ATP-independent substrate reduction by nitrogenase P-cluster variant

Article abstract of DOI:10.1073/pnas.1202429109

The P-cluster of nitrogenase is largely known for its function to mediate electron transfer to the active cofactor site during catalysis. Here, we show that a P-cluster variant (designated P*-cluster), which consists of paired [Fe₄S₄]-like clusters, can catalyze ATP-independent substrate reduction in the presence of a strong reductant, europium (II) diethylenetriaminepentaacetate [Eu(II)-DTPA]. The observation of a decrease of activity in the rank ΔnifH, ΔnifBΔnifZ, and ΔnifB MoFe protein, which corresponds to a decrease of the amount of P*-clusters in these cofactor-deficient proteins, firmly establishes P*-cluster as a catalytically active metal center in Eu(II)-DTPA-driven reactions. More excitingly, the fact that P*-cluster is not only capable of catalyzing the two-electron reduction of proton, acetylene, ethylene, and hydrazine, but also capable of reducing cyanide, carbon monoxide, and carbon dioxide to alkanes and alkenes, points to a possibility of developing biomimetic catalysts for hydrocarbon production under ambient conditions.

Full text of DOI:10.1073/pnas.1202429109

ATP-independent substrate reduction by nitrogenase
P-cluster variant
Chi Chung Lee, Yilin Hu1, and Markus W. Ribbe1
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900
Edited* by Keith O. Hodgson, Stanford University, Menlo Park, CA, and approved March 23, 2012 (received for review February 10, 2012)
The P-cluster of nitrogenase is largely known for its function to
mediate electron transfer to the active cofactor site during catalysis.
Here, we show that a P-cluster variant (designated P*-cluster), which
consists of paired [Fe4S4]-like clusters, can catalyze ATP-independent
substrate reduction in the presence of a strong reductant, europium
(II) diethylenetriaminepentaacetate [Eu(II)-DTPA]. The observation
of a decrease of activity in the rank ΔnifH, ΔnifBΔnifZ, and ΔnifB
MoFe protein, which corresponds to a decrease of the amount of
P*-clusters in these cofactor-deficient proteins, firmly establishes
P*-cluster as a catalytically active metal center in Eu(II)-DTPA–driven
reactions. More excitingly, the fact that P*-cluster is not only capable
of catalyzing the two-electron reduction of proton, acetylene, eth-
ylene, and hydrazine, but also capable of reducing cyanide, carbon
monoxide, and carbon dioxide to alkanes and alkenes, points to
a possibility of developing biomimetic catalysts for hydrocarbon
production under ambient conditions.
circular dichroism (MCD) analyses (7–9) show that the [Fe4S4]-
like cluster pair (designated P*-cluster) is in fact a precursor to the
P-cluster, which can be reductively coupled into a [Fe8S7] structure
upon incubation with dithionite, MgATP, and either Fe protein
alone (in the case of ΔnifH MoFe protein) (12) or Fe protein plus
NifZ (in the case of ΔnifBΔnifZ MoFe protein) (13).
The capture of these cofactor-deficient forms of MoFe protein
affords an unprecedented opportunity to examine the catalytic
competence of P-cluster species without the interference of the
cofactor. Moreover, the distinct P-cluster contents of these MoFe
proteins permit a comparison between the catalytic capacities of
P- and P*-clusters, as well as a correlation between the structure
and function of the P-cluster species. Here, we present a study of
the substrate-reducing activities of ΔnifH, ΔnifBΔnifZ, and ΔnifB
MoFe proteins in Fe protein/ATP-free, europium (II) dieth-
ylenetriaminepentaacetate [Eu(II)-DTPA]-driven reactions. The
observation of a decrease of activity in the rank ΔnifH, ΔnifBΔnifZ,
and ΔnifB MoFe protein, which corresponds to a decrease of P*-
cluster content in these proteins, firmly establishes P*-cluster as the
catalytically active metallocenter in these reactions. More excit-
Fischer–Tropsch CN− CO CO2
|
|
|
itrogenases are a family of metalloenzymes that catalyze the
N
nucleotide-dependent reduction of dinitrogen to ammonia ingly, the fact that P*-cluster is not only capable of catalyzing the
under ambient conditions. The best-characterized member of this two-electron reduction of proton, acetylene, ethylene, and hydra-
enzyme family is the molybdenum (Mo)-nitrogenase of Azoto- zine, but also capable of reducing cyanide, carbon monoxide, and
bacter vinelandii, which consists of two redox-active component carbon dioxide to alkanes and alkenes, points to a possibility of
proteins (1). One of the proteins, termed iron (Fe) protein
developing novel biomimetic catalysts for hydrocarbon production
(encoded by nifH), is a γ2-dimer that contains a [Fe4S4] cluster under ambient conditions.
