Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile

Article abstract of DOI:10.1002/anie.201502731

The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2-1.3?). In combination with FTIR analyses of NHase following hydration in H₂¹⁸O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO^-) atom of αCys114-SO^-, followed by nucleophilic attack of the S(SO^-) atom by a βArg56-activated water molecule to release the product amide and regenerate αCys114-SO^-.

Full text of DOI:10.1002/anie.201502731

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International Edition: DOI: 10.1002/anie.201502731
German Edition: DOI: 10.1002/ange.201502731
Reaction Intermediates Very Important Paper
Time-Resolved Crystallography of the Reaction Intermediate of Nitrile
Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as
a Catalytic Nucleophile
Yasuaki Yamanaka, Yuki Kato, Koichi Hashimoto, Keisuke Iida, Kazuo Nagasawa,
Hiroshi Nakayama, Naoshi Dohmae, Keiichi Noguchi, Takumi Noguchi, Masafumi Yohda, and
Masafumi Odaka*
Abstract: The reaction mechanism of nitrile hydratase
(NHase) was investigated using time-resolved crystallography
of the mutant NHase, in which bArg56, strictly conserved and
hydrogen bonded to the two post-translationally oxidized
cysteine ligands, was replaced by lysine, and pivalonitrile was
the substrate. The crystal structures of the reaction intermedi-
ates were determined at high resolution (1.2–1.3 Š). In
combination with FTIR analyses of NHase following hydra-
tion in H218O, we propose that the metal-coordinated substrate
is nucleophilically attacked by the O(SOÀ) atom of aCys114-
SOÀ, followed by nucleophilic attack of the S(SOÀ) atom by
a bArg56-activated water molecule to release the product
amide and regenerate aCys114-SOÀ.
oxidized cysteines (cysteine-sulfinic acid (Cys-SO2H), and
cysteinesulfenic acid (Cys-SOH)), one cysteine thiol, and
a water molecule.[4] Although Cys-SO2H and/or Cys-SOH
modifications are found in a variety of proteins, such as
peroxiredoxins,[5] hydrogenases,[6] and NADH peroxidases,[7]
and play diverse roles in biological systems,[8] NHase is the
first protein that has shown to possess both modified cysteine
residues as the metal ligands.[4a,b] FTIR analyses[9] and
a combination of EPR, MCD, and low-temperature absorp-
tion spectroscopy with DFT calculations have indicated that
both Cys-SO2H and Cys-SOH are deprotonated.[10] This
unique coordination geometry is common among NHase
family proteins, including the Fe-type[4a] and Co-type[4b]
NHases and thiocyanate hydrolase (SCNase).[11] Fe-type
NHases[12] and SCNase[13] lose their catalytic activities upon
N
itrile hydratases (NHases, E.C. 4.2.1.84) catalyze the
hydration of nitriles to the corresponding amides[1] and are
the most successful industrially applied microbial catalysts.[2]
NHases have been used for the production of acrylamide
(greater than 95 kilotons per year worldwide), nicotinamide,
and 5-cyanovaleramide and may impact the bioremediation
of organic nitrile pollution.[3] NHases are composed of a- and
b-subunits. In the a-subunit, the catalytic center contains
a singular non-heme Fe3+ or non-corrin Co3+, which is
coordinated by two deprotonated backbone amides, two
alteration of the oxidation states of Cys-SO2 and Cys-SOÀ.
