single crystals of Fe -HAO were grown in an
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Fig. 2. Single-crystal continuous-wave EPR spectroscopy of the HAO reaction with a crystal slurry reveals a substrate-based radical intermediate. (A) Time-
lapse in crystallo reaction at room temperature with samples analyzed by X-band EPR spectroscopy at 70 K. (B) HPLC–UV-vis analysis of the single-crystal
reaction mixture. Elution profiles are shown with the absorbance at 280 nm. (C) UV-vis spectra of the HPLC fractions eluted with solvent containing 2%
acetonitrile and 0.1% formic acid. (D) High-resolution mass spectrometry of the HPLC fractions. Peak 1 (blue) was identified as 3-HAA, and peak 2 (red)
was QUIN.
anaerobic chamber. A resting-state structure at 1.60-Å resolution
reveals two solvent-derived ligands completing a distorted octa-
hedral complex (Fig. 3A). To study substrate binding, HAO
crystals grown anaerobically were incubated in mother liquor
containing 3-HAA (1 mM). Turnover conditions were studied by
soaking crystals in air-saturated solutions with 1 mM 3-HAA. In
all cases, similarly sized crystals were used, and after various
incubation periods between 20 and 1,800 s, they were flash
cooled by submersion in liquid nitrogen. Representative high-
quality crystal structures of distinct reaction cycle intermediates
by hand-mixing 3-HAA with HAO crystals and flash cooling in
liquid nitrogen. In this E•S structure refined to 1.90-Å resolution
(Protein Data Bank [PDB] entry: 6VI6), 3-HAA is a mono-
dentate ligand with its C3-hydroxyl moiety bound to the catalytic
II
iron (Fig. 3B). The bond distance between Fe and C3-hydroxyl
II
group is 2.3 Å, while the C2-amino group is 3.2 Å from the Fe
ion (SI Appendix, Table S2). The previously observed loop
movements that enhance the hydrophobicity of the Fe center
2
and facilitate O binding upon bidentate chelation of the sub-
strate are not observed in this intermediate (16). Only one of
three loops composed of residues 139 to 148 partially shift to the
previously defined “closed” form (Fig. 3D and Movie S1). Thus,
this is a previously unobserved E•S structure. We interpret this
state as a transient enzyme–substrate intermediate and abbrevi-
(
≥80% ligand occupancy) are presented (for details, see SI Ap-
pendix, Table S1). The optimal time points were determined by
screening several thousand crystals, from which hundreds of
datasets were collected with good reproducibility. Although the
time points obtained from UV-vis absorbance microspectroscopy
and X-ray diffraction are independent of each other, the catalytic
turnover rate matches well because crystals of similar size were
used. For this reason, single-crystal UV-vis microscopy presum-
ably features the structurally defined intermediates populated at
the same reaction times.
I
ate it as (E•S) . The capture of this intermediate provides in-
formation as to how 3-HAA interacts with the catalytic Fe center
step-by-step at the initial stages of catalysis. Anaerobic incuba-
tion of 3-HAA for 30 s or longer yielded the expected bidentate
E•S complex structure (Fig. 3C), which was originally reported
at 3.20-Å resolution (12), and now updated to 2.61-Å resolution
(PDB entry: 6VI7). The bidentate structure is referred to as the
(E•S)II intermediate, and the reported loop movements relative
Under anaerobic conditions, the earliest enzyme–substrate
complex (E•S) structure was obtained after approximately 20 s
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Fig. 3. A two-step 3-HAA binding process. (A) The ligand-free structure in resting enzyme, (B) the transient (E•S)
-HAA, and (C) the equilibrium (E•S)II complex observed after incubation periods of 30 s with 3-HAA. The light blue 2Fobs – Fcalc maps and green omit Fobs
calc maps are contoured at 1.0 σ and 3.0 σ, respectively. Atom color code: gray, carbon (protein residues); yellow, carbon (ligand); blue, nitrogen; red, oxygen;
intermediate exhibits
I
intermediate after ∼20-s exposure to
3
–
F
magenta, iron. (D) The crystal structure of the substrate-free enzyme is in a fully open form (blue), and the transient monodentate (E•S)
I
a partially closed conformation (pink), whereas the structure of the bidentate (E•S)II complex is in a fully closed conformation (red). Significant loop flexibility
is observed at three specific loop regions consisting of highly conserved residues 21 to 28 of loop A, 40 to 47 of loop B, and 139 to 148 of loop C.
to the ligand-free enzyme structure that increase the hydropho-
bicity of the iron center are observed, which, in turn, increases
the binding affinity of dioxygen (16) (Fig. 3D).
