Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

Article abstract of DOI:10.1073/pnas.1213159109

A doubly substituted form of the nitrogenase MoFe protein (α-70 ^Val→Ala, α-195^His→Gln) has the capacity to catalyze the reduction of carbon dioxide (CO₂) to yield methane (CH₄). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH₄ within 20 min. The catalytic rate depends on the partial pressure of CO₂ (or concentration of HCO₃^-) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H₂C = CH-CH ₃) through the reductive coupling of CO₂ and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO₂ sequestration and formation of olefins.

Full text of DOI:10.1073/pnas.1213159109

Carbon dioxide reduction to methane and coupling
with acetylene to form propylene catalyzed by
remodeled nitrogenase
a
a,b
c
a,1
Zhi-Yong Yang , Vivian R. Moure , Dennis R. Dean , and Lance C. Seefeldt
a
b
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322; Instituto Nacional de Ciência e Tecnologia da Fixação Biológica de
c
Nitrogênio, Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba Parana 81531-980, Brazil; and Department of
Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved October 19, 2012 (received for review July 30, 2012)
A doubly substituted form of the nitrogenase MoFe protein
amino acid residues that provide the first shell of noncovalent
interactions with the active site FeMo cofactor such that CO
becomes a much more robust substrate with catalytic formation
of methane and short chain alkenes and alkanes (26). Related to
these observations, we have also reported that the native nitro-
Val→Ala
His→Gln
(
α-70
tion of carbon dioxide (CO
mized conditions, 1 nmol of the substituted MoFe protein catalyzes
the formation of 21 nmol of CH within 20 min. The catalytic rate
depends on the partial pressure of CO
, α-195
) has the capacity to catalyze the reduc-
2
) to yield methane (CH ). Under opti-
4
4
−
2
(or concentration of HCO
3
)
genase has the capacity to reduce CO at relatively low rates to
2
and the electron flux through nitrogenase. The doubly substituted
MoFe protein also has the capacity to catalyze the unprecedented
yield CO (27). Here, we report that a remodeled nitrogenase
MoFe protein can achieve the eight-electron reduction of CO to
2
2 3
formation of propylene (H C = CH-CH ) through the reductive cou-
CH . Further, it is shown that CO reduction can be coupled to
4
2
pling of CO and acetylene (HC≡CH). In light of these observations,
2
2 2
the reduction of other substrates (e.g., acetylene, C H ) to form
we suggest that an emerging understanding of the mechanistic
features of nitrogenase could be relevant to the design of synthetic
longer chain, high value hydrocarbons (e.g., propylene).
catalysts for CO
2
sequestration and formation of olefins.
Results
CO
2 4
Reduction to CH by Remodeled Nitrogenase. When the wild-
metalloenyzme
| multi-electron reduction | hydrocarbon
type nitrogenase was tested for reduction of CO to yield CH ,
no CH above background could be detected over the course of
4
2
4
arbon dioxide (CO
2
) is an abundant and stable form of
20 min (Fig. 2). Earlier work has demonstrated that several
amino acids having side chains that approach FeMo cofactor
play an important role in controlling substrate binding and re-
C
carbon that is the product of respiration and burning of fossil
fuels. As a result of these activities, the atmospheric concentra-
Val
His
tion of CO
century and contributing to global warming (1). There is strong
interest in developing methods for sequestering CO either by
capturing it or by chemically converting it to valuable chemicals
2–5). Of particular interest are possible routes to reduction of
CO by multiple electrons to yield methanol (CH OH) and
methane (CH ), which are renewable fuels (2). The reduction of
2
, a greenhouse gas, has been rising over the last
duction (20, 28). Among these residues are α-70 , α-195 , and
Gln
α-191 (Fig. 1). Variant forms of the MoFe protein having α-70
substituted by Ala, α-195 substituted by Gln, or α-70 and α-191
2
both substituted by Ala showed no appreciable capacity for re-
(
duction of CO to yield CH . In contrast, a doubly substituted
2 4
Ala Gln
2
3
MoFe protein, α-70 /α-195 , was found to catalyze the for-
4
mation of CH from CO , forming up to 16 nmol CH /nmol
4
2
4
CO
compounds able to catalyze these reactions (6–13). In biology,
only a few enzymes are known to reduce CO (14–18), and none
of these can catalyze the eight electron reduction to CH
2
is difficult, with a limited number of reports of metal-based
MoFe protein over 20 min (Fig. 2). The formation of CH4
depended on the presence of CO , Fe protein, MoFe protein,
2
2
and MgATP . The rate of CH production was found to increase
4
4
.
with increasing partial pressure of CO up to 0.45 atm (Fig. S1).
