Geometric and electronic structures of the Ni(I) and methyl-Ni(III) intermediates of methyl-coenzyme M reductase.
ABSTRACT Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni-F(430) cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the Ni(I) active state (MCR(red1)) on Me-SCoM to form a Ni(III)-methyl intermediate, while computational studies indicate that the first step involves the attack of Ni(I) on the sulfur of Me-SCoM, forming a CH(3)(*) radical and a Ni(II)-thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the Ni(I) (MCR(red1)), Ni(II) (MCR(red1-silent)), and Ni(III)-methyl (MCR(Me)) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCR(red1). Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni-C bond ( approximately 2.04 A) in the Ni(III)-methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the Ni(III)-methyl bond in the subsequent catalytic step. The XAS data provide insight into the role of the unique F(430) cofactor in tuning the stability of the different redox states of MCR.
Article: Unusual coenzymes of methanogenesis.Annual Review of Biochemistry 02/1990; 59:355-94. · 27.68 Impact Factor
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ABSTRACT: Methyl-coenzyme M reductase (= component C) from Methanobacterium thermoautotrophicum (strain Marburg) was highly purified via anaerobic fast protein liquid chromatography on columns of Mono Q and Superose 6. The enzyme was found to catalyze the reduction of methylcoenzyme M (CH3-S-CoM) with N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP = component B) to CH4. The mixed disulfide of H-S-CoM and H-S-HTP (CoM-S-S-HTP) was the other major product formed. The specific activity was up to 75 nmol min-1 mg protein-1. In the presence of dithiothreitol and of reduced corrinoids or titanium(III) citrate the specific rate of CH3-S-CoM reduction to CH4 with H-S-HTP increased to 0.5-2 mumol min-1 mg protein-1. Under these conditions the CoM-S-S-HTP formed from CH3-S-CoM and H-S-HTP was completely reduced to H-S-CoM and H-S-HTP. Methyl-CoM reductase was specific for H-S-HTP as electron donor. Neither N-6-mercaptohexanoylthreonine phosphate (H-S-HxoTP) nor N-8-mercaptooctanoylthreonine phosphate (H-S-OcoTP) nor any other thiol compound could substitute for H-S-HTP. On the contrary, H-S-HxoTP (apparent Ki = 0.1 microM) and H-S-OcoTP (apparent Ki = 15 microM) were found to be effective inhibitors of methyl-CoM reductase, inhibition being non-competitive with CH3-S-CoM and competitive with H-S-HTP.European Journal of Biochemistry 04/1988; 172(3):669-77. · 3.58 Impact Factor
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ABSTRACT: The structure of a derivative of coenzyme F430 from methanogenic bacteria, the bromide salt of 12,13-diepi-F430 pentamethyl ester (5, X = Br), was determined by X-ray structure analysis. It reveals a more pronounced saddle-shaped out-of-plane deformation of the macrocycle than any hydroporphinoid Ni complex investigated so far. The crystal structure confirms the constitution proposed for coenzyme F430 (2) and shows that in the epimer 5, the three stereogenic centers in ring D, C(17), C(18), and C(19), have the (17S)-, (18S)-, and (19R)-configuration, respectively. Deuteration and 2D-NMR studies independently demonstrate that native coenzyme F430 (2) has the same configuration in ring D as the epimer 5. Therefore, our original tentative assignment of configuration at C(19) and C(18)  has to be reversed. This completes the assignment of configuration for all stereogenic centers in coenzyme F430, which has the structure shown in Formula2.Helvetica Chimica Acta 10/2004; 74(4):697 - 716. · 1.38 Impact Factor
Geometric and Electronic Structures of the NiIand Methyl-NiIIIIntermediates of
Methyl-Coenzyme M Reductase†
Ritimukta Sarangi,*,‡Mishtu Dey,§and Stephen W. Ragsdale*,§
Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025, and
Department of Biological Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-0606
ReceiVed January 23, 2009; ReVised Manuscript ReceiVed February 25, 2009
ABSTRACT: Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological
methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR
contains a Ni-F430cofactor, which can exist in different oxidation states. The catalytic mechanism of
methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On
the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve
a nucleophilic attack of the NiIactive state (MCRred1) on Me-SCoM to form a NiIII-methyl intermediate,
while computational studies indicate that the first step involves the attack of NiIon the sulfur of Me-
SCoM, forming a CH3•radical and a NiII-thiolate species. In this study, a combination of Ni K-edge
X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been
performed on the NiI(MCRred1), NiII(MCRred1-silent), and NiIII-methyl (MCRMe) states of MCR to elucidate
the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to
reveal a five-coordinate active site with an open upper axial coordination site in MCRred1. Ni K-pre-edge
and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a
long Ni-C bond (∼2.04 Å) in the NiIII-methyl state of MCR. The formation and stability of this species
support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the NiIII-methyl bond
in the subsequent catalytic step. The XAS data provide insight into the role of the unique F430cofactor in
tuning the stability of the different redox states of MCR.
Methyl-coenzyme M reductase (MCR)1from methano-
genic archaea (1) catalyzes the terminal step in biological
methane synthesis. Using coenzyme B (CoBSH) as the two-
electron donor, MCR reduces methyl-coenzyme M (methyl-
SCoM) to form methane and the heterodisulfide product,
CoBS-SCoM (2, 3). MCR contains an essential redox active
nickel tetrapyrrolic cofactor called coenzyme F430at its active
site (4, 5), which is active in the reduced NiIstate (MCRred1).
All of the biologically generated methane, amounting to 1
billion tons per annum globally, is formed by MCR.
Furthermore, recent evidence indicates that anaerobic meth-
ane oxidation is also catalyzed by MCR and occurs by a
reversal of the methane synthesis reaction (6, 7). The central
role of MCR in the synthesis of this important fuel, which
is also a potent greenhouse gas, makes it important to
understand the catalytic mechanism of methane formation.
Two limiting mechanisms for MCR-catalyzed methane
formation have been proposed (Figure 1). In mechanism I,
based on the NiIIcrystal structure and model chemistry (8-11),
NiIperforms an SN2 attack on the methyl group of methyl-
SCoM to form a NiIII-methyl intermediate that undergoes
one-electron reduction to form a NiII-methyl species, which
then undergoes protonation to form methane, and a CoMS•
radical. Condensation of the CoMS•radical with CoBSH
generates a CoBSSCoM•radical anion that reduces NiIIto
the active NiIstate. Mechanism II, which is based on density
functional theory calculations (12, 13) and considers NiIII
not to be a feasible intermediate, proposes that NiI-MCRred1
†This work has been supported by NIH Grant 1P20RR17675 to
S.W.R. We are grateful for funding from a Department of Energy grant
(DE-FG02-08ER15931) to S.W.R.