3-HAA, as well as the O in the ternary complex structure of
2
HAO with inhibitor 4-Cl-3-HAA (12) (SI Appendix, Fig. S4). The
2 I
electron density around the Fe center obtained with (E•S•O )
crystals cannot be modeled with the structures derived from
crystals incubated for longer time intervals (SI Appendix, Fig.
Oxygen Activation at the Nonheme Iron Center. The first oxygenated
intermediate structure (E•S•O ) was obtained with crystals
2
S3). Therefore, this first reactive intermediate after O exposure
2
I
is most consistent with the active-site iron of HAO coordinated
flash-cooled at 30 to 50 s after air exposure of the anaerobically
substrate-soaked crystals. At this time interval, we analyzed
multiple similar structures; the best X-ray diffraction dataset for
this intermediate was determined to 1.95-Å resolution (PDB
entry: 6VI8). Refinement against either the (E•S) or (E•S)
by 3-HAA and an O2 ligand (Fig. 4A). Starting from the
(
2 I
E•S•O ) , the aforementioned three loops adopt a partially
open conformation, and the active site is no longer in the fully
closed state (Movie S1).
I
II
2 I
In the (E•S•O ) intermediate, 3-HAA is a monodentate li-
yielded positive Fobs − Fcalc omit electron density in the antici-
pated O2 binding site adjacent to the metal center and the
gand, which binds the Fe ion with its hydroxyl group in the po-
sition opposite to His95. The O binds to the Fe in the position
2
3
3
-HAA molecule (SI Appendix, Fig. S3), indicating that only
-HAA and one oxygen (H O or OH) coordination cannot sat-
opposite Glu57 in an end-on manner. Compared to the bidentate
2
(E•S) complex, the C3-hydroxyl group of 3-HAA in the
II
isfy all of the electron density observed at the active site. The
positive density contoured at 3.0 σ is the same site occupied by
nitric oxide in the ternary complex structure of HAO with
(E•S•O ) structure is slightly rotated out of the plane of the
2
I
ring, moving toward Glu110. The O–O bond distance was refined
to 1.4 Å, which is consistent with a superoxo species (37). Depending
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Fig. 4. Dioxygen binding to the enzyme–substrate complex and O–O bond cleavage. (A) A ferric superoxo (E•S•O
2
)
I
or ferrous substrate radical (E•S•O
2
)II (30
to 50 s), (B) alkylperoxo (E•S•O
2
)III (1 min), and (C) seven-membered lactone (E•Int)II (3 to 5 min). The light blue 2Fobs – Fcalc maps and green Fobs – Fcalc omit
maps are contoured at 1.0 σ and 3.0 σ, respectively. Atom color code: gray, carbon (protein residues); yellow, carbon (ligand); blue, nitrogen; red, oxygen;
magenta, iron.
on the oxidation state of the Fe, this species can adopt two reso-
II complex for a
ferric superoxide anion or a ferrous-bound substrate radical, as amino group is in H-bond distance from the proximal iron-bound
previously proposed in the study of homoprotocatechuate 2,3-diox- oxygen atom, potentially indicating a role as an acid catalyst to
densities around the catalytic iron center (SI Appendix, Fig. S5).
In the structure of the (E•S•O III intermediate, the substrate
nance structures, either the (E•S•O
2
)
I
or (E•S•O
2
)
2
)
ygenase (HPCD) (37). Since the two resonance forms cannot be
structurally distinguished at this resolution, one or both species could
populate in the crystal. The out-of-plane rotation of the hydroxyl
group of 3-HAA indicates that the organic substrate is partially ac-
tivated for a superoxide attack.
facilitate the O–O bond cleavage.