2
The bacterial Mo-dependent nitrogenase enzyme catalyzes the A fit of these data to the Michaelis–Menten equation gave a Km
multielectron/proton reduction of dinitrogen (N
2
) to two ammonia for CO of 0.23 atm and a V
max 4
of 21 nmol CH /nmol MoFe
2
(
1
NH
3
) at a metal cluster designated FeMo-cofactor [7Fe-9S-1Mo- protein over 20 min. In a Bis-Tris buffer at pH 6.7, sodium bi-
C-R-homocitrate] (Fig. 1) in a reaction that requires ATP hy- carbonate (NaHCO ) could also serve as a substrate for CH
3
4
drolysis and evolution of H , with a minimal reaction stoichiometry formation, with a determined Km of 16 mM for NaHCO and
2
3
shown in Eq. 1 (19–22).
Vmax of 14 nmol CH /nmol MoFe protein over 20 min (Fig. S2).
4
The rate of electron flow through nitrogenase (called electron
+
À
N2 + 8H + 16ATP + 8e → 2NH3 + H2 + 16ADP + 16Pi: [1]
flux) can be regulated by altering the ratio of Fe protein to MoFe
protein (Fe protein:MoFe protein), with a low ratio corre-
sponding to low electron flux and a high ratio corresponding to
high electron flux. Under all conditions, the majority of electrons
Given that nitrogenase is effective at catalyzing the difficult
2
multielectron reduction of N , it was of interest to determine
whether this enzyme might also catalyze the reduction of CO to
passing through nitrogenase in the presence of saturating CO
were found to reduce protons to make H with relatively low rates
of associated CH formation (Fig. 3). However, as the electron
2
2
the level of CH . Nitrogenase is known to have the capacity to
2
4
reduce a variety of other small, relatively inert, doubly or triply
bonded compounds, such as acetylene (HC≡CH) (19, 23). It has
been shown that an alternative form of nitrogenase, which con-
tains V in place of Mo in the active site cofactor, has the re-
markable capacity to reduce CO and couple multiple CO
molecules, yielding short chain alkenes and alkanes such as
4
Author contributions: D.R.D. and L.C.S. designed research; Z.-Y.Y. and V.R.M. performed
research; and L.C.S. wrote the paper.
The authors declare no conflict of interest.
ethylene (C
2
H
4
), ethane (C
2
H
6
), propylene (C
3
H
6
), and propane
This article is a PNAS Direct Submission.
1
(C
3
H
8
) (24, 25). In contrast, the Mo-nitrogenase is only able to
To whom correspondence should be addressed. E-mail: lance.seefeldt@usu.edu.
reduce CO at exceedingly low rates (24). However, we have
found that the MoFe protein can be remodeled by substitution of
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1213159109/-/DCSupplemental.
1
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1
3
−
1
7 was observed (Fig. S3). When H CO
3
was the substrate,
a peak having the same retention time was found to have a m/z
1
3
of 17, which can be ascribed to the molecular mass of CH4.
−
This result demonstrates that HCO
for CH formation rather than some other component in the
reaction mixture.
When the CO
nitrogenase was performed in the presence of 0.30 mg/mL deox-
yhemoglobin, the amount of CH formed was lowered by ∼25%
Fig. S4). Deoxyhemoglobin binds CO very rapidly (rate constant
3
2
or CO is the substrate
4
2
reduction reaction catalyzed by the remodeled
4
(
5
−1 −1
k ∼ 2 × 10 M ·s ) and with a high affinity (dissociation constant
d
K ∼ 50 nM) and would, therefore, bind any CO released into
solution during CO reduction by nitrogenase (27). Although CO is
2
expected to be an intermediate along the reaction pathway from
CO
2 4
to CH , a relatively small lowering of the rate of formation of
CH from CO when deoxyhemoglobin is included in reaction
4
2
2
mixture indicates that CO reduction follows primarily a non-
dissociative mechanism.
Fig. 1. The FeMo cofactor with some key amino acid residues. Colors are
Mo in magenta, Fe in rust, S in yellow, C in gray, O in red, and N in blue. The
central atom is C (gray), and the structure and numbering of Fe atoms are
based on the protein database file PDB: ID code 1M1N.