À
Based on the crystal structures, three distinct reaction
mechanisms had been proposed.[4c,d,14] Two mechanisms
involve direct or indirect attack of the active water ligand
whereas the other involves a substrate-coordinated inter-
mediate. Studies on many model complexes mimicking the
NHase catalytic center,[4c,d,15] and on the pH and temperature
dependence of the kinetic parameters[16] have suggested the
mechanism that substrate coordination to the metal is
followed by nucleophilic attack of a water molecule activated
by an unidentified base. Previously, we investigated the
reaction mechanism of an Fe-type NHase from Rhodococcus
erythropolis N771 (ReNHase) using time-resolved X-ray
crystallography and a tert-butylisonitrile (tBuNC) substrate
analogue, and proposed that the O atom of Cys-SOÀ functions
as a nucleophile to activate a water molecule that attacks the
substrate coordinated to the Fe3+ atom.[17] However, it is
unknown whether this proposed mechanism accurately
reflects the catalytic mechanism for a nitrile. Recently, the
substrate coordination was demonstrated using single-turn-
over stopped-flow spectroscopy.[18] The crystal structure of the
Co-type NHase from Pseudonocardia thermophila JCM3095
(PtNHase) in complex with boronic acids (R-BOOHs)[19]
suggests that the SOÀ group functions as a nucleophile to
attack the coordinated nitrile carbon to form a cyclic
intermediate. Following this observation, Hopmann pre-
sented a unique mechanism involving disulfide bond forma-
tion between the Cys-SOÀ and the cysteine thiol ligands.[20]
[*] Dr. Y. Yamanaka, Dr. K. Hashimoto, Dr. K. Iida, Prof. K. Nagasawa,
Prof. M. Yohda, Prof. M. Odaka
Department of Biotechnology and Life Science
Graduate School of Technology, Tokyo University of
Agriculture and Technology, Koganei, Tokyo 184-8588 (Japan)
Prof. M. Odaka
Department of Life Science
Faculty and Graduate School of Engineering and Resource Science
Akita University, Akita City, Akita 010-8502 (Japan)
E-mail: modaka@gipc.akita-u.ac.jp
Prof. K. Noguchi
Instrumentation Analysis Center
Tokyo University of Agriculture and Technology
Koganei, Tokyo 184-8588 (Japan)
Dr. Y. Kato, Prof. T. Noguchi
Division of Material Science, Graduate School of Science
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602 (Japan)
Dr. H. Nakayama, Dr. N. Dohmae
Global Research Cluster Collaboration Promotion Unit, RIKEN
2-1, Hirosawa, Wako-shi, Saitama 351-0198 (Japan)
À
However, this mechanism remains uncertain because R
BOOHs have been previously shown to act as general
trapping agents for sulfenic acids to form similar adducts
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201502731.
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[21]
À À À À
(R B O S R’) with sulfenic acids in aqueous media.
presence of the respective substrates (Supporting Informa-
tion, Figure S1).
Moreover, structural evidence for disulfide bond formation
is lacking.
The catalytic reaction was initiated using light illumina-
tion at 293 K and proceeded for up to 700 min. Throughout
the experiments, the overall structures remained nearly
unaltered, except for the region above the Fe3+ center.
Snapshots of the region surrounding the catalytic center at 0,
5, 25, 50, 120, and 700 min are shown in Figure 2. The omit
In the present study to elucidate the catalytic mechanism,
we monitored the hydration of pivalonitrile (PivCN) in
crystals of the mutant ReNHase, bR56K, using time-resolved
X-ray crystallography. bArg56 is conserved among all known
NHase family proteins and is the only residue that forms
hydrogen bonds with both oxidized cysteines.[4a,b,11] The kcat
and Km value of bR56K are 4.6 ” 102 times smaller and
approximately three times larger than those of the wild-type,
respectively.[22] Herein, we present the first crystal structures
of the reaction intermediates of NHase at high resolution (1.2/
À1.3 Š; see the Supporting Information, Table S1). Together
with the results of FTIR analyses using 18O-labeled water, we
propose the first catalytic mechanism of NHase based on solid
structural evidence.
ReNHase is inactivated by the nitrosylation of the Fe3+ ion
in the dark but immediately re-activated by photoinduced
denitrosylation.[23] Because nitrosylated ReNHase is
extremely stable, all crystals were prepared in their nitro-
sylated form in the dark. Crystals of nitrosylated bR56K were
soaked with PivCN in the dark. The structure of nitrosylated
bR56K in complex with PivCN (PDB code: 3WVE) is
essentially identical to that of wild-type ReNHase in complex
with tBuNC (PDB code: 2ZPE),[17] except for the mutated
site (Figure 1). An NO molecule is observed 2.0 Š from the
Fe3+ atom, as observed in the structure of the nitrosylated
Figure 1. Stereoview of the superposed structures of the region sur-
rounding the catalytic center of wild-type ReNHase in complex with
tBuNC (PDB ID: 2ZPE) and the bR56K mutant in complex with PivCN
(PDB code: 3WVE). Both enzymes are in the nitrosylated state.
