Sulfido and cysteine ligation changes at the molybdenum cofactor during substrate conversion by formate dehydrogenase (fdh) from rhodobacter capsulatus

Article abstract of DOI:10.1021/ic502880y

Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO^-) to CO₂ oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Mo-ligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.

Full text of DOI:10.1021/ic502880y

Article
pubs.acs.org/IC
Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor
during Substrate Conversion by Formate Dehydrogenase (FDH) from
Rhodobacter capsulatus
Peer Schrapers,† Tobias Hartmann, Ramona Kositzki, Holger Dau, Stefan Reschke,
,∇
‡,∇
,‡
†
†
‡
§
,†
Carola Schulzke, Silke Leimku
̈
hler,* and Michael Haumann*
†
Institut fu
̈
r Experimentalphysik, Freie Universitat
Institut fur Biochemie und Biologie, Molekulare Enzymologie, Universitat
Institut fur Biochemie, Bioanorganische Chemie, Ernst-Moritz-Arndt-Universitat
S Supporting Information
̈
Berlin, 14195 Berlin, Germany
Potsdam, 14476 Potsdam, Germany
Greifswald, 17487 Greifswald, Germany
‡
̈
̈
̈
§
̈
*
ABSTRACT: Formate dehydrogenase (FDH) enzymes are
attractive catalysts for potential carbon dioxide conversion
applications. The FDH from Rhodobacter capsulatus (RcFDH)
binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD)
−
cofactor, facilitating reversible formate (HCOO ) to CO2
oxidation. We characterized the molecular structure of the
active site of wildtype RcFDH and protein variants using X-ray
absorption spectroscopy (XAS) at the Mo K-edge. This
approach has revealed concomitant binding of a sulfido ligand
(
Mo=S) and a conserved cysteine residue (S(Cys386)) to
Mo(VI) in the active oxidized molybdenum cofactor (Moco),
retention of such a coordination motif at Mo(V) in a
chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation.
The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding
chaperone. This process still functioned in the Cys386Ser variant, showing no Mo−S(Cys386) ligand, but retaining a Mo=S
bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Mo−
ligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported
by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group
exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network
including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.
INTRODUCTION
Protein crystallography has revealed that the metal-depend-
ent FDHs from prokaryotic organisms belong to the so-called
dimethyl sulfoxide (DMSO)-reductase family of enzymes,
■
The enzymatic conversion of carbon dioxide (CO ) has gained
increasing interest in recent years, because such processes may
be valuable for the sequestration of CO from the atmosphere,
to combat the greenhouse effect, for providing important
feedstock to the chemical industry (such as carbon monoxide),
and for the generation of products such as formate (HCOO )
to serve in renewable energy applications.
of enzymes, which catalyzes the conversion between HCOO
2
9,13,14
as characterized by a complex active-site cofactor, in which two
pyranopterin molecules, each carrying a terminal guanine-
nucleotide moiety, are coordinated via their dithiolene-sulfur
functionalities to the redox-active metal center in an often
trigonal-prismatic geometry, resulting in the prototypic bis-
MGD structure (Figure 1). The two apical ligands
complementing the metal coordination sphere have been
proposed to be of crucial functional relevance for the proton
and electron transfer reactions during the enzyme’s formate
2
−
1
−7
The diverse class
−
8
and CO , is denoted as formate dehydrogenase (FDH).
2
Particularly interesting among the FDHs are metalloenzymes,
bearing functionalized second- or even third-row transition
metals, namely molybdenum (Mo) or tungsten (W), at their
active sites (for recent reviews, see refs 5 and 9−11). Important
discriminators between the metal-containing FDHs are the
degree of preservation of their catalytic activity in the presence
15−17
conversion chemistry.
However, both their chemical
identity and exact coordination configuration have remained
ambiguous in many cases.
In the Mo-FDH from Escherichia coli and in the W-FDH
from Desulfovibrio gigas, the Se atom of a selenocysteine
of O and the reversibility of the conversion chemistry to
2
facilitate not only substrate oxidation, but also CO reduction
to generate the formate product.
Received: December 2, 2014
Published: March 24, 2015
2
12
©
2015 American Chemical Society
3260
DOI: 10.1021/ic502880y
Inorg. Chem. 2015, 54, 3260−3271

Inorganic Chemistry
Article
conversion have been formulated using computational
approaches: one involving ligation of the Se/S(Cys) to the
metal throughout the cycle, and the other relying on a transient
15−17
exchange of the Se/S(Cys) ligand by the substrate.
In the present study, we have focused on characterization of
the metal site structure in the FDH protein from Rhodobacter
capsulatus (RcFDH), using X-ray absorption spectroscopy
+
(
XAS). The RcFDH is a NAD -dependent enzyme and
2
8,29
possesses a molybdenum cofactor (Moco),
four [4Fe4S]
clusters and one [2Fe2S] cluster in the α-subunit, one [4Fe4S]
cluster and one FMN in the β-subunit, and one [2Fe2S] cluster
28
in the γ-subunit. Previous biochemical and functional studies
have revealed a bis-MGD cofactor in the RcFDH and a cysteine
(
Cys386) homologous to the metal-binding SeCys in the E. coli
28
FDH. Significant O -tolerance was demonstrated for
RcFDH, similar to other Mo-FDHs containing a putative
cysteine ligand.
Figure 1. Metal ligation in bis-MGD cofactors in protein crystal
structures. The shown structure represents the molybdenum cofactor
in an FDH from Escherichia coli (PDB code 2IV2, 2.3 Å resolution,
2
28
30−33
RcFDH further shows reversible substrate
1
8
reduced state), exemplifying the overall bis-MGD cofactor geometry.
Se(Cys140) (cys) and His141 (his) in E. coli correspond to S(Cys386)
and His387 in RcFDH. The ligand X (cyan) has initially been modeled
conversion with increased CO reduction activity compared to
2
28
the E. coli FDH. The RcFDH thus exhibits versatile catalytic
properties, but a crystal structure of this type of enzymes is not
available.
1
8,18,21
as an O atom
in the structure shown and in the structures of the
oxidized E. coli enzyme (PDB codes 1KQF, 1.6 Å resolution, and
FDO, 2.8 Å resolution) and was later reinterpreted as an S atom.
The structure of the tungsten FDH from Desulfovibrio gigas shows an
Se(Cys) ligand and a vacant X-site (PDB code 1HOH, 1.8 Å
resolution). The structure of molybdenum polysulfide reductase
from Thermus thermophilus shows an S(Cys) ligand and X is an O
atom (PDB code 2VPW, 3.1 Å resolution). The structure of
molybdenum nitrate reductase from Cupriavidus necator shows an
S(Cys) ligand and X is an S atom (PDB code 3ML1, 1.6 Å
The biosynthesis and insertion of the bis-MGD cofactor is a
2
1
1
34−36
complex process
involving in the case of RcFDH two
28,37
system-specific chaperones, namely, FdsC and FdsD.
FdsC
19
was shown to bind the bis-MGD cofactor and a role of FdsC in
2
8,37
bis-MGD insertion into RcFDH was suggested.
However,
25
when FdsC is absent during RcFDH expression, the enzyme
was shown to be inactive, whereas, in the purified protein, bis-
MGD was present. This suggested a further role of FdsC in bis-
23
resolution). Mo/W−S(MGD) bond lengths range between 2.3 Å
and 2.5 Å, Mo/W−Se/S(Cys) bond lengths are 2.1−2.6 Å, and Mo/
W−X bond lengths are 2.2−2.4 Å in these structures. Color code:
Mo/W, magenta; S, yellow; Se/S(Cys), orange; O, red; N, blue; P,
green; C, gray; protons were not resolved and therefore were omitted
in the drawing.
MGD modification/activation before the insertion into the
28,37
FdsA subunit of RcFDH.