Several earlier studies revealed multiple inhibitor and sub-
strate binding sites on FeMo-cofactor, including at least two
binding sites for CO (29–33) and acetylene (31, 34, 35). Two
adjacent binding sites can explain the earlier reports that two or
three CO molecules can be reduced and coupled to form C2 and
C3 hydrocarbon products (24, 26). It was therefore of interest to
test whether the doubly substituted MoFe protein could couple
flux increased, the proportion of electrons passing to CO re-
2
duction increased, reaching a maximum at a molar ratio of ap-
proximately 50 Fe protein per MoFe protein. At this highest flux,
two or more CO molecules to yield short chain hydrocarbons.
2
the molar ratio of H
2
formed per CH
4
formed was ∼250:1. Given
re-
Under the assay conditions examined, no C2 or C3 hydrocarbon
that proton reduction is a two-electron reduction and CO
2
products were detected above the background when CO
sole C substrate. However, when a small amount of acetylene
was added when CO was used as substrate, the C3 hydrocarbon
propylene (H C = CH-CH ) was detected as the major product
and propane (H C-CH -CH ) as a minor product. Propylene
2
was the
duction to CH is an eight-electron reduction, up to 2% of the
4
total electron flux passing through nitrogenase goes to CO re-
2
2
duction to methane under these conditions.
The use of 1 C- or C-enriched bicarbonate (HCO3−) as
2
13
2
3
3
2
3
substrate and product analysis by gas chromatography-mass
formation only occurred in a reaction with all components for
a complete nitrogenase assay, revealing that propylene is formed
by nitrogenase turnover. Interestingly, it was found that pro-
pylene formation was favored under relatively low electron flux
conditions (4 Fe protein:1 MoFe protein), with higher electron
spectrometry (GC-MS) confirmed that CH
4
formation was de-
1
2
−
rived from added CO . When H CO was the added substrate,
2 3
a peak having the same retention time as methane showed a
mass over charge (m/z) peak of 16, whereas no peak with m/z of
flux favoring CH
4
formation at the expense of propylene for-
mation under 0.45 atm of CO and 0.014 atm of acetylene (Fig.
2
4
). Under the optimal electron flux condition, the amount of
propylene formed increased with increasing acetylene partial
pressure up to 0.027 atm and then decreased rapidly at higher
acetylene concentrations (Fig. 5) likely due to inhibition of CO2
reduction by acetylene. The results indicate there is an optimal
concentration ratio between CO
2
and acetylene to achieve re-
ductive coupling of the two molecules at a given electron flux. All
possible combinations of electron flux and acetylene and CO
2
concentration have not been examined.
Formation of propylene from the reductive coupling of one
CO
H CO
2
and one C H
2
2
was confirmed by GC-MS analysis. When
were used as substrates, the propylene
elution peak displayed a molecular ion peak with m/z of 42,
1
2
−
12
3
and
2 2
C H
1
2
which is ascribed to C H . Trace amounts of a fragment with
a m/z of 43 was observed because of natural abundance of
and H. When H CO
lecular ion peak with m/z of 43 was detected at the same re-
3
6
1
3
C
2
13
−
12
3
was used together with
2 2
C H , a mo-
tention time as propylene, consistent with the coupling of one
1
3
12
13
12
12
CO
Discussion
The discovery reported here that remodeled nitrogenase is able
2
with one
2 2 3 2
C H to form CH - CH= CH (Fig. S5).
Fig. 2. CH
4 2
formation as a function of time for different MoFe proteins. CO
Ala
reduction to CH
4
is shown as a function of time for the wild-type (○), α-70
Ala Ala Ala Gln
to reduce CO by eight electrons to CH (Eq. 2) makes it
unique among known enzyme catalyzed reactions. Further, the
ability to reduce CO and couple it to acetylene to form pro-
pylene (Eq. 3) makes nitrogenase unique among all reported
catalysts (5).
2
4
Gln
(◇), α-195
(△), α-70 /α-191
(□), and α-70 /α-195
(■) MoFe pro-
teins. The partial pressure of CO
protein was 0.5 mg/mL, and Fe protein was 3 mg/mL The reaction temper-
ature was 30 °C. The complete assay for α-70 /α-195
done in triplicate, with SE bars shown.
