13C and 63,65Cu ENDOR studies of CO dehydrogenase from oligotropha carboxidovorans. Experimental evidence in support of a copper-carbonyl intermediate

Article abstract of DOI:10.1021/ja406136f

We report here an ENDOR study of an S = 1/2 intermediate state trapped during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR spectra of this state confirm that the ^63,65Cu nuclei exhibits strong and almost entirely isotropic coupling to the unpaired electron, show that this coupling atypically has a positive sign, a_iso = +148 MHz, and indicate an apparently undetectably small quadrupolar coupling. When the intermediate is generated using ¹³CO, coupling to the ¹³C is observed, with a_iso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase, in conjunction with complementary computational studies, leads us to conclude that the intermediate contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme.

Full text of DOI:10.1021/ja406136f

Article
pubs.acs.org/JACS
13C and 63,65Cu ENDOR studies of CO Dehydrogenase from
Oligotropha carboxidovorans. Experimental Evidence in Support of a
Copper−Carbonyl Intermediate
Muralidharan Shanmugam,† Jarett Wilcoxen,‡ Diana Habel-Rodriguez,§ George E. Cutsail III,†
Martin L. Kirk,*,§ Brian M. Hoffman,*,† and Russ Hille*,‡
†Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, United States
‡Department of Biochemistry, University of California, Riverside, California 92521, United States
§Department of Chemistry, The University of New Mexico, Albuquerque, New Mexico 87131-0001, United States
S
* Supporting Information
ABSTRACT: We report here an ENDOR study of an S = 1/2 intermediate state trapped
during reduction of the binuclear Mo/Cu enzyme CO dehydrogenase by CO. ENDOR
spectra of this state confirm that the 63,65Cu nuclei exhibits strong and almost entirely
isotropic coupling to the unpaired electron, show that this coupling atypically has a
positive sign, aiso = +148 MHz, and indicate an apparently undetectably small
quadrupolar coupling. When the intermediate is generated using 13CO, coupling to the
13C is observed, with aiso = +17.3 MHz. A comparison with the couplings seen in related,
structurally assigned Mo(V) species from xanthine oxidase, in conjunction with
complementary computational studies, leads us to conclude that the intermediate
contains a partially reduced Mo(V)/Cu(I) center with CO bound at the copper. Our results provide strong experimental support
for a reaction mechanism that proceeds from a comparable complex of CO with fully oxidized Mo(VI)/Cu(I) enzyme.
INTRODUCTION
■
Carbon monoxide dehydrogenase (CO dehydrogenase) from
Oligotropha carboxidovorans1 is a member of the xanthine
oxidase family of molybdenum-containing enzymes2,3 that
catalyzes the oxidation of CO to CO2 (Scheme 1), transferring
Scheme 1
Figure 1. Active site of CO dehydrogenase from O. carboxidovorans.
The molybdenum coordination geometry is approximately square-
pyramidal, with the pyranopterin cofactor present as the dinucleotide
of cytosine (R).
the reducing equivalents thus obtained into the quinone pool of
the organism.4 The reaction is essential for the organism when
suggesting coupling to the carbon atom derived from
substrate.7
growing on CO as the sole source of both carbon and energy.
The enzyme has been characterized crystallographically5,6
and is found to be an (αβγ)2 heterohexamer with a pair of
[2Fe-2S] clusters in its small subunits (CoxS, 17.8 kDa), FAD
in its medium subunits (CoxM, 30.2 kDa), and a unique
binuclear Mo(VI)/Cu(I) cluster in the large subunits (CoxL,
88.7 kDa) of oxidized enzyme. The binuclear cluster is the site
at which CO is oxidized and has the structure shown in Figure
1, with the two transition metals linked by a μ-sulfido bridge.6
In the course of reduction of the enzyme by excess CO, the
binuclear cluster exhibits a Mo(V) EPR signal with g = [2.0010,
Both the μ-sulfido ligand and the Cu(I) ion of the binuclear
cluster can be removed by treating the enzyme with cyanide,
and a reconstitution protocol has been developed using Cu(I)·
thiourea as the copper source. Alternatively, when Ag(I)·
thiourea is used the Ag(I) ion is reconstituted into the active
site. The Ag(I)-substituted enzyme remains active and is
reduced by CO with a limiting rate constant of 8.2 s−1, as
compared with 51 s−1 for the native Cu-containing form of the
enzyme.8 In the course of reduction by CO, the Ag(I)-
substituted form of the enzyme also exhibits a characteristic
EPR signal, with g = [2.0043, 1.9595, 1.9540] and with strong
3
1.9604, 1.9549] and large hyperfine coupling to the I = /2
63,65Cu nuclei, A(63,65Cu) = [117, 164, 132] MHz.7 The signal
exhibits no proton superhyperfine structure and is unchanged
when the sample is prepared in D2O, but modest line-
broadening is observed when 13CO is used as substrate
Received: June 18, 2013
Published: October 22, 2013
© 2013 American Chemical Society
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Figure 2. Possible structures for the EPR-active species seen with CO dehydrogenase (A−C) and structures of various relevant EPR-active Mo(V)
species observed with the related enzyme xanthine oxidase (D−G).
coupling to the I = 1/2 107,109Ag nuclei with A(107,109Ag) = [82,
79, 82] MHz.