* To whom correspondence should be addressed. R.S.: e-mail,
firstname.lastname@example.org; phone, (650) 926-4621; fax, (650) 926-4100.
S.W.R.: e-mail, email@example.com; phone, (734) 615-4621; fax,
‡SLAC National Accelerator Laboratory.
§University of Michigan.
1Abbreviations: CoBSH, coenzyme B; DFT, density functional
theory; ENDOR, electron nuclear double resonance; EPR, electron
paramagnetic resonance; EXAFS, extended X-ray absorption fine
structure; MCR, methyl-coenzyme M reductase; methyl-SCoM, methyl-
coenzyme M; PDB, Protein Data Bank; TD-DFT, time-dependent
density functional theory; XAS, X-ray absorption spectroscopy.
FIGURE 1: Schematic diagram showing the proposed structures upon
conversion of the active MCRred1to MCRred1-silentand MCRMe. The
NiIII-PS state is analogous to the NiIII-methyl state and is formed
by the reaction of MCRred1with bromopropane sulfonate.
Biochemistry 2009, 48, 3146–3156 3146
10.1021/bi900087w CCC: $40.75
2009 American Chemical Society
Published on Web 02/25/2009
reacts with the sulfur of Me-SCoM to generate a methyl
radical and a NiII-thiolate complex. The methyl radical is
proposed to abstract a hydrogen atom from CoBSH to
generate a CoBS•radical that reacts with bound CoM to
generate NiIIand the same CoBS-SCoM•radical anion
proposed in mechanism 1. Finally, reduction of NiIIand
generation of the CoBS-SCoM product occur as in mech-
anism I. The major distinction between the two mechanisms
lies in the intermediate generated in the first step of catalysis:
NiIII-methyl or methyl radical and NiII-SCoM. However,
so far, none of the proposed intermediates have been trapped
during reactions with the natural substrates; presumably,
these intermediates form and decay too fast to be observed
by stopped-flow and rapid freeze quench EPR methods.
Much of our understanding of the structure and mechanism
of MCR catalysis is based on crystal structures of various
inactive NiIIstates (MCRsilent, MCRox1-silent, and MCRred1-silent)
(8, 10, 14), since the crystal structures of the active states of
MCR have not been reported. In all states, the central Ni
atom is coordinated by four corphin ring nitrogens and a
lower axial glutamine oxygen atom. The upper axial ligands
in MCRox1-silentand MCRsilentare the thiol group of coenzyme
M and the sulfonate oxygen of the heterodisulfide product,
respectively (8, 10, 14). In MCRred1-silent, two structures have
been proposed: a five-coordinate site lacking an upper axial
ligand and a six-coordinate site with the thiol group of
coenzyme M as the upper axial ligand (10). EXAFS studies
have also been performed on various NiIIforms of MCR,
which are consistent with crystal structures; however, high-k
EXAFS studies have not been reported (8, 15).
In this study, NiIand NiIII-methyl species, which are the
starting state and a putative intermediate state in the MCR-
catalyzed reaction, respectively, have been trapped and
characterized. Although a NiIII-methyl intermediate has not
yet been identified during the reaction with the natural
substrate Me-SCoM, its formation has been demonstrated
in the reaction of MCRred1with methyl bromide (16) and
methyl iodide (17). Analogous NiIII-alkyl species are formed
by the reaction of MCRred1 with corresponding alkyl
halides (18-20). Furthermore, this species has been shown
to react with HSCoM (and other thiolates) to generate the
NiI-MCRred1state and methyl-SCoM (or other alkyl thio-
ethers). The similarity between the rate constants for methane
formation from the MCRMe species with HSCoM and
CoBSH (1.1 s-1) and the steady-state kcat for methane
formation from natural substrates (4.5 s-1at 25 °C) is
consistent with the catalytic intermediacy of the methyl-Ni
species (17). Thus, there is significant evidence supporting
the catalytic relevance of the MCRMeintermediate. Although
the crystal structure of active MCRred1is not known, much
of our understanding about the active site structure in
MCRred1 comes from Ni K-edge EXAFS studies. Two
structures have been proposed: a five-coordinate site with
an open upper axial ligand (8) and a six-coordinate site with
an oxygen atom as the upper axial ligand (15). However, in
both studies, the k range for the reported EXAFS data was
2-12 Å-1, limiting the resolution ((0.16 Å) and the ability
to identify and distinguish the equatorial and axial ligands.
The goal of this study was to determine high-resolution
structures of the active NiIstate with accurate metrical
parameters. Although direct structural information about the
NiIII-methyl state is not available, EPR, ENDOR, and
HYSCORE spectroscopic studies have determined the elec-
tronic structure of the active site Ni center (16, 17). These
studies describe MCRMeto be formally NiIIIwith a methyl-Ni
bond formed by the oxidative addition reaction of methyl
iodide with the NiI-MCRred1complex. The presence of a
large13C hyperfine coupling indicates that the methyl group
is coordinated to the paramagentic NiIIIcenter by a covalent
bond, with a Ni-C bond distance of approximately 1.9-2.0
Å (16, 17), although the actual bond distance cannot be
precisely determined by analysis of these hyperfine couplings.
Similarly, EPR studies on the adduct between propane-
sulfonate and MCR demonstrated the presence of a Ni(III)-C
bond with an approximate bond distance of 2 Å (21).
However, the active site geometry of the MCRMestate has
not been determined by any structure determination technique.
In the work described here, Ni K-pre-edge and edge X-ray
absorption spectroscopy and EXAFS investigations have
been combined with time-dependent density functional theory
(DFT) calculations to determine the local geometric structure
at the active sites in MCRred1-silent, MCRred1, and MCRMe.