2
was 0.45 atm, the concentration of MoFe
2
Ala
Gln
MoFe protein was
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reduce CO
2
to yield different reduction products including for-
−
mate (HCOO ), carbon monoxide (CO), formaldehyde (CH O),
2
methanol (CH
3 4
OH), and methane (CH ) (2, 6–13). Common
features of homogeneous catalysts for CO reduction to CH are
2
4
low reaction rates (e.g., turnover frequencies) and limited
number of turnovers (e.g., turnover number) before inactivation
of the catalyst (37, 38). Further, for electrochemical reductions,
a high overpotential is required, with production of H as a waste
2
of electron flux (39, 40). The nitrogenase catalyzed reduction of
CO to CH reported here is comparable in turnover frequency
2
4
−1
(
approximately 1 min ) and turnover number, with notable
slowing of the reaction beyond 20 min. Nitrogenase also diverts
2
most of its electron flux to H formation, with only a small per-
centage going to CO reduction. In contrast to the electrochemical
2
catalysts, nitrogenase catalyzes these reactions at modest electro-
chemical potentials (dithionite is the reductant used in these ex-
periments), however, it does require considerable energy input
from the obligate hydrolysis of ATP.
No other known single enzyme can catalyze CO
2
reduction to
4
CH . Methanogenic bacteria convert CO to CH , but this is
4
2
accomplished by the action of a consortium of enzymes func-
tioning as part of a metabolic pathway (16). In acetogenic bac-
Fig. 3. Electron flux dependence for CO
2 4 4
reduction to CH . The ratio CH
formed to H formed is shown as a function of the electron flux through the
2
Gln
teria, CO
enzymes including CO dehydrogenase, which catalyzes the re-
versible interconversion of CO and CO (41). Like nitrogenase,
CO dehydrogenase also uses a complex metal cluster to achieve
this reaction. Other enzymes have been shown to reduce CO to
as reported here
2
is converted to acetate by the action of several
Ala
α-70 /α-195 MoFe protein for assays quenched after 20 min at 30 °C. The
partial pressure of CO was 0.45 atm, the concentration of MoFe protein was
.5 mg/mL, and Fe protein was varied from 0.5 to 6 mg/mL
2
2
0
2
formate or methanol (17, 18), but none to CH
for nitrogenase.
4
+
À
CO2 + 8H + 8e → CH4 + 2H2O
[2]
The reduction of CO catalyzed by nitrogenase can be con-
2
À
+
sidered in the context of our current understanding of the
CO2 + HCꢀ ≡ ꢀ CH + 8e + 8H → H2C = CH À CH3 + 2H2O
mechanism for the reduction of the physiological substrate N by
2
[
3]
six electrons to two ammonia molecules with two additional
electrons being used to evolve H (20, 28, 42). Several recent
2
Propylene is an especially important hydrocarbon, being the
starting point for the synthesis of a variety of polymers (36). A
limited number of metal-based catalysts have been shown to
studies have added to earlier work in building a probable
mechanism for how N might be reduced at the active site FeMo
2
cofactor. One important insight relevant to the current discus-
sion is the observation of metal-bound hydrides (determined as
two hydrides bridging between Fe atoms) as an integral part of
the FeMo-cofactor reactivity toward N
2
(43–45). These metal
bound hydrides (M-H ) have been proposed to participate in the
initial reduction of N to the proposed intermediate diazene
HN = NH). Further reduction of the metal bound diazene to
−
2
(
two ammonia molecules is proposed to involve successive addi-
tion of electrons and protons. An important observation re-
garding this mechanistic feature is that during N
proposed reaction intermediates (diazene HN = NH or hydra-
zine H N-NH ) are not detected in appreciable quantities, in-
2
reduction, the
2
2
dicating that intermediates remain bound to the active site until
the final products are released. This phenomenon is likely
explained by stabilization of key intermediates along the reaction
pathway through appropriate functional groups, thereby mini-
mizing kinetic barriers in going from N to two ammonias. The
2
observation reported here that nitrogenase can achieve the
multielectron reduction of CO to CH suggests that nitrogenase
2
4
can also stabilize key intermediates along this reaction pathway
through appropriate functional groups. Metal hydrides have
been suggested to be involved in the initial steps of CO
duction catalyzed by metal complexes (39), suggesting that ni-
trogenase might also achieve the two electron reduction of CO
by hydride insertion, in a process parallel to the one proposed for
the initial steps in N reduction. Whether partial reduction
intermediates (e.g., formate or formaldehyde) are leaked from
nitrogenase during CO reduction is technically challenging to
determine because accurate measurement of formate or form-
aldehyde in solutions containing dithionite is complicated by
interference from dithionite.
