CO2 Reduction Catalyzed by Nitrogenase: Pathways to Formate, Carbon Monoxide, and Methane

Article abstract of DOI:10.1021/acs.inorgchem.6b00388

The reduction of N₂ to NH₃ by Mo-dependent nitrogenase at its active-site metal cluster FeMo-cofactor utilizes reductive elimination of Fe-bound hydrides with obligatory loss of H₂ to activate the enzyme for binding/reduction of N₂. Earlier work showed that wild-type nitrogenase and a nitrogenase with amino acid substitutions in the MoFe protein near FeMo-cofactor can catalytically reduce CO₂ by two or eight electrons/protons to carbon monoxide (CO) and methane (CH₄) at low rates. Here, it is demonstrated that nitrogenase preferentially reduces CO₂ by two electrons/protons to formate (HCOO^-) at rates >10 times higher than rates of CO₂ reduction to CO and CH₄. Quantum mechanical calculations on the doubly reduced FeMo-cofactor with a Fe-bound hydride and S-bound proton (E₂(2H) state) favor a direct reaction of CO₂ with the hydride ( direct hydride transfer reaction pathway), with facile hydride transfer to CO₂ yielding formate. In contrast, a significant barrier is observed for reaction of Fe-bound CO₂ with the hydride ( associative reaction pathway), which leads to CO and CH₄. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (α-70^Val→Ala, α-195^His→Gln) are found to significantly alter the distribution of products between formate and CO/CH₄.

Full text of DOI:10.1021/acs.inorgchem.6b00388

Article
pubs.acs.org/IC
CO2 Reduction Catalyzed by Nitrogenase: Pathways to Formate,
Carbon Monoxide, and Methane
Nimesh Khadka,† Dennis R. Dean,‡ Dayle Smith,§ Brian M. Hoffman,*,∥ Simone Raugei,*,⊥
and Lance C. Seefeldt*,†
†Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, United States
‡Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, United States
§Intel Corporation, Hillsboro, Oregon 97124, United States
⊥Pacific Northwestern National Laboratory, Richland, Washington 99352, United States
∥Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States
S
* Supporting Information
ABSTRACT: The reduction of N2 to NH3 by Mo-dependent nitrogenase at its
active-site metal cluster FeMo-cofactor utilizes reductive elimination of Fe-
bound hydrides with obligatory loss of H2 to activate the enzyme for binding/
reduction of N2. Earlier work showed that wild-type nitrogenase and a
nitrogenase with amino acid substitutions in the MoFe protein near FeMo-
cofactor can catalytically reduce CO2 by two or eight electrons/protons to
carbon monoxide (CO) and methane (CH4) at low rates. Here, it is
demonstrated that nitrogenase preferentially reduces CO2 by two electrons/
protons to formate (HCOO−) at rates >10 times higher than rates of CO2
reduction to CO and CH4. Quantum mechanical calculations on the doubly
reduced FeMo-cofactor with a Fe-bound hydride and S-bound proton (E2(2H)
state) favor a direct reaction of CO2 with the hydride (“direct hydride transfer”
reaction pathway), with facile hydride transfer to CO2 yielding formate. In
contrast, a significant barrier is observed for reaction of Fe-bound CO2 with the hydride (“associative” reaction pathway), which
leads to CO and CH4. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in
the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (α-
70Val→Ala, α-195His→Gln) are found to significantly alter the distribution of products between formate and CO/CH4.
bound to one or more of the sulfides of FeMo-co, hence the
notation E4(4H).9
INTRODUCTION
■
Nitrogenase catalyzes the reduction of dinitrogen (N2) to two
ammonia (NH3) molecules,1 the largest contribution of “fixed
nitrogen” in the global biogeochemical nitrogen cycle.2 The
reduction of N2 by Mo-dependent nitrogenase occurs at the
FeMo-cofactor (7Fe-9S-1Mo-1C-1R-homocitrate) contained
within the nitrogenase molybdenum iron protein (MoFe
protein) (Figure 1a).3 Early kinetic studies indicated that
FeMo-co must accumulate four electrons and protons (the
E4(4H) state in the Lowe and Thorneley kinetic model) before
the first step of N2 reduction,4 with the electrons delivered one
at a time by the nitrogenase Fe protein,5 before N2 can be
reduced, and that N2 binding coincides with the evolution of
one equivalent of H2.6 Recently, analysis of the reactivity of
proteins with amino acid substitutions, coupled with advanced
spectroscopic studies of trapped intermediates isotopically
labeled at the metal ions and substrates, has identified the
intermediates postulated in this kinetic model.7−10 In particular,
the reactive E4(4H) state has been revealed to contain two
[Fe−H−Fe] bridging hydrides bound to a particular 4Fe-4S
FeMo-co face (Figure 1b).7,8,11−13 For charge balance, the E4
state is also assumed to contain two bound protons, likely
These experiments have further established that E4(4H)
necessarily undergoes a loss of H2 during N2 binding/reduction,
with the consequence that N2 reduction requires the delivery of
a total of eight electrons/protons to the MoFe protein.12,13
This kinetically and thermodynamically reversible equilibrium
binding of N2 to E4(4H) with H2 release has been proposed to
involve reductive elimination (re) of the two hydrides forming
H2, with concomitant reduction of N2 to a diazenido-level,
metal-bound state M-(2N2H) (Figure 2, right side).9 Four
subsequent electron/proton addition steps ultimately lead to
the formation of two NH3 molecules and return FeMo-co to
the resting (E0) state.