Probing the Catalytic Mechanism by Viewing O–O Bond Cleavage and
O-Atom Transfer: Observation of a Monooxygenated, Seven-Membered
Lactone Intermediate. In the 3- to 5-min reaction time range, a high-
resolution intermediate structure was obtained and refined to
2
The next oxygenated intermediate (E•S•O )III was trapped in
crystals flash cooled 1 min after mixing, and the structure was
refined to 2.30-Å resolution (PDB entry: 6VI9). A notable dif-
1
.59-Å resolution (PDB entry: 6VIA). Structures obtained in this
time range show extended, nonplanar, excess electron density in
the active site, which is not amenable to fitting by 3-HAA or the
intermediates described above. The two anticipated candidates
from computational studies of aromatic dioxygenation of extradiol
ference between the (E•S•O
the latter structure exhibits a much closer distance between the
distal oxygen of the iron-bound O and the C3 of 3-HAA,
shortened from 3.1 to 1.8 Å (SI Appendix, Table S4), suggesting
the formation of a nascent C–O bond (Fig. 4B). (E•S•O
2
)I, II and (E•S•O
2
)III species is that
2
dioxygenases are an epoxide (40), labeled as (E•Int) , and a seven-
I
2
)
III is
membered lactone, labeled as (E•Int) (41–43). In modeling this
II
II
consistent with an Fe -alkylperoxo intermediate in which the
distal oxygen atom of O forms a covalent bond with the organic
intermediate, the seven-membered lactone (E•Int) provided
II
2
substantially better fit into the observed electron density than
substrate. In this structure, the C1-carboxyl and the C3-hydroxyl
groups of the substrate become more extensively puckered rel-
ative to the phenyl ring, suggesting a loss of aromaticity. Addi-
tionally, compared with the end-on binding mode in (E•S•O )
(E•Int) or any other intermediates (SI Appendix, Fig. S6).
I
(E•Int) and (E•Int) were proposed as resonance structures
II
III
with a low energy barrier for transition (42); however, the ob-
2
I
served electron density was better fitted by (E•Int) (Fig. 4C).
II
and (E•S•O ) , the iron–oxygen molecule geometry is more
Accordingly, the intermediate in (E•Int) coordinates the iron ion
2
II
II
bent, and the Fe–O–O angle decreases from 128.1° to 101.6°.
The refinement of models with 3-HAA and other reaction
pathway intermediates resulted in obvious negative and positive
in a monodentate fashion with its hydroxyl group at a distance of
2.3 Å to the Fe ion and 2.3 Å to Glu110. The proximal oxygen
III
from O remains bound to the Fe with an Fe –OH bond length
2
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of 2.2 Å. The lactone intermediate is stabilized by H-bonding in-
teractions between the newly incorporated oxygen and Glu110.
Visualizing the Formation of the Dioxygenase Product Enol Tautomers
and Isomerization. The subsequent step in the dioxygenation is
ring opening by the Fe-bound hydroxyl, attacking the C3 position
of the lactone intermediate (42). This step is thought to be an
energetically favorable step (44). We obtained the first product-
bound structure at 2.10-Å resolution (PDB entry: 6X11), after
aerobic soaking for 9 min (SI Appendix, Table S1). As described
Notably, Arg47 is H-bonded with the distal oxygen atom of the O
2
during catalysis until this step. Previous mutant studies have shown
that Arg47, along with Arg99 and Glu110, plays a critical role in
catalysis (12). The intermediate structures in Fig. 4 show that
Arg47 is crucial to stabilizing the bound oxygen and directing it
toward C3 until O–O bond cleavage. Asp49 forms a salt bridge
with Arg47, thereby limiting its free rotation. Additional views of
below, this intermediate, abbreviated as (E•P) , is best described
I
as a 3E,5Z,2t,4c-enol tautomer of ACMS (Fig. 5A). The dioxy-
genated product is a monodentate ligand attached to the iron ion
the seven-membered lactone intermediate with Fobs – Fcalc omit with its nascent carboxyl group. The electron density of (E•P) is
I
maps are shown in SI Appendix, Fig. S7. It is worth noting that this
distinct from all other intermediates described above (SI Ap-
seven-membered ring-containing intermediate was obtained from pendix, Fig. S8). The torsion angles of the ACMS ligand requires
multiple crystals with a resolution between 1.59 and 1.75 Å, sug- assignment of the C2–C3 and C4–C5 bonds as single, and the
trending of electron density reveals the best fit with a
E,5Z,2t,4c-enol tautomer (SI Appendix, Fig. S2). Fitting with
gesting that this is structurally the most accessible intermediate
during the catalytic reaction in crystallo.