2
re-
2
Fig. 4. Propylene formation from CO
2
and acetylene as a function of
2
electron flux. The amount of propylene formed as a function of electron flux
Ala
Gln
is shown for the α-70 /α-195
was 0.45 atm and C was 0.014 atm. The concentration of MoFe protein
was 0.5 mg/mL, and Fe protein was varied from 0.25 to 3 mg/mL. The
MoFe protein. The partial pressure of CO
2
2
2 2
H
reactions were incubated at 30 °C for 60 min. Inset shows the CH
as a function of electron flux.
4
production
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Yang et al.

coupling of CO
2
to alkynes yielding oxygenated hydrocarbons
(
e.g., carboxylic acids) has been reported for metal catalysts (10,
4
7) but, to our knowledge, the production of olefins is unique to
the reactions reported here.
In summary, the findings presented here initiate the un-
derstanding of how nitrogenase can reduce and couple CO2
4
by multiple electrons to the industrially interesting CH and
propylene. The findings presented here begin to shed light
on these reactions and provide insights into the broader con-
text of how N is reduced to ammonia. Future studies should
2
look toward understanding how reaction barriers are lowered
through stabilization of key reaction intermediates, which
should provide guiding insights that can be used in the design
of more robust catalysts for the reduction of CO
hydrocarbons.
2
to various
Materials and Methods
Reagents and Protein Purification. All reagents were obtained from Sigma-
Aldrich or Fisher Scientific and were used without further purification, unless
3
specified otherwise. Sodium bicarbonate (NaHCO ) was from Avantor Per-
formance Materials. Sodium dithionite was purified to approximately 99%
purity according to a published procedure (48). Gases were purchased from
Air Liquide: CO and acetylene. Methane gas was obtained from household
2
Fig. 5. Propylene formation from CO
propylene formation on the partial pressure of acetylene at a partial pres-
2
and acetylene. The dependence of
natural gas line with an estimated purity of 97%. Propane gas was obtained
from a propane fuel tank with an estimated purity of 86%. All other gases
were purchased from Air Liquide. Azotobacter vinelandii strains DJ1260
Ala
Gln
sure of CO
2
of 0.40–0.45 atm is shown for the α-70 /α-195 MoFe protein.
The concentration of MoFe protein was 0.5 mg/mL, and Fe protein was
0
.5 mg/mL. The reactions were incubated at 30 °C for 60 min.
Val
Gln
Ala
(
(
wild-type, WT, or α-70 ), DJ997 (α-195 ), DJ1310 (α-70 ), DJ1316
Ala Gln Ala Ala
α-70 /α-195 ), and DJ1495 (α-70 /α-191 ) were grown, and the corre-
sponding nitrogenase MoFe proteins were expressed and purified as de-
scribed (49). All MoFe proteins in this study contain a seven-His tag addition
near the carboxyl-terminal end of the α-subunit. The purification of these
proteins was accomplished according to a published purification protocol
Another key finding reported here is the need to remodel the
protein environment around FeMo cofactor to activate the re-
duction of CO to CH . Earlier studies have illustrated that the
2
4
(
50). Protein concentrations were determined by the Biuret assay using BSA
protein environment immediately surrounding FeMo cofactor
control both the size of compounds that can be substrates and
the reactivity of FeMo cofactor toward those compounds (46).
as standard. The purities of these proteins were >95% based on SDS/PAGE
analysis with Coomassie staining. Manipulation of proteins and buffers was
done in septum-sealed serum vials under an argon atmosphere or on a
Schlenk vacuum line. All gases and liquid transfers used gas-tight syringes.
2
We earlier found that CO could be reduced to CO by the wild-
type MoFe protein (27), but very little further reduction products
are detected, suggesting that CO had limited access to the active
2
2
Carbon Dioxide Reduction Assays. Using CO as substrate, assays were con-
part of FeMo cofactor in the wild-type enzyme and that the re-
action cannot go forward beyond CO. The inability to go beyond
CO could be due to steric constraints imposed by the active site
on subsequent intermediates in the reaction pathway or the lack
of functional groups to stabilize reaction intermediates. Such
possibilities are supported by the requirement for amino acid
ducted in 9.4-mL serum vials containing 2 mL of an assay buffer consisting of
approximately 100 mM sodium dithionite, a MgATP regenerating system
(13.4 mM MgCl
0.4 mg/mL creatine phosphokinase) in a combination buffer of 33.3 mM
Mops, 33.3 mM Mes, and 33.3 mM TAPS at pH 8.0 except for the CO partial
pressure dependence study, which was done in 100 mM Bis-Tris buffer, pH
.7. After making the solution anaerobic, addition of CO and equilibration
2
, 10 mM ATP, 60 mM phosphocreatine, 0.6 mg/mL BSA, and
2
6
2
substitutions to achieve CO reduction beyond CO all of the way
2
between the gas phase and liquid phase for approximately 20 min, the MoFe
protein were added. Then the assay vials were ventilated to atmospheric
pressure. Reactions were initiated by the addition of Fe protein and in-
cubated at 30 °C. Reactions were quenched by the addition of 400 μL of 400
mM EDTA at pH 8.0 solution. When using NaHCO3 as the substrate for re-
duction or coupling assays, the reaction mixture was made by mixing a
100 mM Bis-Tris buffer (pH 6.7) containing all components as described
to CH and the fact that the unsubstituted MoFe protein has an
4
exceedingly poor capacity to reduce CO (24, 26).