The ∼0.1 Å resolution (k ) 17 Å-1) EXAFS data presented
here provide a precise description of the active site geometry
in the active MCRred1state with the ability to differentiate
between the axial Ni-O(Gln) bond distance and the
Ni-N(F430) distances. This ability to discriminate the axial
and equatorial bond distances was not present in previously
reported EXAFS data, which extended up to k ) 12
Å-1(8, 15). The results presented in this study also provide
the first experimentally determined atomic-level description
of the Ni center in the proposed intermediate methyl-Ni
state with precise Ni-first neighbor bond distance measure-
ments. The trends in the Ni K-pre-edge and edge energy
positions are used to determine the changes in bonding and
ligand field. Together, the XAS and EXAFS studies are used
to provide insight into the electronic structures of the active
sites and how they relate to the mechanism of formation of
methane by MCR.
Sample Preparation. (i) Material and Organisms. Metha-
nothermobacter marburgensis was obtained from the Oregon
Collection of Methanogens catalogue as OCM82. All buffers,
mediUM ingredients, and other reagents were acquired from
Sigma-Aldrich and, unless otherwise stated, were of the
highest purity available. Solutions were prepared using
nanopure deionized water. N2(99.98%), H2/CO2(80%/20%),
and ultra-high-purity (UHP) H2(99.999%) were obtained
from Cryogenic Gases (Grand Rapids, MI). Ti(III) citrate
solutions were prepared from a stock solution of 200 mM
Ti(III) citrate, which was synthesized by adding sodium
citrate to Ti(III) trichloride (30 wt % solution in 2 N
hydrochloric acid) under anaerobic conditions and adjusting
the pH to 7.0 with sodium bicarbonate. The concentration
of Ti(III) citrate was determined routinely by titrating against
a methyl viologen solution.
(ii) M. marburgensis Growth, HarVest, and MCRred1
Purification. MCRred1was isolated from M. marburgensis
cultured on H2/CO2(80%/20%) at 65 °C in a 14 L fermentor.
Culture media were prepared as previously described (18)
with a slight modification of the sulfur and reducing source.
Instead of H2S used previously, 50 mM sodium sulfide was
Ni K-Edge X-ray Absorption Spectroscopy on MCRred1and MCRMe
Biochemistry, Vol. 48, No. 14, 2009 3147
added at a flow rate of 1 mL/min during the entire growth
period. MCRred1 was generated in vivo and purified as
described previously (18). This purification procedure rou-
tinely generates ∼70% MCRred1as determined by UV-visible
and EPR spectroscopy.
(iii) Preparation of MCRred1and MCRMeSamples. MCRred1
was prepared in 50 mM Tris-HCl (pH 7.6) containing 30%
glycerol. The MCRMesamples were prepared in the anaerobic
chamber by incubating MCRred1with excess methyl iodide
in 50 mM Tris-HCl (pH 7.6). The reaction mixture was split
into three aliquots. One sample was transferred to a cuvette
to monitor the conversion of the NiI-MCRred1complex to
NiIII/NiIIby UV-visible spectroscopy; a second aliquot was
frozen in liquid nitrogen in an EPR tube to measure the
concentration of MCRMespecies, and a third sample was
loaded in 1 mm lucite cells with 37 µm Kapton windows
for X-ray absorption studies, frozen in liquid nitrogen, and
maintained under liquid N2 conditions until data were
collected. An important point to note is that the reaction of
MCRred1to MCRMealways goes to at least 100% conversion,
if not more. This observation has been made repeatedly, and
in these studies, we observed a 3% increase in the MCRMe
concentration compared to the starting MCRred1concentration
that was used.
X-ray Absorption Spectroscopy. Ni K-edge X-ray absorp-
tion spectra of MCRred1-silent, MCRred1, and MCRMe were
measured at the Stanford Synchrotron Radiation Laboratory
on 16-pole, 2.0 T, wiggler beamline 9-3. A liquid N2-cooled
Si(220) double-crystal monochromator was used for energy
selection. A Rh-coated harmonic rejection mirror and a
cylindrical Rh-coated bent focusing mirror were used. An
Oxford Instruments CF1208 continuous-flow liquid He
cryostat was used to maintain the sample temperature at ∼10
K throughout the course of data measurement. Data were
measured up to k ) 18 Å-1in fluorescence mode using a
Canberra Ge-30 element array detector. Internal energy
calibration was accomplished by simultaneous measurement
of the absorption of a Ni foil placed between two ionization
chambers situated downstream of the sample. The first
inflection point of the Ni foil was assigned to 8331.6 eV.
All samples were closely monitored for photoreduction.
However, the Ni active site in all three states of the protein
was resistant to photoreduction. Spectra presented here are
a 10-scan, 24-scan, and 28-scan average for MCRred1-silent,
MCRred1, and MCRMe, respectively. The energy-calibrated
data were averaged and processed by fitting a second-order
polynomial to the pre-edge region, which was subtracted
from the entire spectrum as background using Pyspline (22).
A three-region spline function of order 2,3 and 3 was used
to model the background atomic absorption and subtracted
from the spectrum. Normalization was accomplished by
dividing the entire spectrum by a polynomial of order 1,
which was fit to the postedge region. The experimental
threshold energy was chosen to be 8340 eV (8, 15). The
intensities and energies of the pre-edge transitions were
quantitated by performing least-squares refinement using
EDG-FIT (23). The pre-edge features were modeled by
using 1:1 Gaussian/Lorentzian Pseudo-Voigt line shapes to
simulate the convolution of instrument and core-hole lifetime
broadening. Additional Pseudo-Voigt line shapes were also
required to mimic the rising-edge transition and shoulders
in the edge region. The data were fit over two different
energy ranges: 8125-8135 and 8127-8140 eV. The least-
squares error and a comparison of the second derivatives of
the data and fit were used to estimate the goodness of the
fit. Standard deviations in energy position and intensity were
used to quantitate the errors in these parameters. Theoretical
EXAFS phase and amplitude parameters were calculated
using FEFF (Macintosh version 8.4) (24-26) and the
published crystal structure of MCRred1-silent (PDB entry
1MRO) (27) as the initial starting model. Data were fit using
EXAFSPAK (23). The metrical parameters obtained by
fitting the data indicated significant differences in the local
structures of MCRred1, MCRred1-silent, and MCRMearound the
central absorbing Ni atom. On the basis of these preliminary
fits, a new set of theoretical EXAFS signals, ?(k), were
calculated, and the data for MCRred1and MCRMewere refit
using the new theoretical parameters generated from their
individual refined models. The structural parameters varied
during the fitting process were restricted to the bond distance
(R) and the bond variance (σ2), which is related to the
Debye-Waller factor, resulting from a combination of static
and dynamic disorder (due to thermal motion) between the
absorber and scatterer pair. The nonstructural parameter, ∆E0,
was also allowed to vary but was restricted to a common
value for every component in a given fit. Coordination
numbers were systematically varied in the course of a fit
but were fixed within a given fit. In the case of MCRred1and
MCRMe, which were estimated to have 36 and 33% of the
MCRred1-silent decay product using UV-vis spectroscopy,
respectively, partial coordination numbers were also explored
during the course of the fit. It should be noted that the
EXAFS fits to the data were performed between k ) 2 and
17 Å-1. Since, the Fourier transform intensity, R′, is
significant between 1.5 and 4 Å, the number of independent
parameters is 26 (using the formula 2δkδR′/π + 2) (28).