FdsC can be substituted
functionally by the homologous FdhD protein from E. coli,
which has been proposed to add a sulfur ligand to the
2
2
37
molybdenum, suggesting a similar role for RcFdsC.
Accordingly, the Cys386 and a sulfido ligand at the
molybdenum were suggested to be involved in the RcFDH
28,37
(
SeCys) residue has been shown to be a metal ligand in crystal
activity.
However, direct structural evidence for such a
1
8−20
structures.
Reinterpretation of the crystal structure for the
coordination has not been obtained so far.
reduced E. coli enzyme, however, favored positioning of the
XAS is well-suited for the determination of metal-site fine
structures, compared to protein crystallography, with respect to
its significantly higher resolution of interatomic distances (a
metal−ligand bond length change of ∼0.02 Å may be
discriminated in high-quality EXAFS data), enhanced discrim-
ination between oxygen and sulfur ligands, and facile access to
metal oxidation states, as well as more-reliable preservation of
high-valent species such as Mo(VI) by avoiding X-ray
photoreduction due to a usually lower specific photon
SeCys at a larger distance to the molybdenum under such
1
8,21
conditions.
A second non-pyranopterin ligand was initially
modeled as an oxygen species, probably reflecting a
molybdenum-oxo (Mo=O) bond, in the Mo-FDH struc-
1
8,20
tures.
The presence of a respective ligand was less clear
19
in the W-FDH. In later revisions of the structures, binding of
a sulfur species to molybdenum was suggested.
9
,19,21
Involve-
ment of a sulfur has gained confidence from biochemical
studies, because the system-specific chaperone FdhD has been
38−44
flux.
This technique has significantly contributed to
22
shown to insert a sulfur ligand in the E. coli Mo-FDHs.
A
unravel the Mo site structures in various bis-MGD enzymes
also in the post-structural era (see, for example, refs 34, 41, and
45−53). Discrimination of oxo (Mo=O) vs sulfido (Mo=S)
ligation at the molybdenum by XAS can be expected, because
of their significantly differing bond lengths (∼1.7 Å vs ∼2.2 Å)
coordination motif with six respective sulfur ligands (two
dithiolenes, a cysteine, and a terminal sulfur) to molybdenum
has been assigned in the crystal structures of two periplasmic
23,24
nitrate reductases.
By comparison, a terminal oxygen ligand
54−58
was suggested for the Mo active site of a polysulfide
observed in synthetic model compounds.
In an XAS study
2
5
reductase. So far, the experimental data has not yet
established an unambiguous relationship between the funda-
on the SeCys containing E. coli FDH, a close interaction
between the Se atom and a dithiolene sulfur has been
9
59
mentally different reaction types of bis-MGD enzymes, the
proposed, which may require reinterpretation, with regard
varying O -tolerance and catalytic bias in FDHs, as well as the
to the crystal structures involving a sulfur ligand.
2
Mo/W metalation, Se/S(Cys) ligation, and oxo/sulfido
coordination. In addition, conserved amino acid residues in
the vicinity of the cofactor in FDHs may be important for
Herein, we report the first Mo K-edge XAS study on RcFDH
wildtype (WT) and protein variants (Cys386Ser and
His387Met) expressed in the presence or absence of the bis-
MGD binding chaperone FdsC. The results imply that a Mo=S
ligand, inserted by the FdsC chaperone, and Cys386
26−28
proton transfer (Figure 1).
Based on the crystal data of
FDHs, two different reaction mechanisms of formate
3
261
DOI: 10.1021/ic502880y
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Inorganic Chemistry
Article
coordination at the Mo center are essential for catalytic activity.
Mo-ligation changes in response to pH variation, metal site
reduction, and amino acid exchange, as well as Cys386
substitution by formate at the reduced Mo center, support a
reaction scheme of formate oxidation involving Mo(VI) to
Mo(IV) redox cycling, substrate binding at molybdenum, and
viable roles of Cys386 and His387 in proton handling.
value was 0.37 Å, and R
0
represented mean values for Mo(VI) species
0
6
4−67
(
R (Mo−O) = 1.87 Å, R (Mo−S) = 2.33 Å),
0
⎛
⎜
R − R ⎞
0
i
BVS =
∑
Ni exp
⎟
⎠
⎝
B
(1)
RESULTS
■
Activity of RcFDH Protein Preparations. XAS at the Mo
K-edge was carried out on purified wildtype (WT) RcFDH
protein, which was prepared in the oxidized and reduced states
at pH values of 7.0, 8.0, and 9.0. The protein was expressed in
the presence or absence of the bis-MGD binding chaperone
MATERIALS AND METHODS
■
Protein Sample Preparation and Activity Assay. Rhodobacter
capsulatus formate dehydrogenase (RcFDH) was expressed and
28
purified as described previously. The variants of RcFDH, Cys386Ser
−
FdsC, treated with the substrate formate (HCOO ), or variants
and His387Met, were obtained by PCR mutagenesis. Redox changes in
+
with amino acid exchanges (Cys386Ser and His387Met) at the
Moco active site were analyzed. The relative catalytic activities
of formate oxidation of the WT, ΔFdsC, C386S, and H387M
RcFDH variants at pH values of 7.0, 8.0, and 9.0 are
summarized in Table 1. RcFDHWT was almost inactive at pH
RcFDH samples were induced by adding either 5 mM NAD as an
oxidant or 10 mM NaDT or 50 mM sodium formate as reductants. For
pH conditions of 8.0 and 9.0, 75 mM potassium phospate was used as
a buffer, while 50 mM Tris/HCl buffer was used for pH conditions of
7
.0. Samples were transferred to ultracentrifugation devices
(
VIVASPIN 20, 50-kDa cutoff; Sartorius AG, Gottingen, Germany)
̈
and sealed with parafilm inside an anaerobic chamber and
concentrated to ∼1 mM of enzyme-bound Mo (4 °C, 3000g overnight
centrifugation). Samples were finally transferred to sample holders (30
μL) for XAS and stored in liquid nitrogen. The activities of RcFDH
WT and protein variants were analyzed with formate as the substrate,
as described elsewhere. Measurements over a wide pH range (5−11)
were carried out using overlapping buffer systems (100 mM Britton/
Robinson, 100 mM potassium phosphate, 100 mM Tris/HCl, 100 mM
CHES, or 100 mM CAPS).
Table 1. Relative Activities for Formate Oxidation of RcFDH
WT and Variants
a
Relative Activity (%)
b
28
RcFDH variant
pH 7.0
pH 8.0
pH 9.0
pH optimum
9.0
WT
6
0
88
0
100
0
ΔFdsC
C386S
H387M
0
0
0
X-ray Absorption Spectroscopy. XAS at the Mo K-edge was
performed at the synchrotron SOLEIL (Paris, France) at the SAMBA
bending-magnet beamline, with the storage ring operated in top-up
48
56
21
7.5
a
Activities were normalized to the maximal WT enzyme activity at pH
b
3
4,50,51
9.0, which was set to 100%. Measured in a pH range of 5.0−11.0,
using overlapping pH-buffer systems.
mode (430 mA), as previously described.
A double-crystal
Si(220) monochromator was used for scanning of the excitation
energy, and two palladium-coated mirrors in grazing incidence mode
were used for focusing of the X-ray beam and for harmonics rejection.