In addition to reducing N2 and protons, nitrogenase can
reduce a number of small, multiply bonded compounds.14,15
Reduction of acetylene (C2H2) to ethylene (C2H4) is
commonly used as an activity assay.16 We recently showed
acetylene reduction involves migratory insertion (mi) of bound
Received: February 16, 2016
© XXXX American Chemical Society
A
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Figure 1. FeMo-co of nitrogenase. (A) FeMo-co with the α-Cys275 and α-His442 ligands and R-homocitrate (right). (B) One FeS face of FeMo-co
in the proposed bridging dihydride E4 state with two protons associated with sulfides. Not shown are several possible binding modes for the
hydrides.
C2H2 into a Fe−H bond to form an Fe−alkenyl intermediate,
followed by reductive elimination of ethylene.17 Substitution of
one or more “gatekeeper” amino acids in the protein
environment surrounding FeMo-co has been shown to relax
the native enzyme’s size restriction on compounds that can gain
access to FeMo-co for reduction and to control the distribution
of electrons between competing substrates.18 Most recently, we
demonstrated that nitrogenase remodeled in this way can
19
−
−
reduce nitrite (NO2 ) to NH3. Intermediates along the NO2
reaction pathway were trapped by a freeze−quench method
and characterized by Q-band electron nuclear double resonance
(ENDOR)/non-Kramers electron spin echo envelop modu-
lation (ESEEM) spectroscopy. A pair of intermediates
consistent with M-NH2 and M-NH3 (M = FeMo-co) species
were trapped and identified as the same as intermediates
trapped during the reduction of diazene (HNNH) and
hydrazine (H2N-NH2).11 From this observation, and the fact
that the two-electron/two-proton trapped state of the FeMo-co
likely contains a metal-bound hydride and a proton (E2(2H)),
−
we proposed a mechanism for NO2 reduction that again
invokes migratory hydride insertion, with the substrate
fragment of the M-NOOH state inserting into the M-H−,
with proton addition and loss of H2O to yield the M-NHO
intermediate (Figure 2, left).19 Subsequent reduction leads to
the observed M-NH2 and M-NH3 intermediates and ultimate
release of NH3.
From these findings, it can be seen that hydrides on FeMo-
cofactor can undergo at least two different types of reaction: (i)
reductive elimination of H2 by the state containing two
hydrides, activating FeMo-co for N2 binding and reaction; (ii)
and migratory insertion into the M-H by substrates, such as
acetylene and nitrite, and most likely some of the N2Hx
intermediate states of N2 reduction.
It has been demonstrated that in a MoFe protein with
substituted amino acids near FeMo-co carbon dioxide (CO2)
can be catalytically reduced by two or eight electrons/protons
to carbon monoxide (CO) or methane (CH4) at low rates.20,21
A better understanding of the mechanism by which nitrogenase
catalyzes CO2 reduction would provide insight into the
challenging and environmentally important reduction of CO2.
Here, we report that wild-type nitrogenase reduces CO2 by two
electrons/protons to make formate (HCOO−) at rates up to 10
Figure 2. Pathways for N2 and nitrite reduction. Proposed pathways
for H2 evolution (top), N2 reduction (right), and nitrite reduction
(left), where M represents FeMo-co and the En state is noted. Hydride
protonation (hp) is proposed to make H2, reductive elimination (re)
activates N2, while hydride migratory insertion (mi) has been proposed
in the reduction of nitrite. Oxidative addition (oa) is shown for the
binding of H2 concomitant with the release of N2 (right).
B
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parameters except turning the decoupling parameter off (data not
shown). A colorimetric assay was used to quantify the formation of
formate using a protocol previously reported.24 In a 9.4 mL serum vial,
3.5 mL of 100% acetic anhydride, 50 μL of 30% (w/v) sodium acetate,
and 1 mL of 2-propanol solution containing 0.5% (w/v) citric acid and
10% (w/v) acetamide were added. To this assay solution was added
500 μL of sample with 5 h of incubation time at room temperature.
Using a UV−visible spectrophotometer, the absorbance was
determined at 514 nm (Figure. S2). The formate standard calibration
curve was made as follows: first, the assay buffer with MoFe and Fe
protein was left to turn over for an hour under an Ar atmosphere;
then, the appropriate concentration of formate was added. From this,
500 μL of samples was used for the colorimetric assay.
times faster than the rates of CO and CH4 formation reported
earlier. To understand the alternative reaction mechanisms, we
have explored possible pathways for CO2 reduction on FeMo-
co using density functional theory (DFT) calculations. These
pathways involve reactions of Fe-hydrides and yield energies
that are consistent with the observed product distribution for
CO2 reduction. In addition, we show that amino acid
substitutions near FeMo-co in the MoFe protein can
significantly alter the product distribution between formate
and CO/CH4.