MATERIALS AND METHODS
■
Carbon monoxide gas was obtained from Air, Inc., at a purity of 99.5%,
Several structures can be envisaged for the EPR-active
Mo(V) form of the native copper-containing CO dehydrogen-
ase, as illustrated in Figure 2. Structure A is the Mo(V)-
containing analogue of a proposed Mo(IV)-containing
intermediate in the reaction of enzyme with CO based on
the 1.1 Å resolution structure of the enzyme complexed with n-
butylisonitrile, in which the inhibitor is found to insert between
the S−Cu bond of the μ-sulfido bridge (Figure 2, Structure
A′).6 The Mo(V) species would be formed from an initial
Mo(IV) species by transfer of one electron from the binuclear
cluster to the other redox-active centers of the enzyme prior to
completion of the reaction (in a manner analogous to
generation of the “very rapid” Mo(V) species seen with
xanthine oxidase9−11). Structure B represents an alternate
reaction intermediate proposed on the basis of density
functional studies of the binuclear center,12,13 with the
Mo(V) species again generated by one-electron oxidation of a
Mo(IV) species. Structure C is a Mo(V)-containing analogue of
the presumed complex of CO with oxidized enzyme that is
believed to represent the initial intermediate in catalysis. Here,
the CO is coordinated to the Cu(I) with a residual electron
remaining in the binuclear center after reaction with a prior
equivalent of CO (in this case, partial reduction of the center
would have occurred by reaction of a prior equivalent of CO).
In all three structures, the molybdenum is formally Mo(V) and
the copper is Cu(I).
and 13CO at 99% enrichment was from Cambridge Isotope
Laboratories. All other reagents were obtained at the highest level of
purity available commercially. O. carboxidovorans (ATCC 49405) cells
were grown at 30 °C, pH 7.0 in a 20 L fermentor (BioFlo 415, New
Brunswick) containing minimal medium and CO as the carbon source
(introduced as a mixture of 50% CO and 50% air). Cells were
harvested in late log phase (OD436 >5), washed in 50 mM HEPES (pH
7.2), and stored at −80 °C until needed. CO dehydrogenase was
purified according to the procedure described by Zhang et al.7 using a
combination of Q-Sepharose and Sephacryl S-300 FPLC chromatog-
raphy. Enzyme was routinely reconstituted with copper using the
method of Resch et al.,14 and the degree of functionality determined
by comparing the extent of enzyme bleaching, as observed at 450 nm,
by CO (which reduces only the fully functional enzyme) with that
seen using dithionite (which reduces both functional and nonfunc-
tional enzyme); this typically exceeded 50%.
ENDOR samples were prepared by making the enzyme (at a
concentration of ∼200 μM) anaerobic by cyclic evacuation and
flushing with O2-scrubbed argon gas for 1 h, followed by reduction
under 1 atm of CO for 30 s prior to freezing in a dry ice−acetone bath
and storage in liquid N2. Accumulation of the EPR-active state was
confirmed by EPR using a Bruker Instruments ER 300 spectrometer
̈
equipped with an ER 035 M gaussmeter and HP 5352B microwave
frequency counter. Temperature was controlled at 150 K using a
Bruker ER 4111VT liquid N cryostat. 35 GHz pulsed EPR/ENDOR
̈
2
spectra were recorded as reported previously.15,16
Spin-unrestricted gas-phase geometry optimizations for all CO
dehydrogenase structures were performed at the density functional
level of theory (DFT) using the ORCA software package (an ab initio,
DFT, and semiempirical SCF-MO package, version 2.8−20, Sep
2010).17 All calculations employed the B3LYP hybrid exchange−
correlation functional and a TZVP basis set for all atoms. DFT
bonding calculations and EPR calculations used a scalar relativistic
Hamiltonian, and the radial integration accuracies for Mo and Cu were
increased as recommended for heavy atoms. The enzyme active site
was represented by a Mo(V) ion with an apical oxo ligand and an
equatorial ene-1,2-dithiolate ligand, either an equatorial oxo or
hydroxy ligand, and an equatorial μ-sulfido ligand bridged to Cu(I)-
SCH3. EPR spin-Hamiltonian parameters were calculated for these two
To discriminate among these structural alternatives and gain
insight into the mechanism of CO oxidation, we have used 35
95,97
GHz pulsed-ENDOR spectroscopy to determine the 13C,
Mo, and 63,65Cu hyperfine coupling tensors and the 63,65Cu
quadrupole coupling interaction for 12,13CO-reduced CO
dehydrogenase. The measured 13C-hyperfine coupling is
compared with reported 13C-coupling for paramagnetic
Mo(V) species observed with other members of the xanthine
oxidase family, and the magnitude and sign of the 63,65Cu
couplings are compared with those of other Cu centers. The
combination of these measurements with DFT calculations
discriminates among the potential structures for the inter-
mediate (Figure 2) and support a spin-delocalized [Mo(V)-(μ-
S)-Cu(I)] structure that has CO coordinated to Cu(I). This
species represents a paramagnetic analog of the initial [Mo(VI)-
(μ-S)-Cu(I)·CO] Michaelis complex of the catalytic cycle, from
which a catalytic sequence can be developed.