2
The X-ray spot size on the sample was set by slits to ∼3 × 1 mm . The
7.0 and most active at pH 9.0, and the RcFDHΔFdsC and
RcFDHC386S variants were inactive in the entire pH range. In
the RcFDHH387M variant, the pH optimum was shifted to a
value close to 8.0, where it showed ∼56% WT activity, and
lower activities were observed at pH 7.0 and 9.0. XANES and
EXAFS spectra at the Mo K-edge of the RcFDH protein
variants were collected and the spectral analysis is presented in
the following.
energy axis was calibrated (accuracy ±0.15 eV) using the first
inflection point at 20003.9 eV in the simultaneously measured
absorption spectrum of a Mo foil as a standard. Fluorescence-detected
XAS spectra were measured using an energy-resolving 36-element
monolithic planar germanium pixel array detector (Canberra), which
was shielded by 10-μm-thick zirconium foil against scattered incident
−1
X-rays. The total incoming count rate was kept below 30 000 s per
element, to avoid detector saturation. Samples were held in a
laboratory-built liquid-helium cryostat at 20 K. XAS spectra (scan
A Sulfido Ligand at the Mo Site in RcFDH. Mo-XANES
spectra of oxidized RcFDH WT protein at pH values ranging
from 7.0 to 9.0 are shown in Figure 2. The spectra of all
−1
range of 19 850−20 770 eV, approximately, up to k = 14.2 Å ; 1−2
scans per sample spot) were corrected for detector deadtime.
Averaging (6−10 scans per sample, 1−2 scans per spot), normal-
ization, and extraction of EXAFS oscillations and conversion of the
energy scale to the wave-vector (k) scale were performed as previously
ox
RcFDHWT samples were relatively featureless, showed only a
small pre-edge absorption peak, and exhibited rather minor
spectral differences at the varying pH values. Comparison of the
mean pre-edge area (Apre‑edge) and K-edge energy (Eedge) of
3
4,39
3
2
described.
k -weighted EXAFS spectra were simulated (S0 = 1.0)
by a least-squares procedure using phase functions calculated with
ox
6
0,61
RcFDHWT with the respective parameters of other molyb-
FEFF7,
in-house software SimX
extending over 10% at both k-range ends). E was refined to 20 014 ±
and Fourier transforms (FTs) were calculated using the
39,62
−1
2
doenzymes and model compounds with known Mo coordina-
(k-range of 1.8−14.2 Å , cos windows
ox
tion and redox level (Table 2) revealed that RcFDHWT
0
2
eV in the fit procedure. The fit quality was judged by calculation of
contained mostly Mo(VI) and suggested on average one or
39
the Fourier-filtered R-factor (RF). We note that, because of the
statistical coupling between coordination numbers (N) and Debye−
Waller parameters (2σ ) in the EXAFS fits, the precision in these
values is in the range of 15%−25%. K-edge energies were determined
at the 50% level of normalized XANES spectra (edge half-height).
Areas of the pre-edge peak feature in the XANES were determined
within an energy range of ∼20 001−20 015 eV after subtraction of a
polynomial spline to remove the main edge rise from the spectra using
fewer short Mo−O bonds at the Moco (such as Mo=O or Mo−
−
O ; we note that the assignment of the longer bonds of ∼1.8 Å
2
−
to Mo−O is formal and reflects the partial double bond and
41
−
O character of the ligand; the expected Mo(VI)−OH bond
length is ∼1.9−2.0 Å (see ref 68)) (Figure 2, inset). The
−
presumably noninteger number of Mo=O/−O bonds and
relatively low edge energy suggested admixtures of molybde-
num species in other oxidation states and/or of cofactor sites
with varying numbers of oxygen ligands at molybdenum and,
63
the program XANDA. Bond valence sum (BVS) calculations were
performed using eq 1 and N (coordination number) and R
i
i
(interatomic distance) values derived from EXAFS or crystallographic
therefore, heterogeneity in the metal coordination in
ox
data (the sum is over all molybdenum−ligand bonds). The used B-
RcFDHWT
.
3
262
DOI: 10.1021/ic502880y
Inorg. Chem. 2015, 54, 3260−3271

Inorganic Chemistry
Article
a
Table 2. Mo K-Edge Energies and Pre-edge Areas
sample
E (K-edge) ± 0.1 [eV] A (pre-edge) ± 0.05 [r.u.]
a
RcFDH
WT(ox) pH 7
20013.0
20013.1
20013.2
20012.5
20012.8
20012.4
20013.3
20012.7
20012.9
20012.9
20012.7
References
20019.3
20017.5
20014.7
20014.3
20013.8
20011.3
20011.0
0.35
0.38
0.40
0.43
0.35
0.36
0.31
0.43
0.45
0.19
0.13
WT(ox) pH 8
WT(ox) pH 9
WT(red) pH 9
WT(formate) pH 7
WT(formate) pH 9
C386S(ox) pH 7
H387M(ox) pH 7
H387M(ox) pH 9
ΔFdsC(ox) pH 7
ΔFdsC(ox) pH 9
b
c
VI
(
O =)Mo
2.13
1.30
0.80
0.92
0.49
0.43
0.40
0.05
4
VI
(
O =)Mo S
3
2
d
VI
(
O =)Mo S
2
3
e
VI
(
O =)(S=)Mo S
2
3
f
O=)MoVIS4
(
g
h
i
O=)MoIVS4
(
(
S=)MoIVS4
Mo(IV)S6
20010.7
a
+
RcFDH was oxidized (ox) by NAD , reduced (red) by NaDT, or
b
formate-reduced at the given pH. Molybdate in aqueous solution (pH
7
c
34 d
.5). The isolated mono-pyranopterin Moco.
The mono-
81
pyranopterin Moco in sulfite oxidase with an S(Cys) ligand at Mo.
e
The mono-pyranopterin Moco in xanthine oxidase with an Mo=S
f
bond (Figure S3 in the Supporting Information). The bis-MPT Moco
in MobA protein.
Figure 2. Mo XANES spectra of RcFDH samples. Spectra were
3
4
g
h
Bisdithiolene Mo=O and Mo=S model
vertically displaced for comparison. Spectra (solid lines) represent
averaged data for RcFDH samples (WT, wildtype; ox, NAD -oxidized;
+
complexes (Figure S2 in the Supporting Information) shifted by 2
i
eV to higher energies. Solid molybdenum sulfide (MoS ) shifted by 2
2
red, NaDT-reduced; ΔFdsC, expressed in the absence of FdsC;
formate, reduced with formate; C386S and H387M, mutated variants)
in a pH-range of 7.0−9.0; bluish lines, five WT(ox) spectra at pH 7.0,
eV to higher energies.
8
.0, or 9.0; broken lines, spectra of reference samples (a)−(h)
for all RcFDH preparations (Table 3) suggested a bond length
spread (1σ) of ∼0.1 Å, which was in agreement with bond
(
spectrum (d) is shown in Figure S2B in the Supporting Information,
and spectra (e) and (g) are shown in Figure S3 in the Supporting
lengths differences observed in crystal structures of bis-pterin
Information) (Table 2); the spectrum of MoS (h) was up-shifted by 2
41,51
2
ligated Mo sites in enzymes and model complexes.
Non-
eV for comparison). The asterisk (*) marks the pre-edge feature and
the horizontal bar (−) the edge half-height. Inset: correlation of pre-
edge areas with edge energies. Open circles (○) denote references
integer numbers of Mo−O bonds (∼1.7 Å and ∼1.8 Å) were
observed. The addition of further Mo−X distances (X = C, N,
O, or S) at ∼2.65 and 2.85 Å further improved the fit quality. In
particular, the inclusion of a short Mo−S bond strongly
(
Table 2 and Figures S2 and S3 in the Supporting Information) with
varying Mo coordination (Table 2); solid symbols represent the mean
values for two RcFDH variants (Table 2; bars mark the value ranges
for pH 7.0−9.0). The linear fit (line) to the reference data was drawn
to guide the eye.
diminished R by a factor of ∼2 (Table 3). A contour plot of
F
the R value revealed a minimum in a coordination number
F
range of ∼0.4−0.6 bond per Mo ion, meaning that only about
every second Mo ion carried such a sulfur ligand. A bond length
of ∼2.17 Å was determined for this interaction (Figure 3).