MATERIALS AND METHODS
■
Calculations. Two models of the FeMo-co and the enzyme
environment of increasing complexity were employed for the DFT
Reagents and Protein Purification. All reagents were obtained
from SigmaAldrich (St. Louis, MO, USA) or Fisher Scientific (Fair
Lawn, NJ, USA) and were used without further purification. Carbon
dioxide was purchased from Air Liquide (Walnut Creek, CA, USA)
and methane from Air Gas (Radnor, PA, USA). Azotobacter vinelandii
strains DJ995 (wild-type), DJ997 (α-195Gln), DJ1310 (α-70Ala), and
DJ1316 (α-70Ala/α-195Gln) were grown, and the corresponding
nitrogenase MoFe proteins having a seven-His tag addition near the
carboxyl-terminal end of the α-subunit were expressed and purified as
previously described.22 Protein concentrations were determined by the
Biuret assay using bovine serum albumin as standard. Handling of
proteins and buffers was done in septum-sealed serum vials under an
argon atmosphere or on a Schlenk vacuum line. All gases and liquids
were transferred using gastight syringes.
calculations. The first model comprises the FeMo-co ligands α-275Cys
,
α-442His (Azotobacter vinelandii numbering), and R-homocitrate, which
were modeled as methylthiolate, imidazole (or 4-methylimidazole),
and dimethyl glycolate, respectively (Figure S3a). The second model
adds residues α-70Val and α-96Arg located on top the FeMo-co reactive
face, which were modeled as tert-butyl and methylguanidinium,
respectively (Figure S3b). The atoms of the protein and FeMo-co that
were truncated were kept frozen to their crystallographic position. In
model 2 the steric confinement exerted by the protein matrix on α-
70Val and α-96Arg during the CO2 insertion in the direct hydride
transfer pathway (see Results) was included, adding two ethane
molecules next to α-70Val and α-96Arg at the location of the backbone
C atoms of α-66Gly and α-98Asn. The C atoms of the ethane molecules
were kept fixed at their equilibrium distance. Model 1 was used for all
of the reactions considered in the present work, whereas model 2 was
employed only for selected reactions as discussed.
Carbon Dioxide Reduction Assay. CO2 reduction to CH4 was
carried out at 0.45 atm CO2 in 9.4 mL serum vials containing 2 mL of
an assay buffer consisting of 100 mM sodium dithionite, a MgATP
regenerating system (13.4 mM MgCl2, 10 mM ATP, 60 mM
phosphocreatine, 0.6 mg/mL bovine serum albumin, and 0.4 mg/
mL creatine phosphokinase) in 100 mM MOPS buffer at pH 7.2.
Addition of CO2 was followed by 10 min of incubation at room
temperature to allow equilibration. MoFe protein was added, and then
the assay vials were ventilated to atmospheric pressure. Reactions were
initiated by the addition of Fe protein and incubated at 30 °C for the
noted times. To each was added 500 μL of 400 mM EDTA at pH 8.0
to quench the reaction. From the gas phase, 300 μL was injected into a
gas chromatograph with a flame ionization detector to quantify CH4.
From the same samples, 50 μL of the gas phase was injected into a gas
chromatograph with a thermal conductivity detector to quantify H2.
CO2 reduction to CO was measured using a hemoglobin (0.3 mg/
mL) binding assay as described before.23 The buffer composition was
the same as used for CO2 reduction to CH4 except 0.3 mg/mL
hemoglobin was added in the assay buffer. A 1.2 mL amount of assay
buffer was transferred to a 9.4 mL serum vial containing 0.45 atm CO2,
and 20 min of equilibration time was allowed. Then, MoFe protein
was added (0.5 mg/mL). A 1 mL amount of CO2-saturated assay
buffer was transferred to a 2.2 mL (l cm path length) quartz cuvette
that had been modified to maintain a defined gas atmosphere. Fe
protein was added to initiate the reaction. The increase in the
absorbance at 418 nm corresponding to the binding of CO to the
hemoglobin was monitored using a UV−visible spectrophotometer.
CO2 reduction to formate was established using a 300 MHz JEOL
The electronic structure of the FeMo-co was described within the
DFT framework using the gradient-corrected Becke25 exchange and
Perdew26 correlation functionals. The Ahlrichs VTZ basis set was
employed for all Fe atoms,27 the Los Alamos National Laboratory
basis set LANL2TZ with an effective core potential was employed for
the Mo atom,28 and the 6-311++G** basis set was employed for all
atoms coordinated to metal atoms and protic and hydridic hydrogen
atoms, and finally the 6-31G* basis29 set was adopted for all of the
other atoms. Harmonic vibrational frequencies were calculated at the
optimized geometries using the same level of theory to estimate the
zero-point energy and the thermal contributions (298.1 K and 1 atm)
to the gas-phase free energy. The protein environment around FeMo-
co was described with a polarizable continuum with a dielectric
constant ε = 4.30 Standard state corrections were applied to solvation
free energies. Calculations for the reference state E0 were performed
for the observed S = 3/2 spin state. For the E2 state, the S = 3/2 spin
state was used, while for the E4 state, the S = 1/2 spin state was used,
consistent with the spin states observed experimentally.9 In agreement
with previous reports,31 we found that the ground state of all the
structures considered are characterized by the highest alternating
antiferromagnetic couplings possible between the Fe centers
compatible with the chosen spin multiplicity.