The maximum number of independent parameters used in
the fits is 13, which is lower than the number of maximum
allowed independent parameters.
DFT Calculations. Gradient-corrected (GGA), spin-
unrestricted density functional theory calculations were
performed using the Gaussian03 (29) package on a 32-CPU
Linux cluster. Geometry optimizations were performed in
each case. The B3LYP (30-32) hybrid functional and the
following basis sets were employed: triple-? 6-311+G*
(33-35) on Ni, 6-311G* (33-35) on S, and 6-31G* (36-38)
on O, C, H, and N. The input structures were based on the
published crystal structure and the EXAFS best-fit results
presented herein. The transaxial glutamine ligand was fixed
at the EXAFS distance for MCRred1DFT calculations. Time-
dependent DFT calculations were performed with the
electronic structure program ORCA (39, 40) to calculate the
energies and intensities of the Ni 1s f 3d pre-edge
transitions. Single-point ground-state calculations were per-
formed using the geometry-optimized coordinates obtained
from the Gaussian03 package. The BP86 functional and the
following basis sets were employed: CP(PPP) (41, 42) on
Ni (core properties basis set as implemented in ORCA) and
TZVP (43, 44) on N, C, H, O, and S. Tight convergence
criteria was imposed on all calculations. The calculated
energies and intensities were convoluted with a Gaussian
function with half-widths of 1.4 eV (45) to account for core-
hole and instrument broadening. Calculations were performed
in a dielectric continuum using the conductor-like screening
Biochemistry, Vol. 48, No. 14, 2009
Sarangi et al.
model (COSMO). A shift of 223.0 eV was applied to the
calculated pre-edge energy positions. This is usually the case
with core-level TD-DFT calculation since DFT does not
calculate core potentials accurately, resulting in the core
levels being too high relative to the valence levels.
Ni K-Edge XAS. The normalized Ni K-edge XAS spectra
of MCRred1, MCRred1-silent, and MCRMeare shown in Figure
2 (46). The inset shows the expanded spectra of the second
derivative of the pre-edge region. The MCRred1-silent data
presented here agree well with the previously published XAS
data of Duin et al. (8). The pre-edge feature observed at
∼8332 eV occurs due to an electronic dipole-forbidden
quadrupole-allowed transition from the Ni 1s orbital to
valence orbitals with significant Ni 3d character (47, 48).
This formally forbidden transition gains intensity from Ni
3d-4p mixing due to deviation of the absorbing Ni center
from centrosymmetry (49). The pre-edge energy position
dominantly reflects the change in the ligand field strength
(LF) felt by the absorbing Ni atom and shifts to a higher
energy with an increase in LF (50, 51). Least-squares fits
reveal that the pre-edge transitions for MCRred1, MCRred1-silent,
and MCRMe occur at 8331.5, 8332.0, and 8332.6 eV,
respectively (Table 1), indicating an increase in ligand field
on going from MCRred1 to MCRred1-silent to MCRMe. In
addition to the shift in the pre-edge energy position, an
increase in the pre-edge intensity is observed for MCRMe
relative to MCRred1and MCRred1-silent(Table 1), indicating
a significant increase in the level of 4p mixing in MCRMe.
A comparison of the Ni K-rising-edge spectra of MCRred1,
MCRred1-silent, and MCRMeis shown in Figure 2. The rising-
edge energy positions (approximated to the first inflection
points) for MCRred1, MCRred1-silent, and MCRMe occur at
8341.0, 8342.2, and 8342.4 eV, respectively.
EXAFS. A comparison of the k3-weighted Ni K-edge
EXAFS data and their corresponding non-phase shift cor-
rected Fourier transforms (FT) of MCRred1, MCRred1-silent, and
MCRMeare shown in Figure 3. The EXAFS data for the
MCRred1-silentstate have been previously reported; however,
here, higher-resolution data to k ∼ 17 Å have been obtained.
The resolution in R space is 0.1 Å for all the data sets
presented here (52). The Fourier transforms indicate that with
the transition from MCRred1-silentto MCRred1there is a sharp
decrease in the first-shell intensity at ∼1.5 Å, in addition to
a small shift to a lower R′ (angstroms) (53). With the
transition from MCRred1to MCRMe, the first-shell peak shifts
to a higher R′ and gains intensity. Beyond the first shell, the
MCRred1-silentFourier transform has significant intensity at
R′ ∼ 2.0 Å, which is absent in both MCRred1and MCRMe
forms. The Fourier transform intensities resulting from the
second- and third-shell single and multiple scattering con-
tributions are very similar in all three forms. Importantly,
the MCRred1and MCRMeEXAFS data differ significantly
from each other and from the MCRred1-silentdata, indicating
that the Ni active sites are different in each state.
The k3-weighted Ni K-edge EXAFS data for MCRred1-silent,
MCRred1, and MCRMe, their corresponding non-phase shift
FIGURE 2: (A) Normalized Ni K-edge XAS spectra of MCRred1-silent
(red), MCRred1(green), and MCRMe(blue). The inset shows the
expanded second-derivative spectrum. (B) First-derivative spectrum
showing the edge inflection points.
Table 1: Ni K-Pre-Edge and Edge Energy Positions and Intensities
8331.5 ( 0.04
8332.0 ( 0.02
8332.6 ( 0.02
5.3 ( 1.8c
aThe spectral broadening is ∼1.4 eV.bThe errors in pre-edge energy
position obtained from a statistical analysis over several best fits are
given. The systematic error due to monochromator energy drift and
calibration is less than 0.04 eV.cThe error in total intensity estimation
due to data processing and statistical estimation of the standard
deviation is (0.5 × 10-2. The error in intensity estimation of MCRred1
is higher due to the presence of low-lying edge transitions.