Comparative EXAFS analysis of two model compounds with
The EXAFS spectra of RcFDHWTox samples are shown in
Figure 3. The averaged spectrum obtained for samples at more
alkaline pH was subjected to a detailed EXAFS simulation
analysis (Table 3). Gradual refinement of the curve-fitting
approach, starting from the inclusion of only two Mo−O/S
distances, by the addition of further metal−ligand interactions,
IV
IV
either S=Mo S or O=Mo S motifs, using the same set of
4
4
2
EXAFS phase functions and 2σ values for Mo=O/S bonds as
for the RcFDH, yielded a Mo=O distance of 1.68 Å, a Mo=S
distance of 2.16 Å, and coordination numbers close to the
expected unity values (Figure S2 and Table S1 in the
Supporting Information). EXAFS analysis of the monopyr-
anopterin cofactor containing Mo(VI) in xanthine dehydrogen-
ase (XDH) at pH 9.5 revealed the expected single Mo=S bond
as judged by the decreasing error sum (R ) finally yielded an
F
excellent fit quality (R < 5%) (Table 3, fits 1−7). Bond valence
F
sum (BVS) analysis has contributed to determination of the
5
9,69,70
metal site structure in various molybdenum enzymes.
6
8,71
The BVS that was calculated on the basis of the coordination
(2.18 Å)
plus one shorter Mo−O bond (1.69 Å) and one
numbers (N) and first-sphere molybdenum−ligand distances
longer Mo−O bond (1.79 Å), besides the two Mo−S bonds
(2.4−2.5 Å) (Figure S3 and Table S1 in the Supporting
Information). Protonation of an oxygen ligand at Mo(VI), for
example, in XDH at acidic pH has been reported to elongate
(R) from EXAFS in parallel increased to a value close to six, in
agreement with predominant Mo(VI) species. The detection of
close to four Mo−S bonds in the range of ∼2.3−2.4 Å indicated
a bis-MGD Moco structure in the RcFDH. Notably, the
68
the Mo−OH bond to ∼2.0 Å. According to these results, we
2
Debye−Waller parameters (2σ ) of the Mo−S(pterin) bonds
attribute the shortest distances to Mo=O (∼1.7 Å) and Mo−
3
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The EXAFS best-fit for RcFDHWTox yielded a total first-
sphere coordination number of bonds shorter than ∼2.7 Å (N)
and a respective BVS that were considerably larger than 6 and
therefore exceeded the values expected for a hexa-coordinated
−
Mo(VI) ion (Table 3, fit 7). The Mo=O/−O bonds
contributed particularly strongly to the N and BVS values.
Background contributions to the low-frequency EXAFS
oscillations of the short Mo−O distances (Figure 3) might
cause an overestimation of their coordination numbers.
However, the analysis of EXAFS spectra obtained using further
enhanced background suppression revealed an optimal fit error
for the processing procedure used for the data in Figure 3 and a
−
simultaneous decrease of the N values of the Mo=O/−O and
Mo=S bonds for excess background suppression (Figure S3 in
the Supporting Information). Selective diminishing of the N
−
value of the Mo=O/−O bonds therefore was not feasible. The
−
relative abundance of Mo=O/−O bonds, compared to the
Mo−S(MGD) bonds, may further be enhanced by contribu-
tions from incomplete Mo centers, in which, for example, only
one pyranopterin and three oxygen ligands were bound to
molybdenum and, therefore, the used N value for the Mo−
S(MGD) bonds (4) therefore was too large (Table 3). The
comparable N values for the Mo=S (∼2.17 Å) and Mo−X
(
∼2.65 Å) bonds suggested that they belong to the same metal
centers. This could be explained if the thiol sulfur of Cys386
was a molybdenum ligand in those centers that also contained a
Mo=S bond. The longer bonds were assigned to C/N/O/S
atoms in the second metal coordination sphere. Testing of
these assumptions still yielded a very good EXAFS fit result
(
Table 3, fit 8), and even restricting the total first-sphere
coordination number to six led to an R value of <10% and thus
F
to a good data simulation and a BVS value close to 6 (fit 9).
ox
Further information on the cofactor species in RcFDHWT
was obtained from analysis of changes in the EXAFS spectra
upon pH variation (Figure 3). We used the simulation
approaches described above (Table 3, fits 7−9) to simulate
the EXAFS spectra at pH 7.0, 8.0, and 9.0 (Table 3, fits 10−12;
also see Table S3 in the Supporting Information, fits 1−8). The
BVS values from the simulation results using a first-sphere
coordination number of six increased slightly with increasing
pH, possibly due to an increased population of incomplete Mo
centers at pH 9, but were in agreement with Mo(VI) at all pH
values. The bond lengths of the Mo−S (MGD) interactions
4
2
Figure 3. (A) EXAFS analysis of oxidized WT RcFDH. The top
portion of panel A shows Fourier transforms (FTs) of EXAFS spectra
at three pH values (inset: experimental EXAFS spectra (black lines)
and fit curves (colored lines) with parameters in Table 3 (fits 10, 11,
−
were pH-independent. The number of Mo−O bonds
increased at the expense of the number of Mo=O bonds at
more acidic pH, possibly reflecting a protonation event in the
vicinity of the cofactor. In parallel, the numbers of Mo=S and
longer Mo−S bonds decreased whereas the number of Mo−X
12)). The bottom panel in panel A shows the experimental spectrum
(
dots) of averaged data in the pH range of 8.0−9.0 and the fit curve
(
solid line, fit 7) and contributions from the indicated Mo−ligand
(
3
X = C/N/O/S) bonds increased at more-acidic pH (Figure
B). This could be explained by detachment of S(Cys386)
distances (colored lines, vertically down-shifted); gray dots, calculated
spectrum for a pure S=MoS (MGD) S(Cys386) structure. Lower inset
4
2
from molybdenum and its location in the second coordination
sphere, mostly at low pH. However, the almost-doubled
shows a contour plot of R for variation of the Mo=S bond length and
F
coordination number (gray to red color changes represent a transition
from low to high fit qualities). (B) Changes of coordination numbers
and Mo−ligand distances for pH increase (arrows; averaged values
from fits 10/2, 11/4, and 12/6 in Table 3 and Table S3 in the
Supporting Information; bars show the parameter ranges from the two
fits).
2
Debye−Waller parameter (2σ ) of the Mo−S distances at pH
7
.0, compared to those at pH 9.0, could also suggest an increase
of the sulfur bond length from ∼2.17 Å to ∼2.3 Å, possibly due
to a conversion of Mo=S to Mo−SH.
The EXAFS parameters, in principle, were compatible with a
ox
variety of Mo site structures in RcFDHWT . Mo(VI) species
with four Mo−S bonds (for example, from dithiolene-like
ligands) and carrying both an oxo and a sulfido ligand have
been proposed as interconversion intermediates in the
O− (∼1.8 Å) bonds and assign the ∼2.17 Å bond to an
unprotonated terminal sulfur (Mo=S) in RcFDH. These
findings indicate a sulfido ligand at the molybdenum in a
literature, but respective crystallized chemical models were
ox
54−58
RcFDHWT fraction of ∼50%.
not reported.