Different locations of the hydridic and protic hydrogen atoms on
the reactive face of the FeMo-cofactor (identified by the atoms Fe2,
Fe3, Fe6, and Fe7) were explored. The two models of the active site
yielded similar reaction thermodynamics (differences within 3 kcal/
mol) as detailed in Figure S4. In particular, models 1 and 2 yielded a
similar lowest free energy E2 state, with a “protic” hydrogen located on
a sulfur atom and a “hydridic” hydrogen located on one Fe center or
bridging two Fe atoms. In the lowest free energy structure, the
hydridic hydrogen is located between Fe2 and Fe6 and the protic
hydrogen is bound to S2B (Figure S3). In model 1, the hydridic
hydrogen is asymmetrically positioned between the two Fe atoms
(Fe2−H and F6−H distances of 1.60 and 1.75 Å), whereas in model 2
the hydridic hydrogen is in a nearly symmetric location (Fe2−H and
F6−H distances of 1.63 and 1.68 Å). Both models also predict that the
protic hydrogen is preferentially bound to a bridging μ-S atom (the
S2B atom in the lowest free energy isomer, Figure S5). These lowest
free energy E2 isomers were employed as a starting point for the
−
NMR spectrometer. To a 9.4 mL vial was added 0.026 mg of H13CO3
under Ar. To this vial was added 2 mL of anaerobic assay buffer,
followed by addition of 1 mg of MoFe protein. The reaction was
initiated by addition of 1 mg of Fe protein. The reaction was allowed
to run for 1 h, after which 500 μL of the liquid phase was transferred
to a sealed NMR tube. A D2O-containing capillary tube was placed
inside the NMR tube. 13C NMR was carried out to ascertain the
−
formation of formate during the reduction of H13CO3 (Figure S1).
The NMR probe temperature was set to 25 °C, and the magnetic field
1
was locked using D2O as solvent. The probe was tuned for both H
and 13C nuclei. Single pulse decoupled NMR was carried out using the
following parameters: X-offset 90 ppm, X-sweep 180 ppm, scan 6000,
pulse X-angle 30, relaxation delay 3 s, and both NOE and decoupling
1
on. The JC−H for formate was also determined using the same
C
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Table 1. Product Accumulation and Electron Distribution for Wild-Type MoFe Protein
product
H2
HCOO−
nmol/nmol/min
CH4
CO
a
gas
Ar
CO2
nmol/nmol/min
% e−
% e−
nmol/nmol/min
% e−
nmol/nmol/min
% e−
b
83 1.55
70 1.8
100
85
ND
9.8 1.5
ND
12
ND
ND
ND
ND
ND
ND
1
0.03
a
nmol product/nmol MoFe protein/min. Rates are the average over the 60 min assay at 30 °C with 1 mg of MoFe protein and 1 mg of Fe protein.
b
CO2 was at a partial pressure of 0.45 atm. ND, not detected.
calculations. All of the calculations were performed with the NWChem
quantum chemistry package.32
addition in which the FeMo-co is activated for the binding/
reduction of N2 through the re loss of H2. As a result of this
reversible equilibrium, H2 inhibits N2 reduction to NH3 by
displacement of the bound N2 (Figure 2, right).13 This
inhibition is recapitulated here by activity measurements that
show inclusion of 0.8 atm of H2 in place of 0.8 atm of Ar along
with 0.2 atm of N2 results in ∼50% inhibition of the NH3
formation rate for wild-type nitrogenase (Figure 3). If CO2
reduction also followed a reversible re mechanism, this process
likewise should be inhibited by the addition of H2. However, as
can been seen (Figure 3, panel B), the inclusion of 1 atm of H2
caused no detectable inhibition of the reduction of CO2 to
formate, relative to the Ar control. Inclusion of 0.85 atm of H2
also had no effect on the rate of reduction of CO2 to CO
compared to the Ar control (Figure 3, panel C). These results
indicate that CO2 reduction catalyzed by nitrogenase does not
involve the reversible re step that is operative in N2 reduction.
Alternative Mechanisms of CO2 Reduction by Hydride
Insertion. With the re mechanism having been eliminated, the
most plausible mechanisms for CO2 reduction catalyzed by
nitrogenase involves migratory insertion of CO2 into the Fe−H
bond of reduced FeMo-co, in analogy to the proposed
RESULTS
■
CO2 Reduction to Formate. Earlier studies revealed that
the wild-type MoFe protein reduces CO2 to CO at a very low
rate (∼0.03 nmol CO/nmol MoFe protein/min at 30 °C),20
a
result repeated here (Table 1). In agreement with those earlier
results, we find that under these conditions most of the electron
flux through nitrogenase goes to the reduction of protons to
make H2, which occurs with a roughly 3 × 103-fold greater rate
(83 nmol H2/nmol MoFe protein/min at 30 °C) than CO
formation, and we detect no CH4. In a subsequent study, it was
found that substitution of two amino acids within the MoFe
protein located near the active site FeMo-co, α-70Val→Ala
/
α-195His→Gln, resulted in a MoFe protein that could reduce CO2
by 8 e−/H+ to methane at a rate of ∼1 nmol CH4/nmol MoFe
protein/min, with most of the electron flux going to proton
reduction (∼62 nmol H2/nmol MoFe protein/min).21 The
reduction of CO2 to CO and CH4 is expected to follow the
same reaction pathway, with CO formed after 2 e−/H+
reduction and CH4 after addition of a further 6 e−/H+.