C (bR56K) green, C (wild-type) gray, N blue, O red, S yellow, Fe orange.
In both ReNHases, the Sg of bM40 adopts two conformations (lower
and upper) with occupancies of lower/upper=0.4:0.6. The movement
of bM40 to the upper conformation has been suggested to result from
the binding of a substrate (analogue) to the substrate binding site.[14]
a and b in the amino acid residues labels indicates a and b subunits.
Figure 2. Time-resolved structures of the region surrounding the non-
heme Fe3+ center of the bR56K mutant ReNHase. Omit maps around
the catalytic center (contoured at 3.0 s in green) are superimposed on
the refined structures. ReNHase in complex with PivCN following light
illumination for A) 0, B) 5, C) 25, D) 50, E) 120, and F) 700 min. The
NO, substrate and the O atom of aCys114-SOÀ were excluded from
the calculations. C gray, N blue, O red, S yellow, Fe orange. a and b in
the amino acid residues labels indicates a and b subunits. PDB codes
for the structures of the bR56 K mutant ReNHase in complex with
PivCN following illumination for 0, 5, 25, 50, 120, or 700 min are
3WVE, 3X26, 3X20, 3WVD, 3X24, and 3X25, respectively.
wild-type in complex with tBuNC.[17] A PivCN molecule
exhibits a direction that is highly similar to that of the tBuNC
molecule in the wild-type-tBuNC complex, despite a position
that is 0.2 Š above that of tBuNC. As observed in the wild-
type-tBuNC complex, bMet40 adopts two conformations
owing to steric hindrance of the substrate. The hydrogen
bonding network surrounding the catalytic center is also
highly similar to that of wild-type ReNHase, even in the
map was superimposed in each structure. Before illumination
(0 min), electron densities corresponding the NO molecule
and the O atom of aCys114-SOÀ were observed at the sixth
ligand site and the above of the S atom of aCys114,
respectively. Furthermore, electron density for PivCN is
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observed in the pocket close to the side chain of bTyr37
(Figure 2A). At 5 min (Figure 2B), the electron density at the
sixth ligand site was slightly attenuated, reflecting the
initiation of the release of the NO molecule. At 25 min
(Figure 2C), the electron density of the substrate at the
original position is attenuated, and in turn, a new electron
density coordinated to the Fe3+ atom was observed. Consid-
ering the loss of the electron density at the initial substrate
position, the new electron density should be that of the
substrate coordinating to the Fe3+ atom. Nearly all of the
electron density derived from the substrate is coordinated to
the Fe3+ atom at 50 min (Figure 2D). At 120 min, the omit
electron density map is similar to that at 50 min (Figure 2E).