The radical form of the lactone intermediate (E•Int)II is a
possible candidate for the species observed in single-crystal EPR
experiments. Fig. 2A shows that the maximal intensity of the free
3
keto tautomers of ACMS or bidentate enol tautomers could not
satisfy the density maps (SI Appendix, Fig. S8). The first oxygen
atom derived from dioxygen is 3.1 Å from the previous covalently
linked carbon in (E•Int)II (O2 and C3; SI Appendix, Table S3),
indicating the C–O bond is cleaved. However, the conformation
2
radical signal is observed at 30 s after O exposure in the EPR
experiments using microcrystals, while (E•Int) occurs at 3 to
II
I
of (E•P) retains some cyclic structural feature, suggesting an
5
min. However, the time difference correlates well with the
intermediate formed immediately following the ring opening
proposed lactone intermediate due to distinct crystal size utilized
in EPR and X-ray diffraction experiments. We noticed that the
smaller the protein crystal was, the faster the reaction took place
in crystallo when the reaction was monitored by an individual
single crystal of HAO under microspectroscope. A radical in-
from the lactone intermediate (E•Int) . This oxygenated inter-
II
mediate also inherits the monodentate coordination status found
in the previous lactone intermediate. These observations are
consistent with findings in other extradiol dioxygenases that the
newly formed carboxyl group binds to the iron in a monodentate
fashion with the products retaining a more cyclized conformation
III
termediate with an Fe -bound hydroxyl anion also explains the
(
29, 45, 46).
lower electron density observed at C3. Also, the possibility of
electron transfer between two resonance states may cause some
electron deficiency at the catalytic Fe center. Hence, the occu-
pancy for Fe and e-lactone was reduced to 0.8, resulting in a
better fit for the overall density. This high-resolution HAO in-
termediate structure provides unambiguous experimental evi-
dence for the involvement of an e-lactone intermediate in the
extradiol dioxygenase catalytic cycle.
The last intermediate characterized is also a product-bound
complex. A 1.84-Å resolution structure of enol tautomers of
ACMS with two distinct conformations in one crystal was
obtained (PDB entry: 6VIB) when the crystal reacted with sub-
strates for a longer time (9 to 12 min). All previous intermedi-
I
ates, including monodentate (E•P) , cannot satisfy the strong
electron density maps near the catalytic Fe center with a rea-
sonable fit (SI Appendix, Fig. S9). When the electron density was
Fig. 5. Visualizing the oxygenated ACMS product in two distinct enol tautomers bound to the catalytic iron ion in the monodentate and bidentate modes.
A) Monodentate product complex (E•P) (9 min) and (B) bidentate (E•P) and (E•P)II (9 to 12 min) product complexes in one structure. The light blue 2Fobs
calc maps and green Fobs – Fcalc omit maps are contoured at 1.0 σ and 3.0 σ, respectively. Atom color code: gray, carbon (protein residues); yellow, carbon
ligand); orange, carbon (3E,5Z,2t,4c-enol tautomer of ACMS); green, carbon (3E,5Z,2t,4t-enol tautomer); blue, nitrogen; red, oxygen; magenta, iron.
(
I
I
–
F
(
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modeled as the oxygenated product in this structure, the newly formed
carboxyl group no longer binds to the Fe ion in a monodentate
imine in the HAO-catalyzed reaction, which is likely due to the
absence of a supporting second coordination sphere residue.
fashion, as found in the previous (E•P) structure. Rather, the
The monooxygenated lactone intermediate, (E•Int) , results
I
II
product is in a bidentate metal-ion coordination state. Fitting
with a bidentate ligand of 3E,5Z,2t,4c-enol tautomer resulted in
positive density maps around the enol group (SI Appendix, Fig.
S8G), suggesting a facile rotation of the enol tail. Upon fitting
from heterolytic O–O bond cleavage from the alkylperoxo in-
termediate, followed by Criegee rearrangement. Notably, a gem
diol and an epoxide are anticipated prior to the formation of the
lactone intermediate, but they are not captured in the HAO
reaction. The gem diol intermediate has been captured in HPCD
and characterized by X-ray crystallography (53). The epoxide has
with both the (E•P)
I
and a conformation with a rotated enol tail,
the extra positive electron density was resolved (Fig. 5B). The
keto tautomers of ACMS were also examined for this intermedi- not yet been trapped and characterized. We have obtained a few
ate but, again, did not yield an acceptable fit for the electron structural datasets that appear to be best modeled for the an-
density. Therefore, not only a monodentate-to-bidentate coordi- ticipated epoxide intermediate. However, the resolution and li-
nation transition was found, but also a distinct conformer from gand occupancy do not meet the standards applied to the other
(
E•P) was also observed in this structure. This conformer is best intermediates. Thus, it remains inconclusive whether the proposed
I
described as 3E,5Z,2t,4t-enol tautomer of ACMS, abbreviated as epoxide is an intermediate in the extradiol dioxygenase pathway.
