Molybdenum nitrogenase catalyzes the reduction and coupling of CO to form hydrocarbons

Article abstract of DOI:10.1074/jbc.M111.229344

The molybdenum-dependent nitrogenase catalyzes the multi-electron reduction of protons and N₂ to yield H₂ and 2NH₃. It also catalyzes the reduction of a number of non-physiological doubly and triply bonded small molecules (e.g. C₂H₂, N₂O). Carbon monoxide (CO) is not reduced by the wild-type molybdenum nitrogenase but instead inhibits the reduction of all substrates catalyzed by nitrogenase except protons. Here, we report that when the nitrogenase MoFe protein α-Val⁷⁰ residue is substituted by alanine or glycine, the resulting variant proteins will catalyze the reduction and coupling of CO to form methane (CH₄), ethane (C₂H₆), ethylene (C₂H₄), propene (C₃H₆), and propane (C₃H₈). The rates and ratios of hydrocarbon production from CO can be adjusted by changing the flux of electrons through nitrogenase, by substitution of other amino acids located near the FeMo-cofactor, or by changing the partial pressure of CO. Increasing the partial pressure of CO shifted the product ratio in favor of the longer chain alkanes and alkenes. The implications of these findings in understanding the nitrogenase mechanism and the relationship to Fischer-Tropsch production of hydrocarbons from CO are discussed.

Full text of DOI:10.1074/jbc.M111.229344

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 22, pp. 19417–19421, June 3, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Molybdenum Nitrogenase Catalyzes the Reduction and
ࡗ
Coupling of CO to Form Hydrocarbons*
Received for publication, February 10, 2011, and in revised form, March 21, 2011 Published, JBC Papers in Press, March 28, 2011, DOI 10.1074/jbc.M111.229344
Zhi-Yong Yang‡, Dennis R. Dean§, and Lance C. Seefeldt‡1
From the ‡Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322 and §Department of
Biochemistry, Virginia Tech University, Blacksburg, Virginia 24061
The molybdenum-dependent nitrogenase catalyzes the
multi-electron reduction of protons and N2 to yield H2 and
2NH3. It also catalyzes the reduction of a number of non-phys-
iological doubly and triply bonded small molecules (e.g. C2H2,
N2O). Carbon monoxide (CO) is not reduced by the wild-type
molybdenum nitrogenase but instead inhibits the reduction of
all substrates catalyzed by nitrogenase except protons. Here, we
report that when the nitrogenase MoFe protein ␣-Val70 residue
is substituted by alanine or glycine, the resulting variant pro-
teins will catalyze the reduction and coupling of CO to form
methane (CH4), ethane (C2H6), ethylene (C2H4), propene
(C3H6), and propane (C3H8). The rates and ratios of hydrocar-
bon production from CO can be adjusted by changing the flux of
electrons through nitrogenase, by substitution of other amino
acids located near the FeMo-cofactor, or by changing the partial
pressure of CO. Increasing the partial pressure of CO shifted the
product ratio in favor of the longer chain alkanes and alkenes.
The implications of these findings in understanding the nitro-
genase mechanism and the relationship to Fischer-Tropsch pro-
duction of hydrocarbons from CO are discussed.
All three nitrogenase systems catalyze the reduction of N2 to
ammonia and the reduction of protons to H2. In addition, all
three systems catalyze the reduction of a range of other small
molecules that contain double and triple bonds, such as acety-
lene (C2H2) to ethylene (C2H4), with different rates and ratios of
products being observed for the three systems (2). Carbon
monoxide (CO) is a well established inhibitor of all three nitro-
genase systems, where it inhibits the reduction of all known
substrates except protons (2, 6, 7). Recently, Lee et al. (8) dis-
covered that the vanadium nitrogenase system can reduce and
couple CO at low rates to form the hydrocarbons ethane, eth-
ylene, and propane. The molybdenum nitrogenase was not
observed to catalyze any detectable reduction of CO to
hydrocarbons.
We have recently shown that the substrate range for the
molybdenum nitrogenase can be expanded to larger substrates
by substitution of an amino acid residue located near the active
site FeMo-cofactor (␣-Val70) (9, 10). In light of our ability to
change the substrate range of nitrogenase and in light of the
observation of CO reduction and coupling by the vanadium
nitrogenase, it was of interest to determine whether CO might
be reduced and coupled to form hydrocarbons in the molybde-
num nitrogenase having amino acid substitutions at the ␣-Val70
residue.