between the two subunits and a MgATP binding site within each
Results
subunit; the other, termed molybdenum-iron (MoFe) protein
(encoded by nifDK), is an α2β2-heterotetramer that houses two
complex metal clusters per αβ-dimer: the P-cluster ([Fe8S7]),
which is located at each α/β-subunit interface; and the iron-mo-
lybdenum (FeMo) cofactor, or FeMoco ([MoFe7S9C-homoci-
trate]), which is buried within each α-subunit (2–4). Catalysis by problem, a strong reductant, Eu(II)-DTPA (14), was used to drive
nitrogenase presumably involves repeated association and disso-
Because the conversion of the P*-cluster to P-cluster (12) requires
the same factors as those for the substrate turnover (i.e., Fe pro-
tein, MgATP, and dithionite), the activity of P*-cluster cannot be
assessed by a conventional nitrogenase assay. To circumvent this
the reactions in the absence of Fe protein, ATP, and dithionite. Eu
ciation between the two component proteins and ATP-dependent (II)-DTPA has been used previously to drive substrate reduction
electron transfer from the [Fe4S4] cluster of the Fe protein, by both a MoFe protein variant (15) and the isolated cofactors of
through the P-cluster, to the FeMoco of MoFe protein, where nitrogenase (16) and, therefore, is an appropriate choice of re-
substrate reduction occurs (5).
ducing agent in this case. Indeed, the EPR spectra of ΔnifH,
ΔnifBΔnifZ, and ΔnifB MoFe proteins show little or no change
following the treatment with Eu(II)-DTPA (Fig. S1), suggesting
that (i) these proteins and their associated clusters remain intact
in the presence of Eu(II)-DTPA; and (ii) the P*-clusters in ΔnifH
and ΔnifBΔnifZ MoFe proteins are not converted to the P-clusters
by Eu(II)-DTPA.
The P-cluster has long been regarded as a “capacitor” that
mediates the electron transfer between the [Fe4S4] cluster of Fe
protein and the cofactor site of MoFe protein during catalysis (1).
Although the question of whether the P-cluster can directly reduce
substrates was raised previously, it has remained a topic of debate
because of the difficulty of distinguishing the contribution of the
P-cluster from that of the cofactor to catalysis. As such, MoFe
protein variants carrying the P-cluster species alone need to be
generated to address the catalytic capacity of P-cluster. Recent
studies of the biosynthesis of nitrogenase have led to the identifi-
cation of three cofactor-deficient forms of MoFe protein (Fig. 1).
One form, designated ΔnifB MoFe protein, contains two intact,
[Fe8S7] P-clusters (6); another, designated ΔnifH MoFe protein,
contains two P-cluster variants—each comprising a pair of [Fe4S4]-
like clusters—in place of the two P-clusters (7–9); and the third,
designated ΔnifBΔnifZ MoFe protein, contains one P-cluster and
one pair of [Fe4S4]-like clusters (10, 11). Combined biochemical,
electron paramagnetic resonance (EPR), X-ray absorption (XAS)/
extended X-ray absorption fine structure (EXAFS), and magnetic
Driven by Eu(II)-DPTA (E0′ = −1.14 V at pH 8), the ΔnifH
MoFe protein can reduce cyanide (CN−) to methane (CH4), eth-
ene (C2H4), ethane (C2H6), propene (C3H6), propane (C3H8), 1-
butene (C4H8), n-butane (C4H10), 1-pentene (C5H10), n-pentane
Author contributions: C.C.L., Y.H., and M.W.R. designed research; C.C.L. performed re-
search; C.C.L., Y.H., and M.W.R. analyzed data; and Y.H. and M.W.R. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: yilinh@uci.edu or mribbe@uci.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1202429109/-/DCSupplemental.