FIGURE 3: Comparison of the k3-weighted Ni K-edge EXAFS for
MCRred1-silent(red), MCRred1(green), and MCRMe(blue) and their
corresponding Fourier transforms.
Ni K-Edge X-ray Absorption Spectroscopy on MCRred1and MCRMe
Biochemistry, Vol. 48, No. 14, 2009 3149
corrected Fourier transforms (FTs), and the corresponding
fits are presented in panels A-C of Figure 4. The EXAFS
best-fit parameters are listed in Table 2. In all fits, the
addition of a shell was justified on the basis of a significant
decrease in the error function. A complete shell-by-shell
analysis is presented in the Supporting Information. The first
shell of the EXAFS data for MCRred1-silentwas fit with four
Ni-N components at 2.09 Å, one Ni-S component at 2.41
Å, and one Ni-O component at 2.26 Å. The second and
third shells (2.0-4.5 Å region in the FT spectra) were fit
with single-scattering (SS) and multiple-scattering (MS)
components from the corphin ring (see Table 2). The SS
from the third shell of the corphin ring contributes between
∼3.8 and 4.2 Å. Long-range (high-R) SS contributions from
light atoms are usually weak and do not significantly
contribute to the EXAFS signal. Inclusion of the SS paths
(for all three data sets) resulted in negative σ2values and
did not improve the goodness of fit parameter. However, MS
components, which can have significant long-range contribu-
tions as a result of the forward focusing effects, could be
included and resulted in a statistically significant decrease
in the goodness of fit parameter (see the shell-by-shell fit in
the Supporting Information). The first shell of the EXAFS
data for MCRred1was fit with four Ni-N components at 2.05
Å and a weak Ni-O component at 2.25 Å. The best fit to
the data furnishes a high σ2value for the first-shell Ni-N
component at 2.05 Å, indicating a high level of disorder in
the Ni-N(corphin) bond distances. The second and third
shells were fit with single-scattering (SS) and multiple-
scattering (MS) components from the corphin ring (Table
2). Since the MCRred1sample was contaminated with ∼36%
of the MCRred1-silentdecay form, a 0.36 Ni-S component
was included and proved to be necessary for obtaining a good
fit. In the case of MCRMe, the first shell of the EXAFS data
was fit with five Ni-N components at 2.08 Å and a weak
Ni-O component at 2.32 Å. Split first-shell fits were also
performed which resulted in a best fit with one Ni-C
component at 2.0 Å, four Ni-N components at 2.08 Å, and
a very weak Ni-O component at 2.32 Å. However, the error
value did not improve significantly to justify the addition of
two independent parameters resulting from the split first shell.
The second and third shells were fit with single-scattering
(SS) and multiple-scattering (MS) components from the
corphin ring. Since the MCRMesample was contaminated
with ∼33% of the MCRred1-silentdecay form, a 0.33 Ni-S
component was included and proved to be necessary for
obtaining a good fit. For all three data sets, the second and
third shell were fit using the same number and type of SS
and MS components.
Ni K-Pre-Edge TD-DFT Calculations. The geometric and
electronic structure of MCRred1-silenthas been well character-
ized using X-ray diffraction and optical spectroscopic
techniques, which indicate a six-coordinate high-spin S ) 1
d8NiIIspecies with a dz2ground state (27, 55, 56). Thus, it
is expected that two 1s f 3d transitions corresponding to
the two singly occupied dz2 and dx2-y2 orbitals should be
present. However, the spectral broadening at the Ni K-edge
is ∼1.4 eV which does not allow the two pre-edge features
to be well-resolved. In addition, the overlap of the pre-edge
with the intense rising-edge transition renders it impossible
to separate out the two 1s f 3d transitions. Thus, the average
energy of the two 1s f 3d transitions is determined to be
The one-electron reduced MCRred1form is an S )1/2d9
species with a dx2-y2ground state, indicating that the dz2hole
becomes occupied upon reduction and loss of the upper axial
ligand (1, 57, 58). The pre-edge energy position in MCRred1
is shifted to lower energy (by 0.6 eV) compared to
MCRred1-silent. This is consistent with a decrease in LF
associated with the decrease in the number of ligands from
six (MCRred1-silent) to five (MCRred1) (see Table 2). It is
important to note here that although the ligand field in
MCRred1has decreased relative to that in MCRred1-silent, the
energy position (8331.5 eV) is comparable to those of other
covalent NiIIcompounds (59, 60), indicating a strong ligand
field at the Ni active site exerted due to the presence of the
equatorial F430 cofactor. In MCRred1-silent, the axial bond
distances are longer by ∼0.04 Å, which might suggest that
the pre-edge energy position of MCRred1-silentshould be lower
than in MCRred1. However, the presence of a strong Ni-S
FIGURE 4: k3-weighted Ni K-edge EXAFS data (inset) and their
corresponding Fourier transforms (FT) for (A) MCRred1-silent[data
(gray) and fit (red)], MCRred1 [data (gray) and fit (green)], and
MCRMe[data (gray) and fit (blue)].
Biochemistry, Vol. 48, No. 14, 2009
Sarangi et al.
ligand in MCRred1-silentcompensates for the increase in ligand
field in MCRred1due to shortening of the equatorial Ni-N
bonds (see Table 1). This increases the ligand field felt at
the Ni center in MCRred1-silentand shifts the pre-edge to a
higher energy than in MCRred1. The rising-edge spectrum of
MCRred1shows two transitions at 8333.5 and 8334.9 eV.
These transitions are usually most intense in four-coordinate
square planar complexes in ideal D4h geometry and lose
intensity as the site symmetry deviates from D4h(61, 62).