We examined 32 snapshots from 120 min to 700 min (The
detailed time points examined are given in the Experimental
Section in the Supplementary Information). However, the
omit map coordinated to the Fe3+ atom was not altered
(Figure 2F). These results unambiguously confirm substrate
coordination to the Fe3+ atom during catalysis. The angle
between the C atom of the pivaloyl group and the C and N
atoms of the CN group is no longer linear; rather, these three
atoms and the sulfenyl oxygen atom of aCys114-SOÀ lie in
a single plane. To identify the electron density coordinated to
the Fe3+ atom at 50 min, PivCN or the pivalamide (Piv-
CONH2) product was included in the calculations, and the
omit map was superimposed on the refined structures. The
refined model of PivCN was distorted unrealistically and did
not fit the observed electron densities in the omit map
(Figure 3A), whereas PivCONH2 fits the density well (Fig-
Oamide(PivCONH2) atom dramatically decreased (Supporting
Information, Figure S2C). Thus, the results clearly demon-
strate that this time point reflects the transient state structure
immediately following the hydration reaction, and that the
nitrile N atom that is coordinated to the Fe3+ atom is
nucleophilically attacked by the O atom of aCys114-SOÀ to
form a cyclic imidate intermediate. The formation of cyclic
intermediate species is consistent with the mechanism pro-
posed based on the structures of the Co-type PtNHase in
complex with R-BOOHs.[19]
Following the generation of the cyclic reaction intermedi-
ate, a water molecule activated by a base attacks the S atom of
aCys114-SOÀ or the C atom of the CN group to produce the
amide product and regenerate the SOÀ group. The water O
atom is trapped in aCys114-SOÀ in the former mechanism,
whereas it becomes the amide O atom of the product in the
latter mechanism. To investigate this step in catalysis further,
we performed the nitrile hydration in H218O using wild-type
ReNHase, followed by FTIR analyses. Figure 4A (blue line)
shows a light-induced FTIR difference spectrum of ReNHase
incubated in H218O prior to the hydration reaction, represent-
ing the structural changes of ReNHase upon photoreactiva-
tion.[9,23,24] Prominent bands at (1156–1149)/1126 and (1040–
Figure 3. Structure surrounding the non-heme Fe3+ center of the
bR56K mutant ReNHase in complex with PivCN following light
illumination for 50 min. Omit map around the catalytic center at
50 min (contoured at +3.0 s in green) is superimposed on the refined
structure. A) The PivCN substrate was included in the calculation;
B) the PivCONH2 product was included in the calculation; and C) the
PivCONH2 product was included but the sulfenyl oxygen of aCys114-
SOÀ was excluded in the calculation. C gray, N blue, O red, S yellow,
Fe orange.
ure 3B). However, the Oamide(PivCONH2)–O(aCys114-SOÀ)
distance is too close (0.7 Š; Figure 3B), and negative electron
density corresponding to one O atom was observed around
the Oamide(PivCONH2) and O(aCys114-SOÀ) atoms (Support-
ing Information, Figure S2B). These results indicate that
Figure 4. Light-induced FTIR difference spectra of NHase in H218O
without treatment (blue line) and following the hydration reaction with
methacrylnitrile in H218O and subsequent NO addition (red line).
a) The 1800–850 cmÀ1 region. The inset shows an NO stretching band
at 1855 cmÀ1 indicative of the release of NO from the non-heme iron
center upon photoactivation. The red arrow indicates the region in
which the spectral change is observed. b) An expanded view (935–
870 cmÀ1) of the SO stretching region of Cys-SOÀ. The dotted blue
line represents a dark-minus-dark spectrum of ReNHase without
treatment, corresponding to the noise level.
when the PivCONH2 product was positioned as shown in the
À
À
omit map, the S O bond in the SO group should be broken.
In contrast, when the O(aCys114-SOÀ) atom was excluded
from the calculation, the negative electron density around the
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1033)/1019 cmÀ1 and a minor signal at 914/907 cmÀ1 are
virtually identical to the asymmetric and symmetric SO2
À
stretching vibrations of aCys112-SO2 and the SO stretch of
aCys114-SOÀ, respectively, which have been previously
assigned for ReNHase prepared in H216O,[9] indicating that
À
the oxygen atoms of the aCys-SO2 and aCys-SOÀ groups
were not exchanged with 18O during incubation in H218O. The
FTIR difference spectrum of ReNHase, which was reacted
once with methacrylonitrile in H218O and then nitrosylated
again (Figure 4A, red line) is also highly similar to that of
ReNHase in H218O without catalysis (Figure 4A, blue line). In
fact, the spectral features and peak frequencies are identical
in the 1890–920 cmÀ1 region. However, a clear difference was
observed in the small Cys-SOÀ peaks at 914/907 cmÀ1 (Fig-
ure 4B, blue line). These peaks disappeared, and new peaks
appeared at 890/883 cmÀ1 (Figure 4B, red line). Although
these signals are extremely small compared with other major
peaks, the intensities are significantly larger than the noise
level (Figure 4B, dotted blue line). The 24 cmÀ1 downshift is
comparable to the predicted 18O downshift of 31 cmÀ1 in an
isolated SOÀ vibration.[9] This downshift of the SOÀ vibration
following the NHase reaction in H218O indicates that the
oxygen atom of the Cys-SOÀ group is replaced with 18O from
water; hence, the O atom of aCys114-SOÀ is exchanged with
the solvent water during the ReNHase nitrile hydration
reaction.