6922–6926
|
PNAS
|
May 1, 2012
|
vol. 109
|
no. 18
www.pnas.org/cgi/doi/10.1073/pnas.1202429109

reduction are considerably lower than those from CN−-reduction
by the ΔnifH MoFe protein (Fig. 2B and Table 1). The same
discrepancy in reaction efficiency and product range is observed
when CO and CN− are reduced by the isolated nitrogenase
cofactors in Eu(II)-DTPA–based reactions (16) and, given the
topological analogy between the P-cluster and cofactor (17, 18), it
could be explained by the previous observation of a stabilizing
effect of CN−-binding on certain redox states of these clusters
(19), such that the clusters are more effective in substrate re-
duction. Nevertheless, the two reactions share a similar product
profile, showing a decrease in the efficiency of product formation
with an increase in product length, as well as a preference for the
formation of unsaturated 1-alkenes over saturated n-alkanes (Fig.
3), both of which are consistent with the earlier proposal of
a common reaction pathway for CN−- and CO-reduction (20).
GC-MS analysis further confirms that CO and CN− are the carbon
sources of hydrocarbons formed in these reactions, because all
products display the expected mass shifts when 12CO and 12CN−
are replaced by 13CO and 13CN−, respectively (Fig. 2 A and B).
The observation that ΔnifH MoFe protein is capable of reducing
CO to hydrocarbons raises the question of whether this protein can
generate hydrocarbons from CO2, particularly given the previous
observation that the wild-type MoFe protein can reduce CO2 to
CO (21). Indeed, when a mixture of carbon dioxide/bicarbonate
(CO2/HCO3−) is used as a substrate, C2H4, C2H6, C3H6, and C3H8
are detected as products in the Eu(II)-DTPA–driven, ΔnifH MoFe
protein-based reaction (Fig. 2C). The same set of products can be
Fig. 1. Schematic presentations of cofactor-deficient MoFe proteins. The
ΔnifB MoFe protein (A) contains two P-clusters (i.e., [Fe8S7] clusters); the
ΔnifBΔnifZ MoFe protein (B) contains one P-cluster and one P*-cluster (i.e.,
paired [Fe4S4]-like clusters); and ΔnifH MoFe protein (C) contains two P*-clus-
ters. The [Fe8S7] and [Fe4S4] clusters are represented by gray ovals and cubes,
respectively; whereas the vacant cofactor sites are represented by white ovals.
(C5H12), 1-hexene (C6H12), n-hexane (C6H14), n-heptane (C7H16),
and ammonia (NH3) under ambient conditions (Fig. 2A and
Table 1). Thus, contrary to the wild-type MoFe protein, which
generates CH4 and NH3 as the sole products of CN−-reduction
in a Fe protein/ATP-driven reaction (1), the ΔnifH MoFe protein
generates hydrocarbons of up to seven carbon-length as additional
products in the same reaction driven by Eu(II)-DTPA. Consistent
with this observation, the CH4:NH3 ratio of the reaction catalyzed
by ΔnifH MoFe protein (1:8) is considerably lower than that by the
wild-type MoFe protein (1:1), suggesting a redistribution of elec-
trons available for CH4 formation toward C-C coupling in the case
of the former.
identified with the expected mass shifts when 12CO2/H12CO3 is
−
When CN− is replaced by CO, an isoelectronic carbonaceous
molecule, a similar yet narrower set of hydrocarbons is generated
by ΔnifH MoFe protein in the Eu(II)-DTPA–driven reaction.
Only CH4, C2H4, C2H6, C3H6, C3H8, and 1-C4H8 are detected as
replaced by 13CO2/H13CO3− (Fig. 2C). However, the rate of CO2-
reduction by ΔnifH MoFe protein is too low to be accurately de-
termined by GC analysis. Additionally, CH4, the major product in
ΔnifH MoFe protein-catalyzed reduction of CN− and CO, cannot
products in this case, and the rates of product formation from CO- be clearly identified in this case (Fig. 2C).
Fig. 2. Hydrocarbon formation by ΔnifH MoFe protein. GC-MS analysis of hydrocarbons generated from the reduction of CN− (A), CO (B), and CO2 (C).
Products were generated from the 12C (Upper)- or 13C (Lower)-labeled CN− (A), CO (B), and CO2 (C). The mass-to-charge (m/z) ratios at which the products
were traced are indicated in the figure.
Lee et al.