−
structures and various postulated CO/HCO3 -bound Mo(V) forms.
Geometry optimizations were performed without constraints as well as
with select bond and dihedral angles in the Oapical−Mo−S−Cu−S
chain constrained to maintain geometries consistent with those seen in
the enzyme crystal structure.
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determined by ENDOR, below. At this temperature, however,
the EPR signals from the [2Fe−2S] centers relax slowly and
overlap that of the binuclear center and their features are much
more prominent owing to the greater degree of reduction of the
iron−sulfur centers (40−50%) as compared to the fraction of
the binuclear center that is paramagnetic (no greater than
15%). The spectrum gives no evidence for magnetic coupling
between the binuclear center and the proximal [2Fe−2S]
cluster, as expected given the conditions the sample was
prepared (i.e., by partial reduction of the substrate).
RESULTS
■
X-band (∼9.5 GHz) EPR spectra of CO-reduced CO
dehydrogenase were collected for samples prepared with
12,13CO in H2O 50 mM HEPES buffer, pH 7.2 at 150 K
(Figures 3 (top) and S1, Supporting Information). The EPR
A preliminary set of X-band and Q-band spectra collected
between 150 and 77 K confirm the above g-tensor but show
upon cooling; this finding is extended in 2 K ENDOR
measurements presented below. (The temperature dependence
is not central to the present paper and will be described in
detail elsewhere.) When the binuclear signal is generated using
13CO, the X-band spectrum shows significant line broadening
(Figure S1, Supporting Information) due to unresolved
hyperfine coupling to the 13C, as previously reported.14
The g values for the EPR signal of the binuclear center, all
being ≤ge, indicate that the signal arises from a Mo(V) rather
than Cu(II) species, consistent with the known constitution of
the binuclear center, with Mo(VI) and Cu(I) in the fully
oxidized state. This conclusion is supported by contrasting the
nearly isotropic 63,65Cu hyperfine tensor with the highly
anisotropic hyperfine coupling observed for Cu(II) systems.18
The conclusion is confirmed below by ENDOR measurements
that determine the signs of the 63,65Cu couplings.
The overlap at 2 K of the EPR signals from the slowly
relaxing [2Fe−2S] centers causes no problem for ENDOR
measurements of the [Mo, Cu] binuclear center, as the 13C and
63,65Cu nuclei being studied by ENDOR are associated only
with the [Mo,Cu] center. 13CO signals are identified by
comparing samples prepared with 12,13CO, and the [2Fe-2S]
centers give no ENDOR response in the vicinity of 63,65Cu
ENDOR transitions.21−24
Figure 3. X-band (top) and Q-band (bottom) EPR spectra of the
[Mo, Cu] center (black) in H2O buffer at 150 and 2 K, respectively.
(Top) 9.45 GHz, modulation amplitude = 5 G; simulation with 150 K
parameters from Table 1 (red). (Bottom) Numerical derivative of 34.8
GHz, two-pulse echo-detected spectrum (π-pulse =80 ns, τ = 600 ns,
repetition time =50 ms). Simulation of the binuclear signal using the 2
K parameters from Table 1 (red). g-scale included for correlation with
ENDOR field-frequency patterns given in Figure 4. Note that the scale
of this simulation is exaggerated relative to the intensity of the signal in
the experimental spectrum to better illustrate line positions. The
“braces” show the field values for the canonical g-values of the FeS
clusters. The asterisk denotes the signal of adventitions Cu(II) in the
signal, and the double asterisk the isotropic signal of FADH• from the
enzyme, with g = 2.00.