These intense edge features can result from the following
effects: long-range multiple-scattering effects that are en-
hanced at the rising edge due to the larger photoelectron
mean free path (63) and a formally forbidden two-electron
shakedown process which becomes allowed in the excited
state (64, 65). While the shakedown transition is enhanced
for compounds that have strong charge transfer transitions
or are very covalent (50, 66), both effects are enhanced in
the D4hsymmetry, in which the central atom lies in the plane
of the equatorial ligands. The presence of the low-lying edge
transition only in the MCRred1form indicates that the NiI
center is closer to the corphin plane relative to the
MCRMehas been shown to be a d7species with an S )1/2
ground state (17, 58). This electronic structure allows for a
total of three Ni 1s f 3d transitions contributing to the Ni
K-pre-edge transition. It has been previously shown on the
basis of EPR data that the ground state is dx2-y2; thus, the
three 1s f 3d transitions are to the ? dx2-y2and R and ? dz2
orbitals. The pre-edge feature for MCRMehas shifted by 1.1
eV to a higher energy compared to that of MCRred1, and the
intensity has approximately quadrupled. This indicates a
dramatic increase in ligand field and Ni 3d-4p mixing. The
pre-edge energy position is higher than that for several other
NiIIIcomplexes (pre-edge energy position of e8332 eV)
supporting the increase in ligand field (59, 60). In addition,
the EXAFS results indicate that the first-shell coordination
has increased from four to five on going from MCRred1to
MCRMe(see Results). Together, the pre-edge and EXAFS
data indicate that MCRMehas an additional strongly coor-
dinating axial light-atom ligand, consistent with previous
EPR/ENDOR (16, 58) studies that show the axial ligand to
be a methyl group. The presence of a Ni-C axial interaction
is also expected to result in a dramatic increase in the Ni
3dz2-4pzmixing and hence the 1s f dz2(R and ?) transition
intensity; the pre-edge data directly indicate the presence of
a methyl group as the upper axial ligand in MCRMe.
Interestingly, although the pre-edge energy position shifts
to a higher energy with the transition from MCRred1-silentto
MCRMe, the edge inflection points are very similar for the
two species (Table 1). This shows that the QNiin MCRMeis
similar to that in MCRred1-silent (67), indicating that the
additional hole in MCRMeis spread over the entire corphin
ring and the site is best described as [Ni(F430)Me]+, where
F430represents the corphin ring and the positive charge is
delocalized (see Discussion for further analysis of the charge
To support the Ni K-pre-edge analysis, TD-DFT calcula-
tions were performed on starting active site structures of
MCRred1-silent, MCRred1, and MCRMe, which were generated
by considering the crystal structure of MCRred1-silentand the
EXAFS data presented herein. Selected DFT parameters are
listed in Table 3. The calculated spectra on the geometry-
optimized structures are presented in Figure 5. The calculated
pre-edge energies are 8118.8, 8119.2, and 8119.6 eV for
MCRred1, MCRred1-silent, and MCRMe. The trend in energy
position is in reasonable agreement with the experimental
data (see Table 1).
Fits to the EXAFS data for MCRMeindicate the presence
of a weak Ni-O component at 2.32 Å. Since the contribution
of a relatively long first-shell light atom to the EXAFS data
is usually weak, the fits cannot be definitive indicators of
the presence of the long Ni-O component. Hence, to test
for the presence of this long Ni-O(Gln) axial interaction,
TD-DFT calculations were also performed on a MCRMe
model with no axial glutamine ligand (MCRMe-NA). The
calculations show that both the relative pre-edge energy and
intensity are in worse agreement with the data for the
MCRMe-NAmodel relative to the MCRMemodel, strongly
Table 2: Ni K-Edge EXAFS Least-Squares Fitting Resultsa
aThe S02value was fixed at 1 for all refinements. The positive ∆E0value is justified on the basis of the choice of threshold energy (54).bEstimated
standard deviations in the distances are (0.02 Å.cThe σ2values have been multiplied by 105.dThe error is given by [(?obsd- ?calcd)2k6]/[(?obsd2)k6].
eThe distance for the Ni-S path was kept fixed in the case of MCRred1and MCRMe.
Table 3: Selected DFT Parameters
bond distance (Å)b
Lo ¨ewdin chargeMulliken population (%)
aThe calculations were performed on simplified models (shown in Figure 6).bThe numbering of the Ni-N bonds is explained in Figure S2 of the
orbitals. In the case of MCRred1, only 3dx2-y2 character is present.eValence Ni 3dyz and 3dxz character due to back-bonding with filled π*-type F430
cThe charge on the entire corphin ring.
dThe combined contribution of the antibonding Ni 3dx2-y2 and 3dz2 to the valence
Ni K-Edge X-ray Absorption Spectroscopy on MCRred1and MCRMe
Biochemistry, Vol. 48, No. 14, 2009 3151
indicating the presence of an axial Ni-O(Gln) bond in
Geometric Structures of MCRred1 and MCRMe. The Ni
K-edge EXAFS results indicate that the active site in
MCRred1-silentis six-coordinate with four Ni-N interactions
at 2.08 Å, one Ni-S interaction at 2.41 Å, and one weak
Ni-O interaction at 2.27 Å. This is in reasonable agreement
with the published crystal structure (PDB entry 1HBO with
a resolution of 1.78 Å) (10, 68). The crystal structure reveals
that MCRred1-silentis a dimer of two R?γ trimers with nearly
identical Ni-porphinoid active sites. The crystal structure
also indicates that the Ni cofactor exhibits a large spread in
the equatorial Ni-N bond distances, ranging from 1.94 to
2.31 Å. However, fits to the EXAFS data presented here
show that the first-shell σ2value is low, indicating an ordered
first shell, which in turn suggests that in solution the
equatorial bond distances of the Ni-porphinoid ring of
MCRred1-silentare more symmetric than in the crystal.
The fit to the Ni K-edge EXAFS data of MCRred1indicates
a disordered first shell with four Ni-N contributions and a
weak axial Ni-O interaction at 2.26 Å. This axial interaction
is longer than that reported in a previous EXAFS analysis
(2.12 Å); however, the EXAFS data presented in the reported
study were limited (k ) 2-12 Å-1) (8), and an accurate
estimate of a weak axial ligand was not possible. Our study
reveals that with the transition from MCRred1-silentto MCRred1,
the axial -SR group is lost and the site becomes disordered.
One interesting aspect of the EXAFS data is that the average
Ni-N bond distance decreases from 2.09 to 2.05 Å with
the transition from MCRred1-silentto MCRred1(69). A similar
shortening of the bond distance was observed in the EXAFS
data for the isolated NiI/NiIIF430cofactor (70, 71). This is
counterintuitive since the larger NiIin MCRred1should lead
to longer Ni-N bond distances. However, this shortening
of the bond can be explained on the basis of back-bonding
from the NiIcenter to the low-lying π* orbitals on the corphin
ring (72). The charge transfer arising due to back-bonding
would be consistent with the low-lying charge transfer
transitions observed in the MCD spectrum of MCRred1and
low-energy transitions in the Ni K-rising-edge region (73).