Nitrogenase is the bacterial enzyme responsible for the bio-
logical reduction of N2 to ammonia, accounting for more than
half of the input of fixed nitrogen into the biogeochemical
nitrogen cycle (1). Three different nitrogenases have been iden-
tified, with each being coded for by unique sets of genes (2, 3).
The three nitrogenase systems are composed of a smaller pro-
tein called the Fe protein2 and a larger protein called the MoFe
protein, VFe protein, or FeFe protein, depending on the metal
composition of the corresponding cofactors. The Fe protein
serves as a reductant of the larger protein, with electron transfer
from the Fe protein being coupled to the hydrolysis of two
MgATP molecules for each electron transferred (4). The nitro-
genase system containing molybdenum is the most widely
occurring and is preferentially expressed if sufficient molybde-
num is present in the cell growth medium, with the other nitro-
genases being expressed secondarily if molybdenum is deficient
(3, 5).
EXPERIMENTAL PROCEDURES
Reagents and Protein Purification—All reagents were
obtained from Sigma-Aldrich or Fisher Scientific and were used
without further purification, unless specified otherwise. CO
was purchased from Matheson Tri-Gas (Basking Ridge, NJ)
with a purity of Ն99.9%. Ethylene (99.9%) was obtained from
Praxair Inc. (Danbury, CT). Methane gas was obtained from a
household natural gas line containing about 3% ethane as an
impurity. Propane gas was obtained from a propane fuel tank
with an estimated purity of 86%. All other gases were from Air
Liquide (Plumsteadville, PA). Azotobacter vinelandii strains
DJ1260 (wild-type, WT, or ␣-Val70), DJ997 (␣-Gln195), DJ1310
(␣-Ala70), DJ1316 (␣-Ala70/␣-Gln195), DJ1313 (␣-Gly70),
DJ1391 (␣-Ala70/␣-His96), and DJ1495 (␣-Ala70/␣-Ala191) were
grown, and the corresponding nitrogenase MoFe proteins were
expressed and purified as described previously (11). All MoFe
proteins in this study contain a seven-His tag addition near
the carboxyl-terminal end of the ␣-subunit. The purification of
these proteins was accomplished by following the previously
developed zinc affinity purification protocol (11). Protein con-
centrations were determined by the Biuret assay using bovine
serum albumin as standard. The purities of these proteins were
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant GM-59087 (to L. C. S. and D. R. D.).
ࡗ This article was selected as a Paper of the Week.
1 To whom correspondence should be addressed: Chemistry and Biochemis-
try Dept., Utah State University, 0300 Old Main Hill, Logan, UT 84322. Fax:
435-797-3390; E-mail: lance.seefeldt@usu.edu.
2 The abbreviations used are: Fe protein, iron protein; MoFe protein, molyb-
denum-iron protein; VFe protein, vanadium-iron protein; FeFe protein,
iron-iron protein.
JUNE 3, 2011•VOLUME 286•NUMBER 22
JOURNAL OF BIOLOGICAL CHEMISTRY 19417

Molybdenum Nitrogenase Reduction of CO
Ͼ95% based on SDS-PAGE analysis with Coomassie Blue stain-
ing. 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.
Carbon Monoxide Reduction Assays—Unless stated other-
wise, CO reduction assays were conducted in serum vials hav-
ing a 9.4-ml total volume and containing 1 ml of an assay buffer
consisting of 200 mM MOPS, pH 7.0, with MgATP and a
MgATP-regenerating system (13.4 mM MgCl2, 10 mM ATP, 60
mM phosphocreatine, 1.3 mg/ml bovine serum albumin, and 0.4
mg/ml creatine phosphokinase). After the solution was made
anaerobic, 150 ␮l of 1 M dithionite solution and the MoFe pro-
tein were added. Reactions were initiated by the addition of Fe
protein and incubated at 30 °C. Reactions were quenched by the
addition of 300 ␮l of 400 mM EDTA, pH 8.0, solution. All assays
were done under 1 atm of CO pressure except for wild-type and
␣-Gln195 MoFe proteins (0.106 atm of CO for these two cases).