Davies ENDOR spectra of the binuclear center of CO
dehydrogenase, recorded at 2 K and taken at g = 1.971, are
shown in Figure 4 (top) for samples reduced by 12,13CO. The
υ+ = 23 MHz branch of the 13C doublet (υ (13C) = |A(13C)/2
υ(13C)|) from 13CO is evident in the latter case. (Note that
assignment as υ− would yield a hyperfine coupling too large to
be compatible with the X-band EPR spectra.) At the field of
observation, υ(13C) is ∼13.5 MHz, yielding A(13C) ∼ 19 MHz
for the 13C-hyperfine coupling. The ENDOR intensity in the
9−16 MHz region for both labeled and unlabeled samples can
be assigned to amide 14N features from the two [2Fe−2S]
clusters, whose EPR signals overlap the Mo(V) EPR signal at
low temperature (Figure 3; bottom). Subtraction of the 12CO
background (black line) from the 13CO spectrum (red line) of
the binuclear cluster more clearly shows the υ+ peak (red
dotted line) but also reveals the υ− branch of the 13C-doublet
(relaxation effects lead to the reduced intensity for υ−). Figure 4
(bottom) shows a 2-D field-frequency plot of the background-
subtracted Davies 13C-ENDOR spectra for the binuclear cluster
of CO dehydrogenase reduced by 13CO, recorded at selected
magnetic fields across the EPR envelope (For the complete 2-D
field-frequency pattern, see Figure S2, Supporting Information).
The frequencies of the 13C doublet remain essentially constant
over the entire absorption envelope of the binuclear center’s
EPR signal, and the pattern is well simulated by the hyperfine
tensor, A(13C) = +[16.7, 16.5, 18.8] MHz. The 13C hyperfine
tensor is thus dominated by an isotropic coupling of aiso(13C) =
spectrum of the binuclear center of the enzyme generated with
12CO (black line) shows a well-resolved structure centered
around g ∼1.96 with complex multiplet features due to
hyperfine interactions with both Mo [I(95,97 Mo) = 5/2; 25%
natural abundance] and Cu [I(63,65Cu) = 3/2; 100% natural
abundance] nuclei. In addition to the [Mo, Cu] cluster, the
enzyme contains two [2Fe−2S] centers. However, at liquid
nitrogen temperatures and above, the EPR signals arising from
the iron−sulfur centers relax very fast and are not detected.
The 150 K X-band spectrum of the [Mo, Cu] center is well
reproduced (red dashed line) with the following spin-
Hamiltonian parameters: g is roughly axial, g = [2.0010,
1.9604, 1.9549]; A(63,65Cu) is dominated by the isotropic
interaction, |A(63,65Cu)| = [117, 164, 132] MHz, corresponding
to |aiso(63,65Cu)| = 138 MHz, with a small anisotropic coupling,
T = [−21, 26, −7], having a maximum anisotropic component,
|Tmax(
63,65Cu)| = 26 MHz.7 A 2K echo-detected Q-band EPR
spectrum of the same sample, Figure 3, lower, clearly shows the
features of the [Mo, Cu] center, as can be seen by comparison
with a simulation computed using 63,65Cu hyperfine parameters
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a
Table 1. Spin Hamiltonian Parameters Used To Simulate
the 2-D ENDOR Patterns of 13C and 63,65Cu Nuclei of Cu−
CO Dehydrogenase
nuclei
A1
A2
A3
aiso
13C
+16.7
+148
117
+16.5
+148
164
+18.8
+148
132
+17.33
+148
b
63,65Cu(ENDOR)
63,65Cu(EPR)
+137.6
a
Simulations employed g = [2.002, 1.958, 1.953], and the hyperfine
b
couplings are in MHz. Quadrupole splitting for 63,65Cu (I = 3/2) are
not observed, their inclusion had no effect on simulations, so were not
incorporated.
Figure 4. (Top) Davies 13C-ENDOR spectra of the binuclear signal
measured at g = 1.971. The red dotted line shows the 13C hyperfine
coupling, obtained by subtracting the spectrum of 12CO (black line)
from that of 13CO (red line) in H2O. The black horizontal bar
connects the 13C peaks centered at υ(13C) and separated by A(13C).
Low intensity of the low frequency partner common in Q-band
ENDOR. (Bottom) Simulated (red dotted lines) and experimental
(black lines) 2-D field-frequency plot of Davies 13C-ENDOR spectra
of the binuclear signal reduced by 13CO. The amplitudes of the
simulated υ−(13C) signals are multiplied by 1/3 to reflect the low-
intensities due to relaxation effects (see text). Conditions: π-pulse
length = 120 ns, τ = 600 ns, repetition time = 40 ms, microwave
frequency =34.795 GHz, T = 2 K.