We therefore consider this motif as less
3
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Inorganic Chemistry
Article
a
Table 3. EXAFS Simulation Parameters for RcFDH Samples
2
3
2
b
N [per Mo]/R [Å]/2σ (× 10 ) [Å ]
c
d
RcFDH
fit
Mo=O, −O
Mo=S
Mo−S
Mo···X
fit error sum, R [%]
bond valence sum, BVS
F
WT
(
ox) avg
1
2
3
4
1.2/1.72/4
1.5/1.71/7
1.3/1.71/5
2.7/2.39/14
3.8/2.36/16
3.6/2.36/15
3.8/2.36/15
23.1
12.9
12.0
8.5
4.0
6.7
6.3
6.7
0.6/2.17/4
0.7/2.17/6
0.5/2.17/3
0.9/2.87/15
0.9/2.87/15
e
e
e
e
e
e
e
e
e
e
e
e
e
0.9/1.68/2
e
0
.7/1.78/2
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
f
g
g
5
6
7
0.9/1.68/2
.7/1.78/2
0.9/1.68/2
.7/1.78/2
0.8/1.68/2
.4/1.78/2
0.9/1.68/2
.5/1.77/2
0.8/1.68/2
.4/1.78/2
0.7/1.67/2
.6/1.77/2
0.9/1.68/2
.5/1.78/2
1.3/1.70/2
.3/1.81/2
0.9/1.71/2
.2/1.86/2
0.8/1.68/2
.8/1.78/2
1.1/1.72/2
.5/2.22/2
0.6/2.17/3
0.6/2.17/3
0.5/2.17/3
0.5/2.17/3
0.4/2.17/3
0.2/2.16/3
0.4/2.16/3
0.6/2.17/3
0.5/2.21/3
0.2/2.15/3
0.5/2.19/3
2 /2.31/10
0.7/2.87/10
0.8/2.86/16
1.7/2.86/18
1.6/2.84/17
1.6/2.84/15
1.8/2.85/27
1.6/2.85/17
1.4/2.84/12
1.5/2.84/13
1.8/2.84/22
1.5/3.04/15
7.7
8.1
3.7
4.2
8.8
6.5
5.7
6.1
8.5
7.9
8.9
7.1
6.8
6.6
6.6
6.2
5.9
6.5
7.2
5.9
6.5
5.9
f
0
2 /2.41/10
f
4 /2.36/16
0
f
4 /2.36/18
e
0
0.5/2.64/3
h
f
8
4 /2.37/17
e
0
0.5/2.63/3
f
i
9
4 /2.37/15
e
0
0.4/2.63/3
f
h
(
(
(
(
(
(
ox) pH 7
10
11
12
13
14
15
4 /2.36/27
e
0
0.2/2.81/3
f
h
h
h
h
h
ox) pH 8
4 /2.37/17
e
0
0.4/2.68/3
f
ox) pH 9
4 /2.37/12
e
0
0.6/2.63/3
f
red) pH 9
formate) pH 7
formate) pH 9
4 /2.40/13
e
0
0.5/2.63/3
f
4 /2.36/22
e
0
0.2/2.67/3
f
4 /2.38/15
e
0
0.5/2.90/3
ΔFdsC
h
h
e
e
e
e
e
e
f
(ox) pH 7
16
17
0.6/1.68/2
.5/1.78/2
0.7/1.69/2
.3/1.79/2
0.2/2.15/3
0.1/2.16/3
4 /2.42/13
1.4/2.79/12
0.7/2.87/16
8.0
7.9
5.5
5.5
f
e
0
1 /2.54/3
f
(ox) pH 9
4 /2.42/16
f
e
0
1 /2.44/3
C386S
ox) pH 7
e
e
e
e
f
(
18
0.6/1.68/2
0.3/2.14/3
4 /2.34/24
1.3/2.82/24
7.6
6.1
0.5/1.78/2
.6/2.41/3
0
H387M
h
h
e
e
e
e
e
e
f
(ox) pH 7
19
20
0.9/1.69/2
.4/1.81/2
1.1/1.74/2
.8/2.20/2
0.4/2.16/3
0.1/2.17/3
4 /2.37/17
1.6/2.88/18
1.9/2.85/24
9.0
8.6
6.3
5.7
e
0
0.4/2.68/3
f
(ox) pH 9
4 /2.38/244
e
0
0.1/2.65/3
a
2
For the Mo=O/S ligands, 2σ values were used, which are close to the physically reasonable lower limit, meaning that the respective N values
2
82,83
correspond to the maximal fitted contribution of the ligand atom to the EXAFS for the given 2σ value.
The results of further EXAFS simulation
b
2
approaches are listed in Table S3 in the Supporting Information. Legend: N, coordination number; R, Mo−ligand distance; and 2σ , Debye−Waller
parameter. Samples are denoted as shown in the text (ox, oxidized; red, reduced); avg, data for averaged spectrum of RcFDH in the pH range of 8.0−
c
d
e
2
9
.0. Fit restraints: Sulfur was used as ligand X in all fits. Data taken from ref 39. Data obtained using eq 1. 2σ values for Mo=O, −O, Mo=S, and
f g
2
the second Mo−S bond were fixed in fits 7−20. Integer N-values were fixed. Respective 2σ values were coupled to yield the same value for both
h
i
shells in the fit. N(Mo=S) = N(Mo−S) of the second Mo−S distance and N(Mo···X) = 2 − N(Mo=S). The summed N value of distances <2.7 Å
was set to 6.
likely in RcFDH. Numerous examples of crystallized Mo(VI)S
also was particularly large at high pH, which could be explained,
for example, by the replacement of one MGD ligand to
molybdenum by two oxo groups in a sample fraction.
4
54−58
compounds carrying two oxo groups exist,
but such a
72
motif has not been observed in crystal structures of bis-MGD
9
containing enzymes. The direct binding of the thiol sulfur of
These considerations suggest two main Moco species in
ox
Cys386 to molybdenum at high pH and partial detachment of
this residue at low pH were suggested by the EXAFS data. In
addition, the Mo=S bond was more prominent at high pH.
RcFDHWT . Their relative amounts were estimated from
comparison of calculated and experimental Mo=S and
Mo=O/−O− coordination numbers (Table S2 in the
−
VI
However, the coordination number of the Mo=O/−O bonds
Supporting Information). At low pH, a (O=)Mo S (MGD)
4
2
3
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Article
structure in the main fraction of centers (∼70%) explained the
XAS data well (Figure 4). To account for the increased Mo=O/
Figure 4. Main molybdenum cofactor species in RcFDHWTox.
Proposed structures of Mo sites (shown in schematic representation)
were constructed based on the EXAFS analysis (Figure 3) and in
analogy to crystal structures of other FDH proteins (Figure 1): (a)
structure of the inactive Moco prevailing at low pH (the Cys386 thiol
group may as well be protonated), and (b) structure of the active
Moco predominant at high pH. Given Mo−ligand distances refer to
data in Table 3 and Table S3 in the Supporting Information (bond
length range in parentheses calculated from the Debye−Waller factor).
For further details, see the text.
−
−
O coordination number at high pH, significant fractions of
such a site may have lost a pyranopterin ligand to yield an
O=) Mo S (MGD) structure. As the main cofactor species
∼50%) at alkaline pH, a (S=)Mo S (MGD) S(Cys386)
VI
(
(
3 2
VI
4
2
structure was identified (Figure 4). Comparison of the XAS
results to the relative enzyme activities (Table 1) implied that
Moco species with oxo groups at molybdenum were likely
inactive whereas species with sulfido and S(Cys386) ligands
represent active RcFDH enzymes.
The Chaperone FdsC Inserts the Sulfido Ligand at the
Molybdenum Active-Site. XAS spectra were collected for
oxidized RcFDH expressed in the absence of the specific
ox
chaperone FdsC (Figure 5A). The XANES of RcFDH
ΔFdsC
differed pronouncedly from the one of RcFDHWTox and was
rather similar to the one of molybdenum sulfide (MoS with
2
the metal coordinated to six S-ligands) in showing a diminished
pre-edge feature, suggesting a decreased number of Mo=O/S
bonds at Mo, leading to homogenization of the overall bond
lengths at Mo and increased coordination symmetry (Figure 2).