Methane (CH4), ethylene (C2H4), and ethane (C2H6) were
quantified by gas chromatography by injection of 200 ␮l of the
gas phase of the reaction vial (400 ␮l for wild-type and ␣-Ala70/
␣-Gln195 MoFe samples) into a Shimadzu GC-8A equipped
with a flame ionization detector fitted with a 30 ϫ 0.3-cm
Porapak N column with nitrogen as the carrier gas. The injec-
tion/detection temperature was set to 180 °C, and the column
temperature was set to 110 °C. The standard curves with high
linearity were created using methane, ethylene, and ethane
gases diluted with argon in 9.4-ml serum vials. Propane (C3H8)
and propene (C3H6) were quantified by gas chromatography by
injection of 100 ␮l of the gas phase of the reaction vial into a
Shimadzu GC-2010 gas chromatograph equipped with a flame
ionization detector fitted with a Rt-Alumina BOND/KCl col-
umn (30 m, 0.32-mm inner diameter, and 5-␮m film thickness)
(Restek, Belafonte, PA). Helium was used as the carrier gas set
at a linear velocity of 45 cm/s. The injection/detection temper-
ature was set to 200 °C, and the column temperature was set to
60 °C. Identification of three- and four-carbon hydrocarbons
was confirmed by mass spectroscopy (GCMS-QP2010S, Shi-
madzu Scientific). After the addition of 300 ␮l of EDTA solu-
tion, the total liquid volumes in the standard vials were the
same as those in CO reduction assay vials.
FIGURE 1. Time course of hydrocarbon production by the ␣-Ala70- and
␣-Gly70-substitutedMoFeproteins. Thequantityof(F)ethylene(C2H4), (f)
ethane (C2H6), (ꢀ) propene (C3H6), and (Œ) propane (C3H8) produced per
MoFe protein (nmol of product per nmol of MoFe protein) as a function of
time is shown for the ␣-Ala70 (A) and ␣-Gly70 (B) MoFe nitrogenases. Assay
conditions are described under “Experimental Procedures.”
presence of Fe protein, MgATP, and reductant (Fig. 1). Four
hydrocarbon products were quantified for both the ␣-Ala70 and
the ␣-Gly70 MoFe proteins: the alkanes ethane (C2H6) and pro-
pane (C3H8) and the alkenes ethylene (C2H4) and propene
(C3H6). For both proteins, the highest yielding product was
ethylene followed by propene, ethane, and propane. The
amounts of hydrocarbons produced were ϳ7 nmol of ethylene
(C2H4)/nmol of MoFe protein, 4 nmol of propene (C3H6)/nmol
of MoFe protein, 2 nmol of ethane (C2H6)/nmol of MoFe pro-
tein, and 1 nmol of propane (C3H6)/nmol of MoFe protein over
60 min. Three of these products (ethylene, ethane, and pro-
pane) were reported for CO reduction by the vanadium nitro-
genase system (8), although the relative ratio of products and
rates of production was different between the two nitroge-
nase systems. For the vanadium nitrogenase, product pro-
duction rates were 140 nmol of ethylene/nmol of VFe pro-
tein, 1.5 nmol of propane/nmol of VFe protein, and 5 nmol of
ethane/nmol of VFe protein over 60 min. No propene prod-
uct was reported for the vanadium nitrogenase, whereas this
was the second most abundant product in the molybdenum
nitrogenase.
Effects of Additional Amino Acid Substitutions—In addition
to the pivotal role of ␣-Val70 in defining substrate binding to the
FeMo-cofactor, we have earlier shown that two additional
amino acids located near the FeMo-cofactor, ␣-Arg96 and
␣-Gln191, can affect substrate binding (12, 13). Substituting
these amino acids by histidine or alanine, respectively, has been
shown to further expand the substrate range of nitrogenase
to include even longer chain alkynes. We therefore examined
the doubly substituted ␣-Ala70/␣-His96 and ␣-Ala70/␣-Ala191
MoFe proteins to test whether they might reduce and couple
CO. Both of the doubly substituted MoFe proteins showed an
altered product profile for hydrocarbon production, with the
yield of the longer chain products propane and propene rising
significantly (Fig. 2). For the ␣-Ala70/␣-Ala191 MoFe protein, 8
nmol of propene were accumulated per nmol of MoFe protein
For the electron flux dependence study, 1 mg of MoFe pro-
tein was used for each assay vial. Molar ratios of Fe protein to
MoFe protein from 4:1 to 20:1 were tested. All these assays were
incubated for 2 h before quenching with EDTA. For time
dependence measurements and CO pressure dependence stud-
ies, the amount of MoFe protein and the electron flux for dif-
ferent mutants was varied.