Figure 5. 2-D field-frequency plot of broad-band Davies ENDOR
spectra of the binuclear signal reduced by 12CO. The black brace
encompasses the frequency range of 1H signals; the red brace
encompasses the frequency range of 95,97Mo signals, with the (*) in
representative spectra identifying resolved 95,97Mo signals. Blue
“goalpost” connects the 63,65Cu peaks at υ+(63,65Cu) ∼ 87 MHz and
υ−(63,65Cu) ∼ 60 MHz; the center frequency is A(63,65Cu)/2, and
separation is 2υ(63,65Cu). Blue dotted lines are simulations of the
63,65Cu spectra, where the amplitudes of the υ− branches are multiplied
by 1/2 to reflect their low intensities. Conditions: π-pulse length = 120
ns, τ = 600 ns, repetition time = 40 ms, microwave frequency = 34.772
GHz, T = 2 K.
+17.33 MHz, with an extremely small anisotropic term that
approximates the dipolar form: T = [−T, −T, 2T] where T =
0.73 MHz. These results are summarized in Table 1.
natural abundance, combined with the additional complexity
arising from strong quadrupolar coupling in the case of 97Mo,
no attempt has been made to simulate this pattern. However,
its overall character indicates that the isotropic hyperfine
coupling for molybdenum, aiso(95,97Mo), is roughly 75−125
MHz.26
The hyperfine tensor measured in the X-band EPR spectrum
from the overlapping splittings of 63Cu (69%) and 65Cu (30%)
predicts the presence of a Q-band 63,65Cu ENDOR response
from the υ+(63,65Cu) manifold in the frequency range ∼75−100
MHz. The Q-band ENDOR spectra taken at 2 K exhibit the
predicted response, with υ+(63,65Cu) ∼ 90 MHz (Figure 5, blue
horizontal bar) corresponding to a hyperfine coupling of
Figure 5 shows a 2-D field-frequency plot of Davies ENDOR
spectra collected over the frequency range of 30−100 MHz at
multiple field settings across the EPR envelope for the
spectrum seen with enzyme reduced by 12CO. At all magnetic
field settings, the spectra exhibit a strong 1H-ENDOR response
arising from weakly coupled proton(s) without well-resolved
1
structure. These H-ENDOR features overlap with ENDOR
features attributable to 95,97Mo (I = 5/2), isotopes that together
account for 25% of the total isotopic abundance. These features
are similar to those that have been seen previously in the
ENDOR spectrum of the formaldehyde-inhibited Mo(V) signal
of xanthine oxidase.25 Because of the poor resolution and low
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a
Table 2. Experimental and Computed EPR Parameters for CO-Reduced CO Dehydrogenase
63,65Cu
gmin
gmid
gmax
aiso
63,65Cu T
13C aiso
13C T
1H aiso
1H T
95,97Mo aiso
75−100
b
b
b
experiment
1.953
1.958
2.002 +148
138
∼0 [-20.7,
17.3
[−0.6, −0.8, N/A N/A
c
c
b
+26.4,-5.7]
1.5]
MoO2 + CO bound to Cu(I)
Mo(O)(HCO3−)
1.911
1.949
1.954
1.950
1.967
1.958
1.959
1.996 167
2.022 281
2.015 90
2.012 137
−13, 2, 11
−17, −7, 24
−13,-8, 21
−15, −7, 21
3.7
18.6
15.8
26.6
−4.5, −5.1,
N/A N/A
59
65
65
65
9.7
−1.0, −1.0,
3
4
4
−1, −1,
2.0
2
Mo(O)(OH)(CO)
−2.6, −1.7,
−5, −7,
4.4
11
Mo(O)(OH) + CO bound to Cu(I)
1.955
−3.5, −1.9,
−5, −7,
(constrained S−Cu−S at 139°)
5.5
11
a
b
c
All hyperfine coupling constants are in MHz. These values derive from the present 2 K ENDOR measurements. from the 150 K X-band EPR
spectrum (ref 7).
|A(63,65Cu)| = 148 MHz, within the range of values expected
from the EPR-derived hyperfine tensor. The signals are
exceedingly sharp compared to those seen for a Cu(II)
protein,27 and there is no evidence in the ENDOR spectra
for the significant quadrupolar coupling to the I = 3/2 nuclear
spin of 63,65Cu seen in Cu(II) proteins.