An examination of CO2 reduction catalyzed by a range of
small molecules that utilize metal hydrides reveals that an
alternative product for CO2 reduction is formate.33−37 The
pathway to CO and CH4 formation is believed to involve the
addition of H to the O atom of CO2, whereas the pathway to
HCOO− formation is believed to involve the addition of H to
the C atom of CO2.38,39 In this context, we tested whether wild-
type nitrogenase could reduce CO2 to HCOO−, a reaction that
had not been reported before.
mechanisms of C2H2 and NO2 reduction.17,19 Figure 4
−
presents two alternative hydride insertion reaction pathways
for the reduction of CO2 to formate based on studies of
inorganic complexes: the “direct hydride transfer” reaction of
CO2 with the FeMo-co Fe-H and an “associative” pathway with
hydride insertion by Fe-bound CO2.40 As incorporated into
Figure 4, the formation of CO and CH4 can be viewed as a
shunt off of the associative pathway. The ∼10:1 product ratio of
formate:CO/CH4 observed for CO2 reduction by wild-type
nitrogenase indicates that the pathways to formate formation
are energetically favorable compared to the pathway to CO/
CH4 formation.
Testing Possible Pathways by Quantum Chemical
Calculations. We used DFT calculations to evaluate the
possible pathways for CO2 reduction catalyzed at FeMo-co,
presented in Figure 4. The starting point for the calculations is
the E2(2H) state, which has accumulated 2 e−/H+ as one
sulfido-bound proton (Figures S3 and S5).
Geometry optimization of model 1 in the presence of CO2 in
the vicinity of the Fe-hydride center of the E2(2H) state (at a
C−H distance longer than 3.5 Å) leads to spontaneous “direct
hydride” transfer to carbon, with the formation of formate
(Figure 5, blue pathway). In turn, according to the adopted
computation model, the newly formed formate deprotonates
the S-H of FeMo-co with the formation of formic acid. Isomers
of FeMo-co differing in the location of the proton and the
hydride provided similar scenarios. Calculations carried out on
model 2, which includes residues α-70Val and α-96Arg above the
reactive face of FeMo-co, revealed that the local environment
around the cofactor limits CO2 access to the catalytic center
Wild-type nitrogenase that was allowed to turn over for 60
min in the presence of 13CO2 as substrate catalyzed the
formation of formate, as established by 13C NMR, which
showed a peak at 171.083 ppm associated with H13CO3
−
(Figure S1). Formate was quantified by a colorimetric assay
(Figure S2). As can be seen from the data in Table 1, the wild-
type MoFe protein reduces CO2 to formate at a substantial rate
(∼10 nmol HCOO−/nmol MoFe protein/min at 30 °C).
Under these conditions, ∼12% of the electron flux through
nitrogenase goes to produce formate, whereas about 1% goes to
form CO. The remaining electron flux (87%) makes H2. Thus,
the present measurements reveal that the conversion of CO2 to
formate represents a significant catalytic process for nitro-
genase.
Does CO2 Reduction Involve Reductive Elimination?
Although the reduction of CO2 to formate involves only 2 e−/
H+ and likely involves binding at the E2 state, the 2/8 e−/H+
reduction to CO/CH4 could occur by a process that parallels
the reduction of N2, namely, a reductive elimination mechanism
(Figure 2, right).10 A key feature of the re mechanism for N2
reduction is the reversible reductive elimination/oxidative
D
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(NBO) framework,41 revealed a strong charge transfer from the
σ(Fe−H) bonding orbital to the π*(CO) antibonding orbital
(see Supporting Information), which results in the heterolytic
Fe−H → Fe+ + H− bond cleavage and the transfer of the
hydride to CO2. The exergonic nature of the direct hydride
−
transfer suggests that the hydricity of the E2 state, ΔGH , is
sufficient to transfer a hydride to CO2 to generate formate; that
is, the hydricity is below that of formate (HCOO− → CO2 +
H−, ΔGH = +24.1 kcal/mol).42
−
In contrast, the “associative” pathway for CO2 reduction,
whereby CO2 first coordinates sideways to Fe2 via the C atom
(Figure S7), was found to be thermodynamically and kinetically
unfavorable. Indeed, the formation of the E2(2H)-CO2 adduct
is endergonic by ΔG0 = +10.8 kcal/mol (ΔG0 = +8.7 kcal/mol,
model 2), and the calculated barrier for the subsequent
hydrogen transfer to CO2 is ΔG⧧ = +11.1 kcal/mol (Figure 5,
blue pathway). The computed overall barrier for CO2 reduction
to HCOOH via the associative pathway is +21.9 kcal/mol.
Upon CO2 binding to Fe2, the Fe2−S bond in the Fe2−S−Fe6
bridge breaks (Figure S7A) and it is reformed when the
product is released. The NBO analysis of the associative
pathway indicates that the hydrogen transfer is driven by a
strong charge transfer from σ(Fe−C) of the Fe-bound CO2
moiety to σ*(Fe−H) and from FeMo-co localized orbitals to
CO2, which results in the heterolytic Fe−H → Fe− + H+ bond
dissociation with the net transfer of a proton to the C atom and
the breaking of the Fe−C bond. During the proton transfer the
two σ(Fe−C) electrons are used to make the C−H bond. The
different behaviors of the Fe-H in the direct hydride transfer
pathway and the associative pathway are truly remarkable: in
the former the Fe-H hydrogen behaves as a hydridic hydrogen
and in the latter as a protic hydrogen.