Figure 5. RcFDH protein variants studied by EXAFS. Shown are FTs
for the indicated samples (experimental data) of EXAFS spectra in the
insets (black lines, experimental data; colored lines, simulations with
best-fit parameters in Table 3). Spectra were (in part) vertically
shifted. (A) Comparison of data for RcFDH expressed in the presence
The edge energy, on average, was ∼0.4 eV lower for
ox
RcFDH
(Table 2), which was in agreement with such a
ΔFdsC
coordination change at a Mo(VI) site, but also with more
(WT(ox), averaged spectrum from data at pH 7.0, 8.0, and 9.0; Figure
reduced molybdenum in some centers. The Fourier transforms
3
) or absence of the maturase FdsC. (B) Data for RcFDHWT reduced
ox
(
FTs) of the EXAFS spectra of RcFDHΔFdsC at pH 9.0 and pH
with formate or NaDT. The bottom curves show the EXAFS
contributions of Mo=S bonds and Mo−O distances assigned to
formate binding to Mo at pH 9.0. (C) Data for RcFDH variants
carrying two different amino acid exchanges.
7
.0 showed a pronouncedly increased main maximum, in
ox
comparison to RcFDHWT , suggesting more homogeneous
sulfur coordination at molybdenum (Figure 5A). The EXAFS
simulations revealed close to five Mo−S bonds in
ox
RcFDH
, which were ∼0.05 Å longer than the Mo−
We attribute the fifth sulfur ligand to the Mo−S(Cys386) bond
ΔFdsC
ox
WT
ox
ox
S (MGD) bonds in RcFDH
. In addition, fewer oxygen
in RcFDH
, which is shorter than in RcFDH
. A closer
4
2
ΔFdsC
WT
ox
ox
ligands were detected for RcFDHΔFdsC than for RcFDHWT
Table 3, as well as Table S3 in the Supporting Information).
look at the EXAFS revealed the predominance of shorter
−
(
Mo=O bonds (∼1.7 Å) at pH 9.0 and of longer Mo−O bonds
3
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Inorganic Chemistry
∼1.8 Å) at pH 7.0. Furthermore, the Mo−S(Cys386) and
Article
(
Formate Binds to Molybdenum. Substrate binding and
Mo−S (MGD) bond lengths were similar (∼2.4 Å) at pH 9.0,
redox chemistry at Moco were studied in RcFDH samples at
4
2
formate
but the former bond was ∼0.1 Å elongated at pH 7.0. These
pH 7.0 or 9.0. The XANES shape of RcFDHWT
was
similar, but the edge energies were ∼0.5 eV (pH 7.0) or ∼0.8
changes point to a protonation event around the Moco at low
ox
ox
eV (pH 9.0) lower, compared to RcFDH
containing
pH similar to RcFDHWT . The fitted coordination numbers of
WT
Mo(VI) (Figure 2 and Table 2). Assuming a downshift of the
Mo=S bonds were insignificantly small and thus, a sulfido
ligand was absent both at pH 9.0 and pH 7.0 (Table 3 and
Table S3 in the Supporting Information). In summary, the XAS
K-edge energy by ∼1 eV per single-electron reduction of
3
4,73
molybdenum in similar coordination geometries
suggested
VI
the formation of ∼25% or ∼40% of Mo(IV) and therefore
data suggest a main (O=)Mo S (MGD) S(Cys386) site
4
2
formate
incomplete metal reduction in RcFDH
. However, the
ox
WT
without an Mo=S bond in RcFDHΔFdsC (Figure 6). In
addition, sites lacking the S(Cys386) ligand or containing
additional oxygen ligands were likely present, as in the WT
enzyme. These results corroborate the insertion of the sulfido
accompanying coordination change at molybdenum (see
below) may lead to an underestimation of the reduced metal
formate
fraction. The EXAFS spectra of RcFDH
and
WT
ox
RcFDHWT at pH 7.0 were rather similar (Figure 5B) and
the fit analysis accordingly revealed similar Mo coordination
with low numbers of Mo=S bonds in both samples (Table 3
and Table S3 in the Supporting Information). Formate
apparently did not change the Mo coordination significantly
2
8,37
group into Moco by the chaperone FdsC.
at pH 7.0. However, the FT of the EXAFS spectrum of
formate
RcFDHWT
at pH 9.0 differed from the spectrum of
ox
RcFDHWT (Figure 5B). According to the fit analysis (Table 3
and Table S3 in the Supporting Information), these differences
were mostly attributable to an additional Mo−O bond of ∼2.2
Å length with a similar coordination number (∼0.5 per Mo) as
the Mo=S bond, the lack of an Mo−S bond of ∼2.6 Å, and an
increased number of longer Mo−X distances. This result
suggested that S(Cys386) was not bound to molybdenum in
formate
formate
RcFDHWT
. The Mo=S bond in RcFDH
was
WT
ox
slightly longer compared to RcFDHWT . Additional Mo=O
formate
WT
bonds were also detectable in RcFDH
. The BVS for
at pH 9.0 was ∼0.8 units lower, compared to
formate
RcFDHWT
ox
the value of RcFDHWT (Table 3 and Table S3 in the
Supporting Information), in agreement with the K-edge
downshift. A consistent interpretation of the XAS results on
formate
RcFDHWT
was obtained by the assumption that only in
those (active) centers, which contained an Mo=S bond (Figure
), an oxygen of a formate molecule became a molybdenum
3
ligand by replacing the thiol sulfur of Cys386, thereby forming
Mo(IV) (Figure 6). These results might favor a monodentate
coordination of formate at the molybdenum. The longer Mo−
ligand distances thus may reflect the further C/O atoms of
formate and/or the Cys386 side chain. More specific
attributions of the longer distances are precluded because of
the limited discrimination of low-Z atoms at long distance to
Mo in the EXAFS analysis.
Chemical reduction of the RcFDH at pH 9.0 by the single-
electron donor sodium-dithionite (NaDT), in the absence of
formate, resulted in a K-edge downshift by ∼0.6 eV and, thus,
Figure 6. Model structures for the Moco in RcFDH. Structures
represent the more relevant Mo site species under the specified
conditions; bond lengths and Mo oxidation states stem from the XAS
analysis. (a) Moco structure in inactive RcFDH expressed without
FdsC (the S(Cys386) ligand more prominent at alkaline pH may be
replaced by a further oxygen ligand at lower pH). (b) A Mo−O bond
at the Moco is attributed to formate binding in WT enzyme (at
alkaline pH). (c, d) Bond lengths elongations in the first Mo ligation
sphere are attributed to Mo(V) formation in NaDT-reduced RcFDH
the formation of ∼60% Mo(V) (Table 2). Otherwise, the
red
XANES of RcFDH
was rather similar to the one of
WT
ox
RcFDHWT (Figure 2). Analysis of the EXAFS spectrum
(
Figure 5B) revealed by ∼0.04 Å enlongated Mo−S(MGD)
red ox
bonds in RcFDH
, compared to RcFDH
(Table 3 and
WT
WT
Table S3 in the Supporting Information), in agreement with
significant metal reduction, and similar Mo=O/S coordination
numbers in both samples. An additional Mo−S bond (∼2.6 Å),
attributed to the S(Cys386) group, was also detectable in
(
at alkaline pH). (e) In the inactive C386S variant, besides of a sulfido
ligand, O(Ser386) may interact weakly with Mo. (f) The Moco
structure in the active H387M variant at low pH is more similar to the
structure in the active WT at high pH, whereas at high pH the
S(Cys386) and Mo=S ligands were partially lost. The observed Mo−S
bond elongation may suggest protonation of the sulfido ligand in the
structures shown in panels (b) and (d). For further details, see the
text.
red
RcFDHWT . However, the shortest Mo−S distance was ∼2.23
Å and therefore ∼0.05 Å longer than the Mo=S bond in
ox
RcFDH
. This may be explained by a closer interaction of
WT
the sulfur with the S(Cys386) group at the Mo(V) species or
red
by Mo−SH formation in RcFDHWT (Figure 6).