A remaining issue involves the regeneration of sulfenic
acid. The mechanism proposed by Hopmann, in which the
thiolato ligand functions as a nucleophile to attack the S atom
of the cyclic intermediate to form a disulfide bond between
aCys109 and aCys114,[21] is unlikely because the distance
between the S atoms of aCys109 and aCys114 remains nearly
unaltered throughout the time-resolved X-ray crystallogra-
phy analysis (Supporting Information, Figure S3), despite that
bArg56 is not essential for the disulfide bond formation.
Alternatively, the water molecule forms hydrogen bonds with
the guanidinium groups of bArg56 (or the e-amino group of
bLys56 in the bR56K mutant) and bArg141 and with the two
main chain amide O atoms of aThr163 and aAla164
(Supporting Information, Figure S1, wat1635 in the wild-
type and wat132 in the bR56K mutant). We note that the
water molecule was not involved in the theoretical analysis
for the disulfide bond intermediate mechanism. The water
molecule is the closest solvent molecule to the S atom of
aCys114 and forms hydrogen bonds with the O atom of
aCys114-SOÀ in both wild-type and bR56K mutant
ReNHases.
Figure 5. Proposed catalytic mechanism of NHase. The steps (A)–(D)
are described in the text.
intermediate extracts a proton from the water molecule,
which facilitates nucleophilic attack of the water O atom on
the S atom of aCys114 and subsequent reactions (Figure 5,
C). Finally, the H atom of the regenerated -SOH group is
transferred to the N atom of the intermediate; and the
product was released and a water molecule is coordinated to
be the resting state (Figure 5, D). The decrease in the kcat
value of the bR56 K mutant would be ascribed to difference
in the pKa values between arginine (pKa 12.5) and lysine (pKa
10.5).
In conclusion, we propose the first NHase reaction
mechanism in which aCys114-SOÀ functions as a catalytic
nucleophile and the O atom of the SOÀ group is exchangeable
based on the high-resolution crystal structures of the reaction
intermediates of the NHase-substrate complex. This is the
novel biological function of the cysteinesulfenic acid modifi-
cation in biological systems. Although the hydrogen bonding
networks surrounding the catalytic center and the protona-
tion state of aCys114-SOÀ should be confirmed by future
studies using neutron crystallography, we have now estab-
lished an outline of the NHase catalytic mechanism.
Acknowledgements
Therefore, we propose the following catalytic mechanism
(Figure 5). First, the substrate replaces the water ligand to
coordinate the metal (Figure 5, A). Second, the O atom of
aCys114-SOÀ attacks the C atom of the substrate CN group to
form the cyclic reaction intermediate (Figure 5, B). The
guanidinium group of bArg56 is likely to activate both the
water molecule mentioned above and the O atom of the cyclic
intermediate. In fact, over the course of the 700 min reaction,
the water molecule (wat132) moves slightly toward the Ne
atom of bLys56 in the bR56K mutant ReNHase (Supporting
Information, Figure S4). Third, following the formation of the
cyclic reaction intermediate, the N atom of the cyclic
We would like to thank Prof. E. I. Solomon and Dr. K. E.
Light for valuable discussion and comments. We also thank
the beamline assistants at the Photon Factory for data
collection at beamlines BL-5A and BL-1A. This work was
supported in part by a Grant-in-Aid for Scientific Research
from the Scientific Research (B) KAKENHI 21350089 (to
M.O.) and (B) KAKENHI 24350082 (to M.O.). This work was
performed with the approval of the Photon Factory Advisory
Committee (approval nos. 2008G640 and 2011G645).
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1024 – 1030; b) M. Tsujimura, M. Odaka, H. Nakayama, N.
Keywords: cysteinesulfenic acid · nitrile hydratase ·
reaction intermediates · reaction mechanisms ·
time-resolved X-ray crystallography
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Received: March 24, 2015
Revised: May 26, 2015
Published online: August 14, 2015
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