A pathway leading to the formation of CO from E2(2H)-CO2
was also found, which starts with the transfer of the protic S-H
hydrogen to one of the oxygen atoms of CO2, which is an
exergonic reaction (ΔG0 = −7.6 kcal/mol) with an activation
barrier of ΔG⧧ = +7.7 kcal/mol (Figure 5, green pathway). The
presence of ionizable residues near the reactive FeMo-co face,
most notably α-96Arg, raises the question of whether one of
these residues can act as a proton relay to the CO2 rather than
the protonated FeMo-co S atom. The size of the present
computational models cannot fully address this question.
However, calculations carried out with model 2 indicate that
protonation from α-96Arg is extremely unlikely, given that this
reaction is endergonic by 13.4 kcal/mol. The formation of CO
proceeds with the transfer of the Fe-H hydrogen (as a proton)
to that oxygen, which results in the spontaneous and very
exoergic dissociation of a water molecule (ΔG0 = −27.8 kcal/
mol), followed by the slightly exergonic dissociation of CO
(ΔG0 = −1.4 kcal/mol). Thus, in this case, the Fe-H again
behaves as a proton donor.
Figure 3. Effect of H2 on N2 and CO2 reduction. Specific activities
(nmol product/nmol MoFe protein/min) are shown for N2 reduction
to NH3 (A), CO2 reduction to formate (B), and CO2 reduction to CO
(C) either with or without the addition of H2. All assays were done
with wild-type MoFe protein at 30 °C as described in the Materials
and Methods. Reaction conditions were: 0.2 atm of N2 + 0.8 atm of Ar
or 0.2 atm of N2 + 0.8 atm of H2 for panel A; 40 mM HCO3− + 1 atm
of Ar or 40 mM HCO3− + 1 atm of H2 for panel B; 0.15 atm of CO2 +
0.85 atm of Ar or 0.15 atm of CO2 + 0.85 atm of H2 for panel C.
For completeness, we also investigated computationally the
release of H2 from the E2(2H) state. We found that formation
of H2 is a facile (ΔG⧧ = +6.3 kcal/mol) and very exergonic
process (ΔG0 = −31.3 kcal/mol; Figure 5, red pathway). This
result is consistent with our recent explanation of why H2
cannot reduce FeMo-co: it is too far “uphill”.9 As with the
direct hydride pathway, the Fe-H behaves as a hydride that is
protonated by S-H, with the overall H2 elimination driven by a
σ(Fe−H) to σ*(S−H) charge transfer.
and introduces a barrier for the direct hydride transfer (ΔG⧧ =
+9.6 kcal/mol, see Figure 5), forcing CO2 to approach the
hydridic hydrogen sideways (Figure S6) with respect to the
FeMo-co face rather than perpendicularly as in model 1. It is
important to point out that while model 1 and model 2 largely
differ in the description of the kinetics of the direct hydride
transfer, they provide comparable reaction free energies (ΔG0 =
−25.8 and −23.0 kcal/mol, respectively).
Experimentally Altering Product Distribution during
CO2 Reduction through MoFe Protein Modification. We
sought to alter the selectivity of CO2 reduction to different
An analysis of the electronic properties of FeMo-co during
the hydride transfer, carried out within the natural bond orbital
E
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Figure 4. Possible pathways for CO2 reduction. CO2 activation at one FeS face of the E2 state of FeMo-co is shown. The E2 state is proposed to
contain a single Fe-hydride and a proton bound to a sulfide shown bound to one face of FeMo-co. Reduction to formate (blue box) can go by either
a direct hydride transfer or an associative pathway. A pathway to formation of CO and CH4 is shown in the green box. Six additional electrons and
protons are added to the bottom structure to achieve reduction to CH4.
products by changing the environment around the active site
FeMo-co by amino acid substitutions (Figure S8). Earlier
studies identified the α-70Val and α-195Gln as key residues that,
when substituted, alter the reactivity of nitrogenase toward
substrate reduction.21,43,44 We therefore assessed how changing
these amino acids would alter the ratio of the products when
reacting with CO2 (Figure 6 and Table S1).
For the wild-type MoFe protein under these conditions,
about 10% of the electrons are utilized for CO2 reduction,
formate being the primary product, with a trace of CO. The
remaining 80% of electron flux is used to make H2. In the α-
70Val→Ala-substituted MoFe protein, the electron flux is shifted
in favor of CO2 reduction to make formate (∼30%), with the
remainder going to make H2. The α-195His→Gln-substituted
MoFe protein shows essentially 100% electron flux going to
make H2. Substitution of both α-70Ala/α-195Gln, however,
results in a protein that shows considerable reduction of CO2 to
formate (∼20%), but with detectable formation of CH4 (∼2%),
the remaining flux going to make H2. These findings reveal that
subtle changes around the active site can indeed alter the
reaction pathway energy landscape, thereby altering the
distribution of electrons passing through nitrogenase to CO2
reduction.