(
E•P)II. The final refinement gave the best fitting of (E•P)
I
and
Nevertheless, the capture of the lactone intermediate indicates
(
E•P)II with an occupancy ratio of 9:7. Chemically, ACMS has that dioxygen insertion by HAO takes place stepwise, rather than
four geometric isomers, each with its own enol tautomers. The concerted. Of note, HAO is an established drug target (17, 18),
conversion of these isomers was demonstrated in an NMR study
of an analog compound (3). Among these isomers and tauto-
mers, there are viable possibilities for multiple rotamers. Thus,
which has attracted significant attention due to its participation
in the tryptophan–kynurenine pathway, which plays a role in
exercise, inflammation, and mental health (54). Enzymes in this
the different conformational combinations lead to the 32 potential pathway are also checkpoint proteins regulating the immune re-
ACMS structures, which include the above two conformations sponse. Cancer cells overexpress this pathway to bypass immune
observed in crystal structures (SI Appendix, Fig. S2). From (E•P)
I
surveillance. The successful trapping and characterization of the
to (E•P)II, there is a single bond (C4–C5) rotation of 60° from 4- lactone intermediate may facilitate structure-aided drug design as
cis to 4-trans conformations. Notably, (E•P) represents a more
the e-lactone may be the most specific target for this dioxygenase.
A denticity change, from one to two, is observed in the product
II
linear product conformation compared to (E•P)
I
and corresponds
to an intermediate of the later catalytic stage. In the bidentate bound complex. Most of the products observed through in crys-
product, the enol group of (E•P) is stabilized by Glu110, while in tallo reaction in extradiol dioxygenases maintain a cyclized con-
I
(
E•P) , the oxygen interacts with Thr39 (Fig. 5).
formation (29, 45, 46), as seen in (E•P) of HAO. A linearized
II
I
product like (E•P)II is less commonly seen bound at the active
site in dioxygenases in the degradation of aromatic molecules
(40). Among the forms of ACMS, QUIN is produced via a
pericyclic reaction from the 3E,5Z,2c,4c form of the enol tau-
Discussion
The results presented here show that the HAO reaction occurs at
a much slower rate in crystallo than in the aqueous phase, leading
to a near-complete view of the HAO catalytic cycle (Fig. 6). The
catalytic pathway is complex, including the events of monodentate
and then bidentate 3-HAA coordination, binding and activation of
dioxygen, O–O bond cleavage, oxygen insertion, Criegee rear-
rangement causing ring expansion, a second oxygen transfer with
ring opening, and finally, an enzyme-assisted change of the product
conformation. These events are observed through a series of high-
quality crystal structures, six of which are not previously observed
intermediate structures of the HAO reaction.
I
tomer. In (E•P) , the iron-bound ACMS intermediate is ob-
served to be in its 3E,5Z,2t,4c conformation, which has easy
access to the QUIN forming reaction trajectory via one single
bond (2,3-bond) rotation (SI Appendix, Fig. S2). The 3E,5Z,2t,4t
state in (E•P)II creates a higher energy barrier to QUIN for-
mation. Glu110 and Thr39 may facilitate such a rotation.
Without the aid of the Fe ion and second sphere residues, the
4,5-bond rotation may not occur as readily. Thr39 becomes in-
volved in the product-bound complex only at the end of the re-
action pathway. Unlike other active-site residues presented in
the structural figures, Thr39 is not strictly conserved in type III
extradiol dioxygenases. Methionine is found in the same position
in some HAO during sequences alignment (16). This variation may
be derived from a need to tune the efficiency of the metal- and
active site residue-assisted conversion of the product conformation.
After release from the active site, the enol tautomer of ACMS
The observation of a monodentate (E•S)
I
followed by a
bidentate (E•S) substrate binding has previously been reported
II
in type I, but not type III, extradiol dioxygenases (47, 48). This
finding in a type III enzyme suggests that majority of extradiol
dioxygenases may employ a similar multistep substrate-binding
mechanism as first found in type I dioxygenases (49). The sub-
strate 3-HAA binds to the Fe center with a deprotonated hy-
droxyl group. An active site histidine in type I enzymes
deprotonates the catechol substrate and induces an asymmetric
monoanionic ligand (50–52). This histidine residue is absent in
HAO. Glu110, however, is found in a similar position.