RESULTS
Reduction of CO by ␣-Val70-substituted MoFe Proteins—
Consistent with the earlier report (8), we find that the wild-type
MoFe protein shows no catalytic production of hydrocarbons
by reduction and coupling of CO when analyzed over 90 min over 60 min. A trace of methane (CH4) was also detected as a
under turnover conditions. In contrast, when the MoFe protein product of CO reduction for both doubly substituted MoFe
residue ␣-Val70 was substituted by alanine (␣-Ala70) or glycine proteins, whereas methane was not detected for any of the sin-
(␣-Gly70), the variant MoFe proteins were found to reduce and gly substituted MoFe protein variants. The doubly substituted
couple CO to hydrocarbons in a reaction dependent on the MoFe proteins demonstrated a non-linear production of prod-
19418 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 286•NUMBER 22•JUNE 3, 2011

Molybdenum Nitrogenase Reduction of CO
FIGURE 4. Electron flux dependence on CO reduction product profile. The
quantity of (ࡗ) methane (CH4), (F) ethylene (C2H4), (f) ethane (C2H6), (ꢀ)
propene (C3H6), and (Œ) propane (C3H8) produced per MoFe protein (nmol of
product per nmol of MoFe protein) is shown as a function of the molar ratio of
Fe protein to MoFe protein for the ␣-Ala70 (A), ␣-Gly70 (B), and ␣-Ala70/␣-His96
(C) MoFe proteins. The time of the assay was 120 min.
FIGURE 2. Time course of hydrocarbon production by the ␣-Ala70/␣-
His96- and ␣-Ala70/␣-Ala191-substituted MoFe proteins. The quantity of
(ࡗ) methane (CH4), (F) ethylene (C2H4), (f) ethane (C2H6), (ꢀ) propene
(C3H6), and (Œ) propane (C3H8) produced per MoFe protein (nmol of product
per nmol of MoFe protein) as a function of time is shown for the ␣-Ala70/␣-
His96 (A) and ␣-Ala70/␣-Ala191 (B) MoFe nitrogenases.
FIGURE 3. Time course of hydrocarbon production by the ␣-Ala70- and
␣-Ala70/␣-Gln195-substituted MoFe proteins. The quantity of (circles) eth-
ylene (C2H4), (squares) ethane (C2H6), (inverted triangles) propene (C3H6), and
(triangles) propane (C3H8) produced per MoFe protein (nmol of product per
nmol of MoFe protein) as a function of time is shown for the ␣-Ala70 (open
symbols) and ␣-Ala70/␣-Gln195 MoFe proteins (closed symbols).
FIGURE 5. CO pressure dependence on CO reduction product profile.
Shown are the methane (CH4), ethylene (C2H4), ethane (C2H6), propene
(C3H6), and propane (C3H8) production per MoFe protein (nmol of product
pernmolofMoFeprotein)undereitherlowCO(0.08atm)orhighCO(1.0atm)
for the ␣-Ala70 (A), ␣-Gly70 (B), ␣-Ala70/␣-His96 (C), and ␣-Ala70/␣-Ala191 (D)
MoFe proteins. Assay time is 60 min. PCO, Partial pressure of CO.
ucts, suggesting possible inactivation of these enzymes during
the course of the assay.
The amino acid ␣-His195 is located near the FeMo-cofactor
and has been implicated in the delivery of protons for the reduc-
tion of nitrogenous substrates (14, 15). We find that when
␣-His195 is substituted by glutamine in combination with the
␣-Ala70 substitution, the rates of product formation for CO
reduction and coupling are greatly decreased (Fig. 3), consistent
with the possibility that ␣-His195 functions to deliver protons
for the reduction of CO.
species binds to the FeMo-cofactor, referred to as the low CO
state. At partial pressures of CO above 0.4 atm, two CO mole-
cules bind to the FeMo-cofactor, referred to the high CO state
(17). It was therefore of interest to determine whether varying
the partial pressure of CO might also alter the product profile
for CO reduction and coupling. As can be seen in Fig. 5, the
partial pressure of CO significantly changes the product profile
for the four MoFe protein variants examined (␣-Ala70, ␣-Gly70,
␣-Ala70/␣-His96, ␣-Ala70/␣-Ala191 MoFe proteins). At lower
CO partial pressure (0.08 atm), the shorter chain C2 products
are favored. At this low CO concentration, the ␣-Ala70/␣-His96
MoFe protein also showed a significant methane production
rate. At high CO partial pressure (1.0 atm), the product profile
shifted in favor of the longer chain C3 hydrocarbons.
Influence of Electron Flux on Product Ratio—Electron flux in
nitrogenase is defined as the rate of total electrons flowing
through the enzyme going to substrates. The flux can be con-
trolled by varying the ratio of Fe protein to MoFe protein, with
a low ratio corresponding to low flux and a higher ratio corre-
sponding to higher flux (2). Increasing electron flux affected the
product profile for the ␣-Ala70, ␣-Gly70, and ␣-Ala70/␣-His96
MoFe proteins (Fig. 4). In each case, however, ethylene re-
mained the most abundant product over the range tested.