PNAS
|
May 1, 2012
|
vol. 109
|
no. 18
|
6923

Table 1. Activities of hydrocarbon formation by cofactor-deficient MoFe proteins in
Eu(II)-DTPA–driven reactions
Activities
Products
ΔnifB MoFe protein
ΔnifBΔnifZ MoFe protein
ΔnifH MoFe protein
From CN‒
CH4
C2H4
C2H6
91 14
468 49
515 51
117 10
182 13
1,280 204
1,230 114
250 20
85
18
20
9
2
3
C3H6
475 51
C3H8
C4H8
C4H10
C5H10
C5H12
NH3
2.7 0.3
ND
ND
ND
ND
36
72
7
2.1 0.3
ND
3
8
1
72
160 19
24
67
13
9
2
9
1
1,193 114
4,648 410
10,180 1131
From CO
CH4
C2H4
C2H6
C3H6
6
1
25
11
4
1.4 0.2
0.5 0.1
3
1
4
83 11
2.7 0.3
0.9 0.1
ND
29
14
5
4
2
1
C3H8
ND
2.7 0.3
Activities are expressed in nanomole product/micromole protein per hour. See Materials and Methods for
assay conditions. ND, not detectable.
Apart from CN−, CO, and CO2, other nitrogenase substrates
can also be reduced by the ΔnifH MoFe protein in the presence
of Eu(II)-DTPA. Like the wild-type MoFe protein, the ΔnifH
MoFe protein can reduce proton (H+) to hydrogen (H2), acet-
ylene (C2H2) to C2H4, C2H4 to C2H6, azide (N3−) to NH3, and
hydrazine (N2H4) to NH3 (Table S1). However, contrary to the
wild-type MoFe protein, the ΔnifH MoFe protein: (i) does not
reduce N2 to NH3; (ii) generates C2H6 in addition to C2H4 from
C2H2; and (iii) reduces C2H4 to C2H6 in the Eu(II)-DTPA–
driven reaction (Table S1). Moreover, the ΔnifH MoFe protein/
Eu(II)-DTPA system is far less efficient than the wild-type MoFe
MoFe protein (Fig. 1). In contrast, the ΔnifB MoFe protein is
much less effective in substrate reduction than either ΔnifBΔnifZ
or ΔnifH MoFe protein, suggesting that the P-cluster is minimally
active in the reactions driven by Eu(II)-DTPA (Fig. 4). Together,
these data strongly indicate that the P*-cluster is the catalytically
active species in Eu(II)-DTPA–based reactions.
Discussion
A previous small angle X-ray scattering study has shown that the
ΔnifH MoFe protein exists in a more extended conformation than
the ΔnifB MoFe protein and that the increase in the size of ΔnifH
MoFe protein is correlated with an increase in the solvent ac-
cessibility of its P*-cluster Fe atoms (22). Such a conformational
change of ΔnifH MoFe protein can be best modeled by having
a 6 Å gap at its α/β-subunit interface, which is absent from the
structure of ΔnifB MoFe protein (22). The gap between the α- and
β-subunits of ΔnifH MoFe protein not only results in a “split” of
the P-cluster ([Fe8S7] structure) into a P*-cluster (paired [Fe4S4]-
−
protein/Fe protein/ATP system in reducing H+, N2H4, N3 and
C2H2 (Table S1), as the rates of product formation from these
substrates by the Eu(II)-DTPA–based system range between
0.5% and 3.5% of those by the Fe protein/ATP-based system.†
Interestingly, although a similar discrepancy in efficiency is also
observed between the ΔnifH and wild-type MoFe protein-cata-
lyzed reactions of CO-reduction, the rate of product formation
by the former is still 20% of that by the latter (see comment
regarding calculations, above), suggesting a preference of CO
over other substrates by the ΔnifH MoFe protein/Eu(II)-
DTPA system.
The distinct reactivity of ΔnifH MoFe protein is clearly asso-
ciated with its unique P*-cluster species. The rates of product
formation by the three cofactor-deficient forms of MoFe protein
are ranked in the order of ΔnifB MoFe protein (containing two P-
clusters) < ΔnifBΔnifZ MoFe protein (containing one P-cluster
and one P*-cluster) < ΔnifH MoFe protein (containing two P*-
clusters), illustrating that the [Fe4S4]-like cluster pair (i.e., P*-
cluster) is a much better catalyst than the [Fe8S7] cluster (i.e.,
P-cluster) in the Eu(II)-DTPA–driven reactions (Table 1 and
Table S1). In particular, the overall efficiencies of CN−- and CO-
reduction by ΔnifBΔnifZ MoFe protein are 39% and 31%, re-
spectively, of those by ΔnifH MoFe protein (Fig. 4), both of which
are close to—albeit lower than—the theoretical number of 50%
that is derived from the half P*-cluster content in the ΔnifBΔnifZ
Fig. 3. Product distributions of reactions catalyzed by ΔnifH MoFe protein.