DISCUSSION
■
The discovery reported here that nitrogenase can reduce CO2
to formate, coupled with the earlier reports of reduction to
CO20 and to CH4,21 makes nitrogenase unique in its ability to
catalyze the reduction of CO2 to a range of products. For
example, CO dehydrogenase and formate dehydrogenase
efficiently catalyze 2 e−/H+ reduction of CO2 to CO and
HCOO−, respectively, but do not catalyze reduction of CO2 to
CH4.45,46 Indeed, few known organometallic complexes can
reduce CO2 to this suite of products,47,48 making the study of
the mechanism of CO2 reduction by nitrogenase important to
F
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Figure 5. Computed free energy diagram for CO2 reduction and H2 formation occurring at the E2 state of FeMo-cofactor. Calculations start with the
E2 state that contains one bridging hydride and one H+ associated with a sulfide. This starting state is assigned a relative free energy of 0 kcal/mol,
and all other free energy changes are relative to this state. Going to the left (red) is the pathway for heterolytic formation of H2. To the right (blue) is
the direct hydride transfer or associative pathway going to a CO2 bound to FeMo-co. Further to the right (green) is the pathway to CO formation.
Reported data were calculated with model 1 except for the activation barrier for the direct hydride transfer, which was calculated with model 2. For a
more detailed comparison between model 1 and model 2, see Figure S3.
N2 to this activated state is associated with the loss of H2 by a
reductive elimination mechanism, with two hydrides combining
to make H2 coupled to the reduction of N2 to a diazene-level
state.9−13 It has been shown that the binding of N2 to the
E4(4H) state with loss of H2 involves the thermodynamically
and kinetically reversible re equilibrium (1):
E4(4H) + N2 ⇆ E4(2N2H) + H2
(1)
which activates FeMo-co for the addition of the first two
electrons and protons to N2, the most difficult step in N2
reduction. Subsequent steps of reduction are likely to involve
migratory insertion of this bound species into M-H on FeMo-
co, ultimately leading to the formation of two NH3. The ability
of FeMo-co to simultaneously hold the partially reduced N2
species juxtaposed to one or more Fe-hydrides offers an
attractive way to achieve the stepwise reduction of N2 all the
way to NH3 without release of intermediates that might be
Figure 6. Product distribution under CO2 for different MoFe proteins.
Shown are the electron distribution to different products with CO2 for
toxic to the cell.
An important consequence of an obligatory loss of H2 during
the wild-type, α-70Ala, α-195Gln, and α-70Ala/α-195Gln MoFe proteins.
reduction of any substrate through a re equilibrium is that
Assay conditions: 1 mg of MoFe, 6 mg of Fe, 0.45 atm of CO2 with an
added H2 will inhibit reduction of the substrate through such a
incubation time of 60 min at 30 °C.
re mechanism by “driving” the equilibrium to the left.13 The
findings presented here, that CO2 reduction to formate and to
gain mechanistic insights into how to reduce CO2. Studies
during the past few years have revealed the presence of Fe-
bound hydrides in reduced states of nitrogenase, as well as their
importance in the nitrogenase N2 reduction mechanism.9
Activation of nitrogenase by the accumulation of 4 e−/H+ on
FeMo-co (E4(4H) state) results in the storage of these reducing
equivalents as two bridging hydrides (Fe−H−Fe) in the central
E4(4H) state. There is ample evidence now that the binding of
CO and CH4 are not inhibited by H2, eliminates the possibility
that CO2 binding and reduction is coupled to a reversible re/
oxidative addition (oa) mechanism in which N2 binding/
reduction is coupled to the re loss of H2. This finding directed
our attention to alternative mechanisms involving proton-
coupled electron transfer or migratory hydride insertion, as is
proposed for CO dehydrogenase and formate dehydrogen-
ase.45,46
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Scheme 1. Thermodynamic Cycle for Determining the Hydride Donor Ability of E2(2H)
Studies on inorganic metal hydride complexes indicate there
are two pathways for the participation of metal hydrides in
reduction of CO2 to formate.40 One is an associative pathway,
in which CO2 binds through its C atom to a Fe of FeMo-co
that has at least one bound hydride (Figure 4); the presence of
a bound hydride increases the π back-donation ability of the
metal and the nucleophilicity of the metal. The bound C then
undergoes mi into the Fe−H bond and accepts a proton bound
to sulfur to release HCOOH. Alternatively (Figure 4), in a
direct hydride transfer mechanism, CO2 does not bind to a
metal ion, but rather the C atom of CO2 associates with the
metal-bound hydride and the hydride is directly transferred to
the C of CO2, to generate formate with transfer of the proton
bound to sulfur, again giving HCOOH.
Both of these mechanisms seem reasonable, and it would be
difficult to distinguish between them from experimental
evidence. Instead, this is an ideal situation where calculations
can distinguish between possible pathways. Exploratory DFT
calculations have been carried out by starting the reaction
pathway from an E2(2H) state, the state with which CO2 is
proposed to initially interact. These calculations employed
simplified models of the nitrogenase active site, which includes
FeMo-co with truncated Cys, His, and homocitrate ligands
(model 1), similar to previous investigations,49,50 and addition-
ally truncated α-70Val and α-96Arg residues (model 2) to
examine the energetics of the possible direct hydride and
associative reaction pathways and, at the same time, to analyze
the chemical bonding and the reorganization of the electron
density distribution upon bond breaking and forming.
units. The extremely high acidity of E0(H) suggests that, in the
formation of E1(H), E0 is first reduced then protonated
probably through a proton-coupled electron transfer reaction.