+
either spontaneously cyclizes to the essential NAD precursor,
QUIN, or is further metabolized by ACMS decarboxylase
(55–59) (SI Appendix, Fig. S1). Interestingly, a previous study has
shown that the autocyclization of QUIN from ACMS released
from HAO is impeded by the presence of HAO compared to the
cyclization rate in the absence of HAO (3). Our product-bound
complex structures provide a rationale for the underlying
mechanism of how the presence of the enzyme slows the spon-
taneous formation of QUIN. We propose that HAO possesses
in situ isomerase activity. By maintaining ACMS in a more stable
conformation, HAO raises the bar for autocyclization and hence
reserves a portion of the metabolic flux for other downstream
enzymes of the kynurenine pathway. Similar isomerase activity
has been found in the dehydrogenase enzyme two steps down in
the pathway (31, 33). It has been found that the ratio of QUIN
relative to the enzyme-controlled metabolism is 1:72 in normal
healthy individuals (60). Overproduction of QUIN within cells
should be avoided since it is excitotoxic at high concentrations
Dioxygen binds to the Fe ion in an end-on mode in HAO.
Based on the structure, the distal oxygen of the superoxide is
directed correctly to proceed via radical recombination with the
radical on the C3 carbon of the substrate, resulting in an alkyl-
2
peroxo intermediate (E•S•O )III with its oxy bridge trans to the
glutamate metal ligand. The alkylperoxo closely resembles the
bond lengths and orientations of the equivalent intermediate
trapped in HPCD (29). One apparent variation between HAO
and HPCD is the metal–ligand interaction during oxygen acti-
vation. The organic substrate is a bidnetate ligand in the super-
oxo and alkylperoxo complexes in HPCD but monodentate in
HAO. The possible reasons for the discrepancy include the dif-
ference between a ketone and an amine of the respective sub-
strate molecules, and the transient coordination of the amine/
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Fig. 6. HAO catalytic reaction cycle defined by the characterized intermediates. The time windows for observing the in crystallo reaction intermediates are
highlighted. The structurally characterized intermediates include monodentate E•S complex, bidentate E•S complex, Fe-bound superoxo, alkylperoxo,
e-lactone, monodentate and then bidentate 3E,5Z,2t,4c-enol tautomer of ACMS, as well as bidentate 3E,5Z,2t,4t-enol tautomer of ACMS. The superposition
of the crystal structures of the substrate-free HAO and the above seven intermediates is shown as an Inset to highlight the conformational change of loops A,
B, and C during the catalytic reactions. Loops of substrate-free, bidentate E•S complex, and subsequent intermediates after dioxygen binding are colored in
blue, red, and yellow, respectively.
and causes excessive stimulation of neuronal cells (61, 62). The
potential isomerization of HAO may represent a natural strategy
to maintain some noncyclized ACMS, highlighting a mechanism
for this bifurcated pathway to fine-tune the conformation of an
unstable metabolic intermediate.
moves to the fully open form along the reaction pathway, the
loop B of the alkylperoxo intermediate is an outlier which moves
to the closed conformation. Thus, the superoxo and alkylperoxo
intermediates not only differ from each other in the iron-bound
2
substrates, 3-HAA and O ; they also have a significantly different
Single-crystal EPR studies have been made to characterize the
stable tyrosyl radical in ribonucleotide reductase, photosystem II,
and the oxidized state of [FeFe]-hydrogenase by using special
sample containers (63–65). Previous methods used a limited
number of crystals, which creates spectra with orientation de-
pendence yielding additional information such as g-tensor pa-
rameters. However, orientation dependence complicates data
interpretation, especially when identifying an unknown inter-
mediate during a time-resolved reaction. In this study, we tested
the minimal number of equivalent single crystals needed in a
regular, quartz X-band EPR tube to surmount the orientation
dependency and created an updated method for detecting in-
termediates in crystallo by EPR spectroscopy during enzymatic
reactions.
conformational state in residues between 40 and 47 (loop B). In
the alkylperoxo intermediate, Arg99 of loop B moves toward the
iron center and is responsible for stabilizing the oxy bridge. This
requirement from the second coordination sphere may explain
the closed conformation of loop B. From the alkylperoxo to the
monooxygenated lactone intermediate, a significant movement
of loop B to the near open form takes place, presumably because
the intermediate stabilization by Arg99 is no longer required.
Subsequent changes from the lactone to the enol tautomers of
the dioxygenated product are minor, and most loop movement
for opening is focused on loop C (residues 139 to 148). It is worth
noting that these loop dynamics during catalysis are rarely ob-
served and difficult to predict. It is also challenging to determine
the active-site dynamics during catalysis in the solution state of
the HAO reaction due to its high turnover rate. Thus, the results
presented in this work provide a case study and comprehensive
view regarding both the active-site chemistry and the protein
conformational dynamics during an enzymatic action.
A common feature during enzymatic action is that many en-
zymes adopt an open conformation at the catalytic center, and
upon substrate binding, the catalytic center becomes closed.