A final notable observation from the current work is the ca-
pacity for reductive coupling of CO with acetylene to yield the
2
3
C olefin propylene by the remodeled MoFe protein. Several
earlier studies have suggested two binding sites on FeMo co-
factor. For example, it has been proposed that two CO molecules
bind to FeMo cofactor in the high CO concentration inhibited
state (29–33). Likewise, two acetylene binding sites have been
implicated from studies combining kinetics and amino acid
substitutions (31, 34, 35). Finally, the finding that CO can be
reduced and coupled to make C2 and C3 hydrocarbons is con-
sistent with two adjacent binding sites (24, 26). Here, we report
above and a stock solution of NaHCO
pH 6.7). For coupling reactions between CO
nent buffer system (33.3 mM Mops, 33.3 mM Mes, and 33.3 mM TAPS at pH
.0) was used. All pH values in this work were nominal values before mixing
and/or equilibration with CO gas or NaHCO solution. Methane (CH ) and
propylene (C ) were quantified by gas chromatography by injection of 500
3
dissolved in 100 mM Bis-Tris buffer
(
2
and C , the three-compo-
2 2
H
8
2
3
4
3 6
H
μL of the gas phase of the reaction vial into a Shimadzu GC-8A equipped
with a flame ionization detector fitted with a 30 cm × 0.3 cm Porapak N
column with nitrogen as the carrier gas. The injection/detection temperature
was set to 180 °C, and the column temperature was set to 110 °C. The
standard curves with high linearity were created by using methane, and
propane gases diluted with argon in 9.4-mL serum vials.
that CO
acetylene, yielding predominately propylene. Up to 8% of the C3
product during CO and acetylene reduction catalyzed by ni-
2 4
can be reduced to the level of CH and coupled to
2
trogenase is the C3 product propane. Addition of ethylene to a
reaction with CO2 did not yield propane. This result clearly
suggests that both CO and acetylene are binding to the active
2
GC-MS Analysis. The production of CH
4
from CO
2 3 6
reduction and C H from
the reductive coupling between CO and C H
2
2 2
was confirmed on a Shimadzu
site and both are being activated within the active site during the
coupling reaction. This observation is best explained by two
adjacent substrate activation sites, which can be populated to
varying extents by changing the electron flux through nitrogenase
and the partial pressures of CO2 and acetylene. Reductive
GC-2010 gas chromatograph equipped with a programmed temperature
vaporization (PTV) injector and a Shimadzu GCMS-QP2010S mass spec-
12/13
trometer by using
3 2
C-enriched NaHCO as CO source. Separation of
methane was achieved with a GC-CARBONPLOT column [30 m, 0.32 mm
inner diameter (ID), and 3.0-μm film thickness] (Agilent Technologies), and
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separation of propylene and propane was achieved with a Rt-Alumina
BOND/KCl column (30 m, 0.32 mm ID, and 5.0 μm film thickness) (Restek).
The injector and column temperatures were set to 35 °C. Ultrapure helium
was used as the carrier gas set at a linear velocity of 50 cm/s for methane
separation and 60 cm/s for propylene separation. For separation of methane
and propylene, 25 μL and 500 μL of headspace gases were directly injected
into the PTV injector, respectively. The mass spectrometer was operated in
electron ionization and selected ion monitoring mode.
ACKNOWLEDGMENTS. This work has been supported by National Institutes
of Health Grant GM 59087 (to D.R.D. and L.C.S.). V.R.M was the recipient of
Brazilian Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior
(CAPES) Mobility Fellowship 0388/11-4.
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