These calculations demonstrate that hydride-bound FeMo-co
can act as a strong H− donor. On the other hand, the same
hydride ligand can act as a proton donor in the less active
associative pathway. Our results highlight several of the
numerous fallacies in a recent misanalysis of nitrogenase
catalysis.49 A hydrogen atom bonded to a metal is defined
formally as a hydride, and the present results show that it is
incorrect to attempt to define an M-H moiety as a hydride or
proton based on atomic point charges derived from electron
density partitioning schemes, rather than reactivity (see
Supporting Information). The calculations reported in the
present paper, together with other recent reports, show little
differences between atomic charges on H atoms bound to Fe or
S, with variation smaller than differences between the different
varieties of the partitioning scheme employed (Tables S2 and
S3).49 Correspondingly, the NBO analysis reported here
indicates that the Fe−H and S−H bonds are covalent with a
small polarization as expected from the difference in electro-
negativity of the atoms involved (see Supporting Information).
Nonetheless, the Fe−H and S−H bonds show dramatically
different reactivity patterns. We also would like to point out
that, as a consequence of the strong covalent character of the
Fe−H and S−H bonds, during the accumulation of e−/H+ to
form E2(2H) and, in turn, E4(4H), electrons are stored neither
on the FeMo-co metal core, in particular on the Fe atoms, nor
in hydridic H− atoms. Rather the added electrons are nearly
equally shared between the atoms involved in the new chemical
bonds, as substantiated by the electron configurations reported
in Table S4.
In contrast to the formation of formate, the reduction of CO2
to CO, and on to CH4, is likely to follow an associative reaction
pathway, involving the binding of CO2 to FeMo-co through the
C (Figures 4 and 5). The common early step in this pathway
for both formate and CO formation is the endergonic
coordination of the CO2 to the Fe of FeMo-co. On the
pathway to CO formation, the H+ associated with the S has
been modeled to interact with one O of the bound CO2, with
the hydride bound to the Fe acting as a proton donor to the O,
resulting in the reduction of the O to water and a bound CO.
The overall barrier to the formation of the intermediate leading
to CO is found to be +18.5 kcal/mol relative to the E2(2H)
state. Thus, the calculations are consistent with the
experimental observations that the wild-type nitrogenase
exhibits a larger formate/CO product ratio during CO2
reduction. The calculations indicate that the direct hydride
transfer pathway will be substantially favorable compared to the
associative pathway that leads to CO and CH4 production. For
comparison, calculations were also done for the conversion of
the E2(2H) state to the E0 + H2 state, revealing an exergonic
process of −31.3 kcal/mol compared to the E2(2H) baseline
state, with an activation barrier of 6.3 kcal/mol.
The calculations reveal a significant barrier between CO2 and
formate along the associative pathway. For this pathway, the
coordination of CO2 to E2(2H) is endergonic by +10.8 kcal/
mol, with the overall activation free energy for forming formate
being endergonic by +21.8 kcal/mol relative to the E2(2H)
state. In contrast, the direct hydride pathway is found to have a
barrier of only 9.6 kcal/mol. Comparison between model 1 and
model 2 indicates that this barrier is basically due to protein
residues above the reactive face of the FeMo-co, which limits
the CO2 access to the hydridic hydrogen. These environmental
effects are demonstrated by the changes in product ratios
introduced by amino acid substitutions in the vicinity of the
active sites on FeMo-co. The lack of a barrier when CO2 has
free access to the hydridic hydrogen is notable, suggesting that
the hydride donor ability of the Fe−H on the E2(2H) state is
considerably more favorable (smaller hydridicity) than that of
formate. Using the experimental hydride donor ability of
formate in aqueous solution, ΔG0(HCOO−) = 24.1 kcal/mol,42
as a reference value, we can obtain a conservative estimate of
the hydride donor ability (hydricity) of E2(2H) from the half-
reactions51 reported in the thermodynamic cycle of Scheme 1
as ΔG0(E2(2H)) = ΔG0 + ΔG0(HCOO−). Using the calculated
(model 1) value ΔG0 = −8.4 kcal/mol for the free energy
change of the hydride transfer reaction from E2(2H) to CO2
yields ΔG0(E2(2H)) = +15.7 kcal/mol.
Following similar arguments (Scheme S1), we can obtain an
estimate of the pKa for the protonated E0(H) state of −9.1 pKa
In summary, wild-type nitrogenase is shown here to be
effective in CO2 reduction to formate. The most favorable
H
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b00388.
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Discussion of the natural bond order analysis, computed
structural and energetic data of other possible E2 isomers,
figures showing the NMR traces for determining formate,
colorimetric formate assay, the structure of the FeMo-co
model used for DFT calculations, tables of products for
different variant MoFe proteins, and the coordinates of
all of the species considered in the computational work
(PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
*Phone (B. M. Hoffman): 847-491-3104. E-mail: bmh@
northwestern.edu.
*Phone (S. Raugei): 509-372-6902. E-mail: simone.raugei@
pnnl.gov.
*Phone (L. Seefeldt): 435-797-3964. E-mail: lance.seefeldt@
usu.edu.
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Notes
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2571−2577.
The authors declare no competing financial interest.
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2008, 4, 1029−1031.
ACKNOWLEDGMENTS
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This work is based upon work supported by the U.S.
Department of Energy, Office of Science, Basic Energy Sciences
(DE-SC0010687 and DE-SC0010834; L.C.S. and D.R.D.), the
Division of Chemical Sciences, Geosciences, and Bio-Sciences
(S.R.), and the NSF (MCB 1515981; B.M.H.).
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