Influence of CO Partial Pressure on Product Profile—Varying
the partial pressure of CO has been shown to result in different
numbers of CO bound to the FeMo-cofactor (6, 16). Under low
DISCUSSION
Earlier spectroscopic studies of CO bound to nitrogenase
concentrations of CO (ϳ0.08 atm partial pressure), a single CO indicate that there are two CO binding sites on the FeMo-co-
JUNE 3, 2011•VOLUME 286•NUMBER 22
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Molybdenum Nitrogenase Reduction of CO
substrate reduction. To test whether ␣-His195 might also be
responsible for delivery of protons for CO reduction, the
␣-Gln195 substitution was introduced into the ␣-Ala70 MoFe
protein. The resulting doubly substituted MoFe protein
showed significantly lowered CO reduction rates for all
products, consistent with the ␣-His195 being responsible for
much, but not all, of the proton delivery for CO reduction in
the MoFe protein.
FIGURE 6. Schematic representation of a possible CO-derived hydrocar-
bon formation at the nitrogenase active site. Black circles represent CO
binding sites, presumably Fe atoms, upon which two CO can bind. Following
reduction to semireduced states (-CHx and -CHy), coupling can occur, result-
ing in formation of a CHx–CHy species. The binding of additional CO mole-
cules can lead to further chain elongation.
One of the goals for Fischer-Tropsch chemistry is finding
factor (6). At low CO concentrations, only one CO is bound, catalysts and conditions that favor the formation of the higher
whereas at higher CO concentrations, two CO molecules are value, longer chain hydrocarbons such as propene and propane
bound (17–19). The observation that CO can be reduced to (23). Here, we report that the introduction of additional amino
two- and three-carbon containing hydrocarbons demands that acid substitutions near the FeMo-cofactor and increasing the
the two CO binding sites on the FeMo-cofactor are close CO concentration can significantly shift the product profile of
enough to allow a coupling reaction that would result in the nitrogenase CO reduction and coupling in favor of production
formation of C2 and C3 hydrocarbon products. This fact, cou- of these longer chain hydrocarbons. Thus, the molybdenum
pled with the parallels between this reaction and the Fischer- nitrogenase offers an experimentally tractable system for exam-
Tropsch reaction (20–23), suggests a possible mechanism for ining mechanistic features that favor the production of longer
CO reduction and coupling by nitrogenase (Fig. 6).
chain hydrocarbons from CO that might be translated to the
Initially, two CO molecules would chemisorb to two adjacent development of small molecule metal complexes that catalyze
metal (Fig. 6, black circles) atoms of the FeMo-cofactor. Avail- such reactions.
able spectroscopic studies on CO bound to FeMo-cofactor
In summary, we report that when the active site of the molyb-
indicate that the CO molecules are bound to iron atoms located denum nitrogenase is exposed by substitution of ␣-Val70, CO
in the waist region of the FeMo-cofactor (19, 24, 25). Reduction can be reduced and coupled to yield the hydrocarbons meth-
of the two metal bound CO molecules by protons and electrons ane, ethylene, ethane, propene, and propane. Further, it is
(hydrogen) with loss of water would result in the formation of found that the relative ratio of products can be manipulated
metal-bound -CHx and -CHy groups. Analogous intermediates by altering other amino acids, electron flux, or the CO
have been proposed in a mechanism for the Fischer-Tropsch concentration.
reaction (22). Either the two metal-bound -CHx groups could
be further reduced/protonated, resulting in release of methane,
or a coupling reaction could result in the formation of a CHx–
CHy species bound to one metal. This species could yield eth-
ylene or ethane, or a third CO could bind to and be reduced at
the open metal site, leading to the formation of C3 or longer
hydrocarbons. Traces of longer chain (C4) hydrocarbon prod-
ucts were detected from molybdenum nitrogenase reduction
and from coupling of CO molecules that were tentatively
assigned by mass spectroscopy as isobutene and n-butane (data
not shown).
Acknowledgment—We thank Dr. Brett Barney, University of Minne-
sota, for helpful discussions.
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JUNE 3, 2011•VOLUME 286•NUMBER 22
JOURNAL OF BIOLOGICAL CHEMISTRY 19421

Enzymology:
Molybdenum Nitrogenase Catalyzes the
Reduction and Coupling of CO to Form
Hydrocarbons
Zhi-Yong Yang, Dennis R. Dean and Lance
C. Seefeldt
J. Biol. Chem. 2011, 286:19417-19421.
doi: 10.1074/jbc.M111.229344 originally published online March 28, 2011
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