However, little is known whether it is in the open or closed
conformation during the intermediary steps of catalysis. Our
success in obtaining seven intermediate crystal structures pro-
vided an outstanding opportunity to address such a question in
an iron-based dioxygenase. Previously, we have shown that the
closed conformation due to 3-HAA binding increases the hy-
drophobicity of the iron center and causes a substantial increase
Experimental Procedures
The materials and routine methods are described in SI Appendix, including
the source of reagents, protein expression, isolation, enzyme activity assays,
kinetic analyses, product analysis by HPLC and high-resolution mass spec-
trometry, crystallization, and X-ray data collection and refinement.
2
of the O affinity of the active site (16). The structural studies in
Single-Crystal UV-Vis Spectroscopy. The catalytic activity was initially dem-
onstrated by using an Agilent 8453 UV-vis spectrophotometer in a reaction
mixture containing seven single crystals in mother liquor with 25% PEG
this work show how the dioxygenase modulates the active-site
conformation throughout the catalytic reaction. Fig. 6, Inset, and
Movie S1 show that the active site changes quickly from the
closed to a primarily, but not wholly, open form after the arrival
of dioxygen at the iron center. While the active site gradually
8
2 2
000, 200 mM MgCl , and 30% glycerol, 0.3 mM 3-HAA, and 1.3 mM O . The
reaction was monitored at room temperature in a quartz cuvette. The λmax
numbers for 3-HAA and ACMS in solution at pH 8.5 are 320 and 360 nm,
Wang et al.
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respectively. At this pH, ACMS is less stable and forms QUIN more rapidly
with much less ACMS observed at 375 nm than expected by the loss of
coordination distances and exogenous ligand occupancy are shown in SI
Appendix, Tables S2–S4. Each dataset was obtained on a distinct single crystal
from one continuous data collection at the beamline. No datasets were
merged during analysis because each crystal and time point are unique.
3
-HAA (55). After 70 min, all seven crystals were found intact by microscopy
(Leica Microsystems).
In situ single-crystal UV-vis absorption spectroscopy was used to charac-
terize the reaction intermediates and turnovers. A fully automated, remote
accessible microscope spectrophotometer at the Stanford Synchrotron Ra-
diation Lightsource (SSRL) beamline 9-2 was employed to collect the UV-vis
absorption spectra of HAO single crystals during reaction or alongside dif-
fraction measurements. A default 50-μm light spot at the sample was used to
monitor the catalytic reaction in crystal. All spectra were measured at 100 K.
Reference of the spectroscopy was taken in the absence of crystal samples. In
order to minimize artifacts from the frozen mother liquor, ice, or the nylon
loop, different orientations of a single crystal were scanned by phi-angle
rotation from 0 to 360° with a step of 10°. The optimal absorption spectra
exhibiting reproducible spectral features were selected for presentation. The
Ligand Refinement and Molecular Modeling. In order to minimize bias, ligand
refinement was initiated at the final stage of structure refinement by ex-
amining ligand-omit difference maps. In every case, 3-HAA was the initial
ligand model. As warranted, subsequent ligand models in the reaction cycle
were fit into the 2Fobs – Fcalc electron density maps and by examining the Fobs
–
Fcalc maps. Phenix eLBOW was applied to generate the geometry restraint
information of 3-HAA–derived superoxo, alkylperoxo, epoxide, e-lactone,
and enol tautomers of ACMS. With the exception of two conformations of
the product in one crystal structure, only the intermediates with ≥80% li-
gand occupancy are included. The light blue 2Fobs – Fcalc maps around the
metal-ligands (His51, Glu57, and His95) and the exogenous ligands are
contoured at 1.0 σ. Finally, the exogenous ligands were removed, and the
resultant ligand-free structures were refined to obtain the Fobs – Fcalc omit
maps (green) contoured at 3.0 σ, which are in agreement with the active-site
ligands in the full models. The detailed ligand refinement and model fitting
are described in SI Appendix, Materials and Method, under X-ray data collection
and refinement.
λmax for 3-HAA and ACMS in crystals are 325 and 374 nm, respectively.
X-band Continuous-Wave EPR Spectroscopy of HAO Single Crystals. Anaerobi-
cally crystallized HAO microcrystals (∼1,000 single crystals) were prepared in
mother liquor containing 20% glycerol. EPR samples were prepared in 4-mm
quartz EPR tubes and rapidly frozen by liquid ethane. X-band EPR spectra
were recorded by a Bruker E560 spectrometer at 9.4-GHz microwave fre-
quency with an SHQE high-Q resonator at 100-kHz modulation frequency.