The frequency of this 63,65Cu signal remains constant as the
field is moved toward g2 and g3, indicating that the hyperfine
interaction is isotropic within experimental error, with
aiso(63,65Cu) = +148 MHz, a slight increase from, aiso(63,65Cu)
obtained from the continuous-wave EPR spectra at higher
temperature, Table 2. The vanishing of the anisotropic coupling
upon cooling to 2 K is compatible with the temperature
variation of the small 63,65Cu hyperfine anisotropy seen at
higher temperatures (see above). This phenomenon may be
assigned to changes in the geometry at Cu(I) with cooling. It is
well-known that considerable changes in the geometry of a
diatomic bound to a metal ion can occur during cooling,28 and
this quite likely is happening to the Cu−CO linkage. However,
the spectra collected toward the high field/low-g-value edge of
the EPR spectrum do show a “tailing” toward higher frequency
that may reflect a heterogeneity in the geometry at Cu(I). For
consistency, the 2 K ENDOR-derived 63,65Cu coupling will be
used in the following discussion.
Signs for the hyperfine tensors, which are not available from
EPR measurements, were determined from the ENDOR
spectra of the binuclear cluster (more specifically, the sign of
gNucANuc) using the Pulse-Endor-SaTuration-REcovery
(PESTRE) protocol, a pulse sequence comprised of multiple
Davies ENDOR sequences.29,30 Figure 6 shows the PESTRE
measurements obtained at g = 1.975 for the υ+ branches of the
13C and 63,65Cu-ENDOR responses, where relatively sharp
ENDOR responses for 13C and 63,65Cu nuclei are observed. In
phase I of the PESTRE traces (no radio frequency
illumination), the υ+ branches of the 13C and 63,65Cu have
reached the steady-state electron spin−echo baseline; in phase
II, radio frequency excitation is applied and converts the spin
populations and spin−echo response to their steady-state
ENDOR values; in phase III (again in the absence of radio
frequency illumination), the electron−nuclear spin populations
give rise to a spin−echo signal denoted as the dynamic
reference level, which relaxes to the BSL. In phase III, the υ+
signals of 13C (red line) and 63Cu (blue line; inset) both relax
to the spin−echo baseline “from below”, behavior that
unambiguously establishes that both A(13C) and A(63,65Cu)
are positive.
Figure 6. PESTRE spectra measured at the υ+ peaks of the 13C and
63,65Cu nuclei (inset; indicated as red and blue lines) of the binuclear
signal. The RF frequencies and the g value at which PESTRE
experiments were carried out are given in the inset: Conditions: π-
pulse length = 120 ns, τ = 600 ns, repetition time = 40 ms, tRF = 20 μs,
microwave frequency = 34.795 GHz, T = 2 K.
DISCUSSION
■
The aim of the present investigation is to determine the
structure of the species that gives rise to the EPR signal seen
upon partial reduction of CO dehydrogenase with CO and to
use this information in conjunction with DFT calculations to
elucidate the reaction mechanism of the enzyme. The
experimental and computational results are summarized in
Table 2, where it can be seen that the most remarkable feature
of this species is the large and positive isotropic hyperfine
coupling to copper, with aiso(63,65Cu) = +148 MHz, with
negligible anisotropic hyperfine coupling to the I = 3/2 nuclear
spin. The small-to-vanishing anisotropic component seen with
CO dehydrogenase contrasts sharply with the large anisotropy
in Cu(II) centers, where large negative isotropic and negative
anisotropic 63,65Cu couplings are typically observed:
aiso(63,65Cu) ∼ Tmax 63,65Cu) ∼ −[150−200] MHz;31−33 for
(
a type 2 Cu(II) center, aiso(63,65Cu) ∼ Tmax 63,65Cu) ∼ −70
(
MHz for a type I Cu.34,35 No less startling, the ENDOR/
PESTRE measurements show that the observed isotropic
63,65Cu hyperfine coupling is positive, opposite in sign to those of
d9 Cu(II) centers with an unpaired electron in a (dσ*) orbital.
The Cu hyperfine couplings in CO dehydrogenase are instead
consistent with a d10 Cu(I) ion whose closed-shell electronic
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configuration acquires a large positive aiso through delocalization
of the electron spin in the “Mo(V) SOMO” over the entire
[Mo(V)−(μ-S)−Cu(I)] unit, into the 4s orbital of Cu.
aiso for the “very rapid” signal is 7.9 MHz,25 while aiso for the
13C of glycol in the glycol-inhibited signal is even smaller at 6.2
MHz (Shanmugam et al., unpublished results). In both cases,
the 13C hyperfine coupling is weak because there is no direct
bonding of carbon to Mo(V) and the Mo−C distance is long
(∼ 3.4 Å), precluding strong overlap between Mo(V) dxy and
carbon 2s orbitals. The DFT results for structure B yield a very
large aiso for 13C of 54 MHz, however, much larger than
observed experimentally. The calculated copper hyperfine also
is highly rhombic, A(63,65Cu) = [19,−94,105] MHz, in sharp
disagreement with the observed isotropic coupling seen here
with CO dehydrogenase. These observations argue against
structure B being responsible for the signal seen with CO
dehydrogenase.