3
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Article
Altered Mo Coordination in the C386S and H387M
Variants. Two variants of the RcFDH with mutations at the
cofactor site were studied, namely Cys386Ser and His387Met.
ox
The similarity of the XANES to the one of RcFDHWT
suggested the presence of predominantly Mo(VI) in both
variants (Figure 2, Table 2). The FTs of the EXAFS spectra of
the variants, however, showed discernible peak amplitude
ox
differences (Figure 5C). For RcFDHC386S , the EXAFS analysis
−
revealed a distribution of Mo=O/−O and Mo=S bonds similar
ox
to that for RcFDH
, in agreement with a bis-MGD structure
WT
also in this variant. Mo−S distances of ∼2.6 Å were barely
detectable, but a high-quality EXAFS fit was obtained including
an additional long Mo−O bond of ∼2.4 Å (Table 3 and Table
S3 in the Supporting Information). The BVS also was in
ox
agreement with a Mo(VI) ion in RcFDHC386S . These
observations suggested that either the side-chain oxygen of
Ser386 or a water species was (weakly) coordinated to Mo in
ox
RcFDHC386S , presumably in those centers that did not contain
a Mo=S bond (Figure 6).
By comparison, the increased pre-edge feature in the
ox
RcFDHH387M variant at pH 9.0 suggested that more oxygen
ligands at the molybdenum were present, compared to this
variant at pH 7.0 and to the WT (Table 2). The EXAFS
ox
analysis showed that, in RcFDHH387M at pH 7.0 (Figure 5C),
the molybdenum coordination by O/S ligands overall was
ox
similar to the one in RcFDHWT , in agreement with a bis-MGD
structure. However, the considerably smaller Debye−Waller
2
parameter (2σ ) of the Mo−S(MGD) bonds implied a
Figure 7. Correlation between XAS parameters and activity of RcFDH
variants. BVS (reflecting the approximate Mo oxidation level) and
N(Mo=S) values refer to EXAFS simulations using a total N value of 6,
and the error bars reflect the respective parameter ranges for the three
simulation approaches in Table 3 and Table S3 in the Supporting
significant geometry change around the metal in the variant
at pH 7.0, including a shortening of the Mo−S(Cys386) bond,
ox
which also was more prominent in RcFDH
RcFDHWT
than in
H387M
ox
(Table 3 and Table S3 in the Supporting
Information). In addition, the increased bias of the short
Mo−O bonds to the Mo=O form at pH 7.0 in the variant was
more similar to the situation at pH 9.0 than at pH 7.0 in the
ox
Information. Dashed lines mark the values for RcFDHWT at pH 9.0,
where the enzyme is most active; the same activities (set to 100%)
were plotted for oxidized and NaDT- or formate-reduced RcFDHWT
for comparison. The given E (K-edge) values were obtained after
subtraction of 20010 eV.
ox
WT protein. In RcFDHH387M at pH 9.0, the sulfido and
S(Cys386) ligands were mostly lost and an additional longer
Mo−O bond (∼2.2 Å) may be attributed to a water species
interacting with molybdenum (Table 3 and Table S3 in the
present in centers that also bear a sulfido group. Accordingly, a
Supporting Information). These results suggest a cofactor
VI
(
S=)(SCys386)Mo S (MGD) configuration of the oxidized
4
2
ox
configuration in RcFDH
at pH 7.0 more similar to the
H387M
ox
cofactor is proposed for active RcFDH. The interpretation of
FDH crystal data has remained ambiguous regarding the Mo
configuration in RcFDHWT at pH 9.0, which could explain the
enhanced relative activity of the variant at lower pH, whereas, at
high pH, the variant was more susceptible to deactivation by
sulfido and S(Cys386) ligand loss (Figure 6).
ligation, although a sulfur ligand (−SH) has been favored after
18,19,21
reinterpretation of structures.
In the corresponding PDB
database entries (2IV2 and 1H0H), the respective ligand was
1
9,21
assigned as “unknown”.
The only structure in the PDB
DISCUSSION
■
database for DMSO-reductase type enzymes assigning con-
comitant sulfur and S(Cys) ligation of molybdenum is for a
Structure of the Active Site in RcFDH. We have
presented the first XAS characterization of the FDH from R.
capsulatus. The XAS analysis of the Mo site structure has
consistently revealed a short Mo−S bond (∼2.17 Å) in those
RcFDH samples that showed catalytic activity (Figure 7).
According to structural data for other molybdoenzymes like
2
3,24
periplasmic nitrate reductase.
However, the rather long
bond (∼2.4 Å) in the latter structure suggests a sulfhydryl
(Mo−SH) rather than a Mo=S ligand as in RcFDH. In an
earlier Se-XAS study of E. coli Mo−FDH, a close interaction
between the Se(Cys) group and a dithiolene sulfur was
6
8,71,74
54−58
59
xanthine oxidase,
this bond corresponds to a terminal unprotonated sulfido group
Mo=S) at the Mo(VI) center in the bis-MGD structure.
and synthetic model complexes,
suggested. Because a sulfido ligand had not been considered,
this interaction may rather reflect a short distance between the
sulfido group and the Se atom. Our XAS data provide the first
direct evidence that a Mo=S motif is an integral part of the
molybdenum cofactor in RcFDH.
(
Maximally, ∼60% sulfuration of the metal site was observed at a
pH of 9.0, where the WT RcFDH exhibits the optimum of
formate oxidation activity. In addition, the numbers of short
sulfido bonds and longer (∼2.6 Å) Mo−S distances per Mo ion
were correlated. In analogy to the situation in crystal structures
Insertion of the Sulfido Ligand at the Moco. In RcFDH
expressed in the absence of the specific bis-MGD binding
2
8,37
chaperone FdsC, but in the presence of FdsD,
a Mo=S
1
8,19,21
of other FDHs,
we therefore suggest that a Mo−
bond was not detectable by XAS and an oxo-ligand was present
in most of the active sites instead. This result strongly supports
S(Cys386) bond, similar to the Mo/W-SeCys binding, is
3
268
DOI: 10.1021/ic502880y
Inorg. Chem. 2015, 54, 3260−3271

Inorganic Chemistry
Article
the previous hypothesis that RcFdsC is involved in the
replacement of the oxo ligand by a sulfido ligand to insert
with Mo=O bonds may reflect incompletely maturated cofactor
species in the RcFDH preparations. In any event, cofactors with
oxygen ligands represent inactive RcFDH enzyme. The
pathways of oxidative Moco modification in the RcFDH, in
28,37
the sulfido ligand at the bis-MGD cofactor.
Previous studies
37
showed that FdsC binds bis-MGD directly. A sulfur-
transferase activity was suggested for the homologous FdhD
relation to its O tolerance, remain to be determined.
2
2
2
protein in E. coli. The detection of almost five similar Mo−S
bonds implied that the bis-MGD structure was preserved in
RcFDHΔFdsC and that S(Cys386) likely still was a molybdenum
ligand in a less-distorted cofactor structure. This geometry
apparently stabilized the bis-MGD structure in the ΔFdsC
variant, leading to diminished oxidative cofactor degradation
compared to the WT enzyme, in agreement with previous
Mechanism of Formate Conversion. Different reaction
schemes have been considered for the formate oxidation
chemistry to explain the differences in the crystallographic
structures of FDHs regarding to the Se/S(Cys) coordination to
1
5−18,21,77
the metal.
Except the mechanism proposed by
18
Boyington et al., they all involve transient detachment of
(H)Se/S(Cys) from the metal due to replacement by the
substrate (unbinding mechanism). We found that (i) the
2
8,37
21
findings.