The temperature was maintained with a cryogen-free 4 K temperature
system. All spectra were collected at 70 K and 16-μW microwave power, with
an average of nine scans. The g values and linewidths reported were
obtained by inspection of the EPR line shape.
The in-tube reaction was prepared by the addition of 5 mM 3-HAA into
HAO crystals to a final volume of 200 μL. The minimum time for sample
handling is 30 s before freezing. After acquiring each spectrum, the EPR
sample was thawed and incubated at room temperature before being rap-
idly frozen. The selected time points for spectral acquisition are 0.5, 1, 2, 7,
Data Availability. The structure models fitted to the electron density maps
have been deposited in the RCSB Protein Data Bank (https://www.rcsb.org).
These data are available through the following PDB entries: 6VI5, substrate-
free enzyme (1.60-Å resolution) (66); 6VI6, monodentate substrate–enzyme
complex (1.90-Å resolution) (67); 6VI7, bidentate substrate–enzyme complex
(
6
2.61-Å resolution) (68); 6VI8, superoxo intermediate (1.95-Å resolution) (69);
VI9, alkylperoxo intermediate (2.31-Å resolution) (70); 6VIA, lactone inter-
mediate (1.59-Å resolution) (71); 6X11, monodentate 3E,5Z,2t,4c-enol tau-
tomer of ACMS–enzyme complex (2.10-Å resolution) (72); and 6VIB,
enzyme–product complex in 3E,5Z,2t,4c-enol tautomer and 3E,5Z,2t,4t-enol
tautomer conformations (1.84-Å resolution) (73). A video clip, which was
constructed by using the University of California, San Francisco, ChimeraX
software (74), is available in Movie S1.
1
7, and 47 min. After completion of the reaction, crystals and aqueous so-
lution were separated by gentle centrifugation (2,200 rpm). Crystals were
dissolved in buffer (200 μL of 50 mM Tris at pH 8.5) with continuous vor-
texing. Protein concentrate was estimated as 185 μM based on the absor-
bance at 280 nm. The aqueous portion of the in-tube reaction was retained
for analysis by HPLC and mass spectrometry.
ACKNOWLEDGMENTS. We thank Drs. Jennifer L. DuBois and J. Marty
Bollinger Jr. for providing the chlorite dismutase plasmid used in the
solution-state kinetics studies, and Dr. Jiafeng Geng for participating in
the kinetic studies. We also thank Dr. Allen M. Orville and Dr. Ana Gonzalez
for assistance with our initial attempts to perform the single-crystal micros-
copy spectroscopic studies. This work was supported by National Science
Foundation Grants CHE-1623856 and CHE-1808637, and partially by the
National Institutes of Health Grants GM108988, GM133721, and MH107985,
and the Lutcher Brown endowment. We thank the staff scientists for
assistance with remote data collections at beamline 9-2 of the SSRL and
beamlines 19-BM, 19-ID, 22-ID, and 22-BM in the Argonne National Labo-
ratory of Advanced Photon Source (APS). Use of the SSRL and APS is
supported by the US Department of Energy (DOE), Office of Science, Office
of Basic Energy Sciences under Contracts DE-AC02-76SF00515 and DE-AC02-
Preparation of the Reaction Intermediates. In order to study the formation of
the binary E•S complex, single HAO crystals were incubated with mother
liquor (0.1 M Tris·HCl, pH 8.5, 0.2 M MgCl
2
, 20% PEG 8000) supplemented
with 1 mM 3-HAA (about 45-fold larger than the K
m
value) in an anaerobic
chamber. The monodentate E•S complex was trapped by flash-cooling
crystals 20 to 30 s after they were briefly dipped in mother liquor aug-
mented with 20% glycerol as a cryoprotectant. The chelated, equilibrium
E•S complex was flash-cooled after 30 or 300 s. Turnover was initiated at
room temperature by incubating crystals in 1 mM 3-HAA and then air-
saturated mother liquor. The crystals were flash-cooled after 50-s, 1-, 3-, 5-,
7-, 9-, or 13-min incubation periods. Crystals were prepared separately, and
flash-cooled directly in liquid nitrogen after being dipped into mother liquor
with 20% glycerol. The diffraction datasets and refined structures with the
highest resolution are presented here. The corresponding data collection
and refinement statistics are summarized in SI Appendix, Table S1. The
0
6CH11357, respectively. The SSRL Structural Molecular Biology Program is
supported by the DOE Office of Biological and Environmental Research and
by the National Institutes of Health, National Institute of General Medical
Sciences (including Grant P41GM103393).
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