In support of this assignment, we observe that the Cu(I)
hyperfine coupling seen here is quite similar to that of Cu(I) in
an inorganic model compound prepared by Gourlay et al.,36
which mimics the structure and spectroscopic properties of the
paramagnetic active site of CO dehydrogenase:
[TpiPrMo(V)(O)(OAr)(μ-S)Cu(I)(Me3tcn)] (where TpiPr
=
hydrotris(3-isopropylpyrazol-1-yl)borate; OAr = 3,5-(di-tert-
butyl)phenolate; Me3tcn =1,4,7-trimethyl-1,4,7-triazacyclono-
nane). The isotropic Cu(I) hyperfine interaction, |aiso(63,65Cu)|
∼ 159 MHz, observed for this model is quite similar in
magnitude to aiso(63,65Cu) = +148 MHz seen with CO
dehydrogenase. Calculations on the model have confirmed
that the remarkably large values of aiso reflect a strong covalent
delocalization of the SOMO through the bridging sulfido.
The hyperfine coupling to 13C in the EPR signal manifested
by 13CO-reduced CO dehydrogenase also is distinctive in being
highly isotropic. The isotropic coupling, aiso(13C) = +17.4
MHz, is intermediate between the values observed to date for
carbon-containing ligands to a paramagnetic Mo(V) center:
aiso(13C) = +43.8 MHz for formaldehyde-inhibited xanthine
oxidase25,33,37,38,41 and |aiso(13C)| = 7.9 MHz for the “very
rapid” Mo(V) intermediate trapped with that enzyme.10,39
Additionally, the anisotropic component seen here with CO
dehydrogenase, T = 0.73 MHz,40 is smaller than that observed
in the formaldehyde-inhibited xanthine oxidase (T = 3.8 MHz)
and somewhat smaller than that observed in the “very rapid”
intermediate (T = 1.15 MHz). We therefore argue that this
small isotropic 13C coupling is unlikely to arise from a species
with a Mo−C bond.
One possible assignment for the state studied here is
structure A of Figure 2. Our recent 1,2H and 13C-ENDOR study
of the formaldehyde-inhibited Mo(V) of xanthine oxi-
dase25,38,41 has shown that it possesses the core of four-
membered ring structure D, with an Mo−C distance of 2.76 Å
in the DFT-optimized geometry.25,41 This structure resembles
that of structure A′ (Figure 2), found by X-ray diffraction of n-
butylisonitrile-inhibited CO dehydrogenase (with a Mo−C
distance of 2.63 Å).6 The planar geometry of the related
structure D, with a short Mo−C distance within the ring, favors
strong covalent spin delocalization via the Mo−O−C linkage or
a strong “transannular hyperfine interaction” between Mo(V)
dxy orbital and carbon 2s orbitals, resulting in an extremely large
hyperfine coupling to the 13C of formaldehyde, with aiso = 44.6
MHz.25 The significantly smaller hyperfine coupling for 13C
seen here with CO dehydrogenase thus argues against structure
A of Figure 2 being responsible for the observed EPR signal.
A second possible assignment of the Mo(V) species studied
here is the five-membered metallocyclic ring of structure B
(Figure 2), the closest established analogue of which is the
species giving rise to the “glycol-inhibited” Mo(V) EPR signal
of desulfo xanthine oxidase (Figure 2, structure E). The 1,2H-
ENDOR results for the “glycol-inhibited” species were
interpreted in terms of a five-membered (Mo−O−C−C−O)
metallocyclic structure.25,38 The paramagnetic Mo(V) species
giving rise to the “very rapid” EPR signal seen with xanthine
oxidase also has a Mo−O−C unit analogous to that of structure
B. Here, the slow substrate 2-hydroxy-6-methylpurine9 is bound
to the Mo(V) ion via the equatorial oxygen atom after being
incorporated into product as a hydroxyl group.10,42 When the
C-8 position of 2-hydroxy-6-methylpurine is labeled with 13C,
It is noteworthy that a recent X-ray crystallography study of
the glycol- and glycerol-inhibited Mo centers of AOR (aldehyde
oxidoreductase) claims a third structural possibility, a direct
Mo−C bond (structure F) for this species.20 However, if for
illustrative purposes we assume that the dipolar interaction
arises solely from a through-space interaction of the 13C with a
point electron spin on Mo(V), the measured value T = 0.73
́
MHz corresponds to a Mo−C distance of ∼2.4 Å. Subtraction
of a 13C local contribution to the observed Tobs = 0.73 MHz
would further increase the resultant Mo−C distance,10 thus
ruling out a direct Mo−C bond in CO dehydrogenase. A
similar analysis10 has helped rule out a direct Mo−C bond in
the “very rapid” Mo(V) signal of xanthine oxidase prepared
with 2-hydroxy-6-methylpurine (13C-8).