This is also consistent with the valence Mulliken populations
obtained from the DFT calculations, which show significant
valence Ni 3dxzand 3dyzcharacter in the valence low-lying
orbitals for MCRred1(see Table 3). Thus, a combination of
the edge data, the pre-edge energy position, and the EXAFS
data reveals a five-coordinate, disordered active site with a
weak axial Ni-O bond in MCRred1(Figure 6).
The Ni K-edge EXAFS data for MCRMeare significantly
different from those of MCRred1-silent and MCRred1. In
particular, the Ni-S contribution seen in MCRred1-silentis not
present and the first shell is composed of five light atom
ligands [simulated using five Ni-N components (see Table
2)], in contrast to four in MCRred1and MCRred1-silent. On the
basis of the Ni K-pre-edge data and the TD-DFT calculations,
the additional light atom ligand is best described as a methyl
group. Since the MCRMesample had ∼33% MCRred1-silent
contamination, fits with 4.67 Ni-N components and split
first-shell fits with 0.67 Ni-C and 4 Ni-N components were
attempted. In the 4.67 Ni-N first-shell case, the fit improved
slightly (F ) 0.26), with very small changes in the σ2values
of the remaining paths. Since the error in EXAFS coordina-
tion number determination is 25%, the fit cannot be dif-
ferentiated from the best fit presented in Table 2. In the 0.67
Ni-C and 4 Ni-N first-shell case, again, the fit improved
slightly (F ) 0.26); however, the two first-shell paths (2.02
and 2.08 Å) were within the resolution of the data (∼0.10
Å), and the first-shell split was not justifiable. However, both
fits are consistent with the best fit presented in Table 2 and
support an axial Ni-C(methyl) interaction. It is important
to note here that the Ni-C(methyl) interaction cannot be
separated from the spread of Ni-N interactions. In addition,
the σ2value of the Ni-N component is on the higher side
(∼591), indicating that there is a spread in the first-shell bond
distances, likely due to the presence of a shorter Ni-C(methyl)
component. On the basis of the resolution of the data, the
lower limit of the Ni-C bond distance is estimated to be
1.99 Å. The Ni-O bond distance from the weak glutamine
ligand is ill-determined from the EXAFS fits. Since the Ni-C
bond distance can be modulated by the transaxial ligand,
DFT calculations were also performed in the absence of the
transaxial ligand. The optimized Ni-C bond distance with
and without the lower axial Ni-O ligand is 2.0 and 2.04 Å,
respectively, which are both consistent with the EXAFS data
and Ni-C bonds in model complexes, which range from
1.95 to 2.04 Å (74). In addition, a more than 3-fold increase
in the pre-edge intensity of MCRMerelative to MCRred1-silent
suggests that the Ni-C bond is shorter than the average
Ni-N distance. Thus, a combination of the Ni K-pre-edge
intensity and energy position, DFT calculations, and the
EXAFS data reveal a six-coordinate active site with a
significantly weak axial Ni-O bond and an axial Ni-C bond
with a bond distance of ∼2.04 Å (average of EXAFS and
DFT bond distances) in MCRMe(Figure 6).
Methyl-coenzyme M reductase catalyzes the reduction of
methyl-coenzyme M (methyl-SCoM) with coenzyme B
(HSCoB) to methane with the subsequent formation of the
heterodisulfide of methyl-SCoM and HSCoB. Several ex-
perimental and theoretical studies have been performed in
an effort to understand the catalytic mechanism of MCR and
the geometric and electronic structures of the intermediates
FIGURE 5: (A) Comparison of the Ni K-pre-edge XAS data (s)
with the TD-DFT calculated spectra (---): MCRred1-silent (red),
MCRred1(green), MCRMe(blue), and MCRMe-NA(gray).
Biochemistry, Vol. 48, No. 14, 2009
Sarangi et al.
in the catalytic cycle, and to date, two mechanisms have been
proposed, which differ in the first step: the reaction of
MCRred1with methyl-SCoM. The first mechanism involves
the formation of an organometallic NiIII-methyl intermediate
starting from MCRred1and methyl-SCoM. This is followed
by protonolysis to generate methane and the heterodisulfide.
The second mechanism involves a direct attack of the NiI
on the S of methyl-SCoM, resulting in a homolytic cleavage
of the thioether bond and formation of a NiII-thiolate species
and a CH3•radical. The methyl radical reacts with HSCoB
to form methane followed by subsequent heterodisulfide
formation and reduction of the NiIIspecies to MCRred1. In
the study presented here, the geometric and electronic
structures of MCRred1and of a stable NiIII-methyl species
(formed from the reaction of MCRred1and MeI) have been
elucidated using X-ray absorption spectroscopy.
The formation and stability of this species support the
intermediacy of an alkyl-Ni species in the catalytic cycle
of MCR, as proposed in mechanism I. The next step in
the catalytic cycle (mechanism I) involves the cleavage of
the NiIII-methyl bond. This can occur either by homolytic
cleavage resulting in NiIIand a CH3•radical or by heterolytic
cleavage involving two-electron transfer from the methyl
group to the Ni center resulting in NiIand a CH3+cation.
The results presented in this study indicate that the Ni-C
bond in MCRMeis long (74), reminiscent of the long CoIII-C
bond in adenosylcobalamin (AdoCbl) (∼2.04 Å) (75) and
in methylcobalamin (1.96-2.08 Å) (76). In AdoCbl-depend-
ent enzymes, the long weak Co-C bond undergoes a
homolytic cleavage forming an Ado•radical, which subse-
quently initiates radical-based substrate rearrangements
(77-80), while in MeCbl, a heterolytic cleavage occurs to
leave a methyl cation and CoI. Thus, the long Ni-Me bond
in MCR observed by XAS could promote homolysis of the
NiIII-methyl bond, which would lead to formation of a
methyl radical or enhance the electrophilicity of the methyl
group, hence increasing its susceptibility toward nucleophilic
attack. In the case of the radical mechanism, the resulting
CH3•radical can then abstract an H•from HSCoB (either
directly or indirectly through another radical intermediate),
yielding CH4and•SCoB, which subsequently would lead to
the formation of CoBS-SCoM and the active MCRred1state
In this study, Ni K-pre-edge, rising-edge, and EXAFS data
analysis have been combined with TD-DFT calculations on
the MCRred1-silent, MCRred1, and MCRMestates of MCR to
elucidate the geometric and electronic structure differences.