That the ΔFdsC variant was inactive further
supports an essential character of the sulfido group.
Interestingly, a sulfido ligand could still be detected in the
C386S variant of the RcFDH lacking the Mo−S(Cys386) bond,
which was expressed in the presence of FdsC (Figure 7).
Seemingly, the presence of a cysteine at position 386 is not
essential for the stability of a sulfido ligand at the molybdenum.
coordination number of the putative Mo−S(Cys386) bond is
unchanged in NaDT-reduced RcFDHWT and that (ii) a longer
Mo−O interaction replaced a Mo−S bond in the formate-
reduced enzyme, whereas (iii) the Mo=S occupancy remained
unchanged under both conditions compared to the oxidized
protein. These results support the unbinding mechanism,
meaning that formate likely is a Mo(IV) ligand and replaces the
cysteine rather than the sulfido ligand, as suggested ear-
The particularly short Mo=S bond in RcFDH
was similar
C386S
75
to that of a synthetic bis-dithiolene complex, suggesting only
a sole apical sulfido ligand in a fraction of the C386S variant. In
a further cofactor species carrying an oxo ligand instead, the
hydroxyl group of Ser386 perhaps could weakly coordinate to
Mo. However, all these cofactor species were fully inactive,
indicating that concomitant sulfido and S(Cys386) ligation is
required to promote activity.
1
5,16,21
lier.
This could be followed by rebinding of the
release
S(Cys386) after single electron abstraction and CO
2
to generate the Mo(V) site (Figure 8). S(Cys386) coordination
Inactive Molybdenum Cofactor Species. The optimum
pH of WT RcFDH for formate oxidation is 9.0, whereas at pH
7
.0 the enzyme is almost inactive. The lower activity at more-
acidic pH apparently was correlated with the parallel loss of
Mo=S/−S(Cys386) bonds and gain in Mo−O bonds (Figure
7). The XAS data have suggested that Mo centers containing
oxygen ligands did not react with formate and were redox
inactive (Figure 7). Accordingly, we attribute such metal sites
to catalytically incompetent enzyme states. The apparent
decrease in Mo=S coordination number, however, according
to the larger bond lengths spread, may also result from bond
elongation to the lower limit of the Mo−S(MGD) range
Figure 8. Tentative reaction cycle of formate oxidation in RcFDH.
Structural motifs of the Mo sites are based on our XAS results.
Essential roles of the sulfido ligand and of the conserved Cys386 and
His387 residues in formate positioning and proton management
during the C−H bond cleavage and redox reactions are suggested as
discussed in the text. The dashed line marks partial double-bond
character; group charges were omitted for simplicity. The position of
the formate proton (H) in the Mo(IV) site is unclear, possible
(
∼2.25−2.50 Å at pH 7.0). In addition, an increasing number
of ∼0.1 Å elongated Mo−O bonds was observed for pH-
lowering in WT, ΔFdsC, and C386S variants. These
observations may point to a protonation event near the
cofactor at lower pH.
−
locations at (H)COO , (H)S(Cys386), (H)N(His387), or Mo−S(H)
are conceivable. The Mo(V) state presumably contains an S(Cys386)
and a sulfido ligand at the metal according to our XAS results; its in-
depth structural characterization requires further studies. The CO2/
formate conversion cycle certainly involves further intermediates,
which, however, were not resolved by XAS and therefore were not
included in the scheme.
Several conserved histidine residues may be located close to
the Mo binding site in RcFDH, similar to FDH proteins from
1
5,18,19,21
other organisms.
His387 in RcFDH is homologous to
His141 in the E. coli Mo−FDH, and this residue was suggested
to function as a proton acceptor during the C−H bond
1
7,21
activation chemistry.
Protonation of the histidine, with a pK
value near neutral pH, thus may cause inactivation of RcFDH in
a reversible fashion. That the H387M variant of RcFDH, in
which the methionine side chain could not be protonated, was
active, even at low pH, supports this view (see below). In
addition, irreversibly inactive cofactor species must be
considered. Protonation events at low pH, for example of the
imidazole side chain and/or the Mo=S/−S(Cys386) ligands,
may facilitate replacement of the Mo=S by a Mo=O and
S(Cys386) detachment, resulting in a (O=)MoS (MGD) site,
to Mo(V) is in agreement with electron paramagnetic
9
,27,78
resonance (EPR) studies on various FDH enzymes.
The
∼0.05 Å elongation of the Mo=S bond detected by XAS in
reduced RcFDH may point to a close interaction between
WT
the S(Cys396) and the sulfido group at Mo(V), but may also
reflect protonation to form a Mo−SH species. Transient
formation of a Mo−S−Se/S(Cys) motif is an ingredient of the
so-called “sulfur-shift” mechanism of formate conversion that
1
1,15,16,79,80
has been previously suggested.
We note that Mo-
4
2
which may even convert to a (O=) MoS (MGD) species with
XAS data are not conclusive on the formation of a S−S bond in
RcFDH. A formate species may coordinate primarily to the
metal, but may also interact with the sulfido or sulfhydryl ligand
at Mo(IV) (Figure 8). Quantum chemical calculations have
3
2
one remote pyranopterin ligand, according to our XAS data. A
remote pterin group has been observed in crystals of DMSO-
7
2,76
reductase under certain conditions.
Alternatively, Mo sites
3
269
DOI: 10.1021/ic502880y
Inorg. Chem. 2015, 54, 3260−3271

Inorganic Chemistry
Article
suggested that also from a thermodynamic point of view, the
unbinding mechanism is favored, because of exothermic
product formation.
to S.L. and Grant No. Ha3265/6-1 to M.H.). C.S. thanks the
European Research Council (ERC) for support (Grant No. Ga-
281257). M.H. thanks the DFG for a Heisenberg Fellowship
1
6,17
Interestingly, the H387M variant showed at least 75% of WT
and the German Bundesministerium fu
Forschung for financial support within the Ro
Cluster (Grant No. 05K14KE1). We thank A. Zitolo, E. Fonda,
and V. Briois at Samba of Soleil for excellent technical support.
The authors thank B. Duffus (University of Potsdam) for
critical reading of the manuscript. The European Union COST
Action Program (No. CM1003) is gratefully acknowledged.
̈
r Bildung und
ntgen-Angstro
activity at an optimal pH that was ∼1.5 units shifted to lower
̈
̈
m
values compared to RcFDH . Furthermore, the structural
WT
parameters of the Mo site in the variant at lower pH resemble
those of the WT enzyme at its alkaline pH optimum, showing
similar sulfido and S(Cys386) coordination to Mo(VI). In turn,
the imidazole nitrogen of the histidine may function as a final
1
7,21
proton acceptor in the formate C−H bond cleavage.
In
contrast, our results suggest that His387 is not essential for the
proton abstraction in RcFDH, but may rather be involved in
hydrogen-bonding interactions serving in substrate positioning
and/or proton stabilization at the (H)S(Cys386) or Mo−S(H)
ABBREVIATIONS
■
BVS, bond valence sum; EXAFS, extended X-ray absorption
fine structure; MGD, molybdopterin-guanine-dinucleotide;
Moco, molybdenum cofactor; NaDT, sodium-dithionite;
RcFDH, formate dehydrogenase from Rhodobacter capsulatus;
XANES, X-ray absorption near edge structure; XAS, X-ray
absorption spectroscopy
15
positions, as proposed by Mota et al. Apparently, these
functions could also be fulfilled (although less effectively) by
the S(Met387) side chain. The increase of its relative activity at
low pH thus could be explained by the absence of His387
protonation in the H387M variant. More-specific assignments
may require a crystal structure of the RcFDH.
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DOI: 10.1021/ic502880y
Inorg. Chem. 2015, 54, 3260−3271