Overall, a comparison of the 13C coupling seen here (Table
2) with previously reported 13C hyperfine tensors for a ligand
to the paramagnetic Mo(V) enzyme species indicates that
structures A and B, as well as any structure with a Mo−CO
bond, are unlikely to represent the structure of the binuclear
center of CO dehydrogenase. Instead we propose that Structure
C of Figure 2, with CO coordinated to the copper of the
Mo(V)−Cu(I) binuclear center best represents the structure
seen in the enzyme.
A structure having a Cu(I)-coordinated CO is consistent
with both of the computational studies of the reaction of CO
dehydrogenase that identify CO coordinated to the copper of a
fully oxidized binuclear center as the starting point for
catalysis.12,13 In the case of the partially reduced complex
examined in the present study, with the molybdenum in the
EPR-active Mo(V) valence state, the enzyme cannot progress
through the catalytic sequence, thus accounting for the
accumulation of the signal in the course of our sample
preparation. Our Mo(V)/Cu(I)·CO species in fact represents a
paramagnetic analogue to the bona fide Michaelis complex for
the reaction, and is thus analogous to the species giving rise to
the well-characterized “Rapid” Mo(V) EPR signals in the
related molybdenum-containing enzyme xanthine oxidase43
(albeit with a substantively different structure).
Although there is no EPR or ENDOR evidence regarding
protonation of the equatorial MoO group in the Mo(V)
state, our DFT calculations (Table 2) indicate that the observed
g-tensor anisotropy is not consistent with a dioxo species such
as seen in the oxidized enzyme.6,7 Coupled uptake of protons
with electrons is a common property of even the simplest
molybdenum complexes,44,45 and there is precedent for
equatorial Mo−OH protons being only very weakly coupled
in the Mo(V) species.46,47 We thus consider it likely that the
partially reduced binuclear center in fact possesses an equatorial
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Article
Figure 7. Reaction mechanism of CO dehydrogenase.
Mo−OH (in contrast to the equatorial MoO of oxidized
AUTHOR INFORMATION
Corresponding Author
russ.hille@ucr.edu; bmh@northwestern.edu; mkirk@unm.edu
■
enzyme6,7) in the Mo(V) state.
CONCLUSION
Notes
■
The authors declare no competing financial interest.
Our ENDOR results provide direct experimental support for
CO coordination to Cu(I) of the binuclear center of CO
dehydrogenase. Upon binding of CO, the reaction progresses
as shown in Figure 7 by nucleophilic attack of the equatorial
MoO oxygen on the Cu-bound CO, with substrate likely
activated to at least some degree by backbonding from the
copper which leads to population of the CO π* orbital. The
DFT calculations of Hofmann and co-workers13 indicate that
this chemistry leads to a set of three species having the
structures shown in brackets in Figure 7. Although presented as
discrete intermediates in the computational work, we consider
it likely that these species are readily interconverted. We further
observe that they involve bonding of the carbon of substrate to
each of the three atoms of the Mo−μ(S)−Cu core and reiterate
that each of the three core atoms contributes significantly to the
redox-active orbital.36 We therefore present these species in
Figure 7 as an ensemble of states from which the reaction
proceeds further. Completion of the catalytic sequence involves
formal reduction of the binuclear cluster and formation of CO2
from this ensemble, with hydroxide from solvent necessarily
being introduced into the molybdenum coordination sphere to
regenerate the equatorial ligand of the molybdenum and
complete the catalytic cycle.
In summary, the ENDOR and computational study
presented here provides direct experimental evidence that the
EPR signal seen in the course of substrate reduction of CO
dehydrogenase arises from a partially reduced, Mo(V)/Cu(I),
binuclear center with CO bound at the copper. This conclusion
provides strong support for a reaction mechanism that begins
with a fully oxidized binuclear center, with CO coordinated to
copper and thereby activated for nucleophilic attack by the
equatorial MoO ligand.
ACKNOWLEDGMENTS
■
We acknowledge the support of the National Institutes of
Health (GM 057378 to M.L.K.), the National Science
Foundation (MCB 0723330 to B.M.H., DGE-0824162 to
G.E.C.), and the Department of Energy (13ER16411 to R.H.).
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ASSOCIATED CONTENT
■
S
* Supporting Information
9.5 GHz EPR spectra of the CO dehydrogenase binuclear signal
reduced with 12,13CO; 2-D 13C-Davies ENDOR spectra of the
binuclear center of CO dehydrogenase collected at selected
magnetic fields across the EPR envelope, 2 K. This material is
available free of charge via the Internet at http://pubs.acs.org
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