It is shown that the Ni K-pre-edge energies (1s f 3d
transition) in all three states of MCR are higher than in most
Ni complexes (e8331.5 eV), which provides direct evidence
for the strong Ni-F430 cofactor interaction in MCR. For
MCRred1-silent, high-k EXAFS data have been used to show
that the geometric structure in solution is very similar to that
obtained from the crystal structure, a six-coordinate active
site with four Ni-N distances of 2.09 Å, one Ni-S distance
of 2.41 Å, and one Ni-O distance of 2.26 Å. The data
provide high-resolution first-shell bond distances ((0.02 error
in bond distance estimation) that have not yet been achieved
by X-ray diffraction measurements. For the MCRred1state,
a better understanding of the five-coordinate, reduced NiI
active site has been achieved, and it is shown that the first-
shell coordination is distorted with a large distribution of
the Ni-N distances. The average Ni-N distance is 2.05 Å,
and the shorter axial Ni-O distance is 2.25 Å. The first
solution structure of an NiIII-alkyl state of MCR, MCRMe,
has been determined, which shows that the active site is six-
coordinate with four Ni-N distances of 2.08 Å, one Ni-C
bond (∼2.04 Å), and a poorly determined lower axial Ni-O
interaction of 2.32 Å.
The EXAFS analysis (combined with the Ni K-edge pre-
edge data and DFT calculations) presented here unambigu-
ously demonstrates the presence of a long Ni-C organo-
metallic bond (74), which is attributed to an upper axial
NiIII-methyl interaction. The EXAFS data reveal that a weak
axial Ni-O interaction is present in all three states of MCR,
demonstrating that this axial ligand does not participate in
strong bonding with the central Ni atom. It is therefore likely
that the lower axial glutamine ligand is present to tune the
redox potential and/or to provide stability to the active site.
The XAS data for the different forms of MCR show a
unique property: the shift in the edge energy positions is
relatively small, indicating that the QNion the Ni is similar
in all the three states of MCR, consistent with the Ni K-edge
EXAFS data. This demonstrates that the Ni-N bond
distances have not changed significantly with the transition
from MCRred1to MCRred1-silentto MCRMe. The similarity in
charge for the three MCR states is a direct consequence of
Pauling’s electroneutrality principle which is manifested in
this system by two different mechanisms. Upon reduction
of MCRred1-silentto MCRred1, the filled 3d orbitals on the Ni
center are destabilized, allowing for back-bonding interaction
between the Ni center and the F430cofactor with a partial
FIGURE 6: Schematic diagram of the predicted active site structures of MCRred1-silent, MCRred1, and MCRMebased on Ni K-edge EXAFS and
Ni K-pre-edge analysis. Relevant first-shell bond distances have been included. Some atoms of the F430cofactor have been omitted for the
sake of clarity. The lower-axial bond distance in MCRMeis ill-determined by EXAFS. The dashed Ni-O bond in MCRred1and MCRMe
indicates a larger than normal EXAFS uncertainty. Asterisks indicate the average Ni-C bond distance, based on TD-DFT calculations and
EXAFS data analysis.
Ni K-Edge X-ray Absorption Spectroscopy on MCRred1and MCRMe
Biochemistry, Vol. 48, No. 14, 2009 3153
flow of charge from the Ni to the F430orbitals, and hence,
the charge on the central Ni atom remains closer to that in
MCRred1-silent(closer to NiIIthan NiI). This is consistent with
the similar Lo ¨ewdin charges on the Ni center in MCRred1
and MCRred1-silentobtained from DFT calculations (Table 3).
Calculations also show a dramatic increase in the Ni 3dyz
and 3dxzhole character due to back-bonding interaction with
the filled π* orbitals on the F430ring. This increases the total
valence Ni character, leading to similar charges on the Ni
center in MCRred1and MCRred1-silent. With the transition from
MCRred1-silentto MCRMe, the additional 3d hole created in
MCRMe(NiIIIin d7configuration) undergoes covalent delo-
calization over the entire active site, resulting in a species
that is best described as [Ni(F430)Me]+. Thus, in this case
also, the charge on the central Ni atom remains closer to
that in MCRred1-silent. Since the total charge on the
MCRred1-silent and MCRMe models chosen for the DFT
calculations are different, a direct comparison of the indi-
vidual fragment charges will be inaccurate; however, the
combined valence Ni 3d character in MCRred1(150%) and
MCRred1-silent(141%) are similar, with only a small increase
for MCRMe. This, combined with the small edge shift with
the transition from MCRred1-silentto MCRMe, indicates similar
charges in the two states. This noninnocent role of the F430
cofactor in tuning its bonding with the Ni center in different
oxidation states is expected to play a direct role in modulating
the geometric and electronic structures of the active site and
therefore plays an important role in the catalytic pathway.
For example, it might be expected that the NiIIIsite in MCRMe
would be very unstable due to a high redox potential and
might spontaneously reduce to form a NiII-methyl species.
The stability of the MCRMe can be attributed to the
noninnocent participation of the F430cofactor in bonding,
which is consistent with the stability observed in reported
biochemical studies performed with this state of MCR. In
the case of MCRred1, the low charge on a formally NiIspecies
would be expected to increase the pKaof the coordinating
anionic nitrogen on the F430ring and destabilize the Ni-N
bond toward dissociation and protonation. Here again, the
participation of the F430ring in noninnocent bonding results
in an increase in QNi and promotes the stability of the
MCRred1species. Thus, the Ni K-edge XAS data indicate
that the F430cofactor plays a critical role in stabilizing the
different forms of MCR and tuning the reactivity of the
SSRL operations are funded by the Department of Energy,
Office of Basic Energy Sciences. The SSRL Structural
Molecular Biology program is supported by the National
Institutes of Health, National Center for Research Resources,
Biomedical Technology Program, and the Department of
Energy, Office of Biological and Environmental Research.
SUPPORTING INFORMATION AVAILABLE
Figures and table showing the FEFF fits to the Ni K-edge
EXAFS data and their corresponding Fourier transforms of
the MCRred1-silentsubtracted MCRred1and MCRMeforms and
the metrical parameters, respectively, and a shell-by-shell
analysis of the EXAFS data for MCRred1, MCRred1-silent, and
MCRMe. This material is available free of charge via the
Internet at http://pubs.acs.org.
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