Specific activities of hydrocarbon formation from the reduction of CN− (A)
and CO (B), which were calculated based on the rates of product formation
in the linear phases (first 15 min) of these reactions (Fig. S2). Alkane and
alkene products are presented in gray and black bars, respectively, in the
figure. The alkene/alkane ratios are 4.9 (C2H4/C2H6), 6.6 (C3H6/C3H8), 6.7
(C4H8/C4H10), and 5.2 (C5H10/C5H12) in the reaction of CN−-reduction (A) and
2.1 (C2H4/C2H6) and 1.9 (C3H6/C3H8) in the reaction of CO-reduction (B).
†These calculations were based on the activities of the same molar amount of wild-type
MoFe protein under conditions identical to those of the ΔnifH MoFe protein-based
reactions, except that Eu(II)-DTPA was replaced by excess Fe protein and MgATP.
6924
|
www.pnas.org/cgi/doi/10.1073/pnas.1202429109
Lee et al.

which comprise a [Fe4S4] cluster that is either positioned near or
bridged to a second metal-containing cluster, thereby permitting
the transfer of electrons from the former to the latter for the
subsequent reduction of substrates at the latter site. By analogy,
the P*-cluster could follow the same mode of action as these two
enzymes, transferring electrons from the standard [Fe4S4] cluster
to the atypical [Fe4S4]-like cluster, where substrate reduction
takes place. Given the capacity of standard, ferredoxin-type
[Fe4S4] clusters in electron transfer (33, 34), such a hypothesis
could account for the ability of P*-cluster to reduce a variety of
substrates by two electrons. On the other hand, a different and
perhaps more intricate mechanism may be used by the P*-cluster
during catalysis, as this cluster is also capable of reducing more
“complex” substrates, such as CN−, CO, and CO2, than those
reduced by only two electrons.
Regardless, the fact that the surface-exposed P*-cluster and
solvent-isolated cofactors can catalyze the ATP-independent re-
duction of CN−, CO, and CO2 points to a vivid possibility of de-
veloping biomimetic catalysts for hydrocarbon production under
ambient conditions. Moreover, the observation that the bio-
synthetic precursors of P-cluster (e.g., the P*-clusters in ΔnifH
MoFe protein) and cofactor [e.g., the L-cluster in NifEN (35–37)
(see below)] show catalytic activities similar to, yet distinct from
those of their mature counterparts implies that nitrogenase
clusters might have evolved through several primordial in-
termediate stages, where they took on different functions, such as
detoxification (e.g., reduction of C2H2 and N3−), hydrogenation
(i.e., reduction of H+), carbon fixation (e.g., reduction of CN−,
CO and CO2), and nitrogen fixation (i.e., reduction of N2).‡ Fi-
nally, given the topological analogy between the P-cluster and
cofactor, it is chemically plausible that these clusters evolved from
a common ancestral precursor (18, 38). This hypothesis raises the
provocative question of whether the P*-cluster in ΔnifH MoFe
protein represents a structural homolog of such a common pre-
cursor, particularly in light of the observed parallelism between
the CO-reducing abilities of the P*-cluster and cofactor. Studies
of the biosynthetic intermediates of nitrogenase, therefore, may
not only shed light on the evolution of nitrogen fixation, but also
unearth other lost functions—such as the reduction of CO to
hydrocarbons—of early biological systems.
Fig. 4. Activities of cofactor-deficient MoFe proteins in hydrocarbon for-
mation. Time-dependent formation of hydrocarbon products from the re-
duction of CN− (A) and CO (B) by ΔnifH (●), ΔnifBΔnifZ (▼), and ΔnifB (○)
MoFe proteins. Activities were expressed in nanomole total reduced carbon/
micromole protein, which were calculated based on the data in Fig. S2.
like structure), but also renders the P*-cluster easily accessible to
both substrates and electrons, an advantage not shared by the
well-shielded, intact P-cluster in ΔnifB MoFe protein in the Eu
(II)-DTPA–driven reactions. It should be noted, however, that
during ATP-dependent substrate turnover, MoFe protein nor-
mally undergoes a conformational change upon the docking of Fe
protein (1, 5), which may render the P-cluster more accessible to
substrates. Moreover, the redox change of P-cluster in this process
could be accompanied by a structural change of the cluster toward
a more “open” conformation, such as that observed in the case of
the two-electron oxidized P-cluster (P2+). Interestingly, theoreti-
cal calculations have used a split [Fe4S4] cluster model to explain
the spin state of P2+ (23), implying that a P*-cluster-like in-
termediate could be generated during the process of substrate
turnover.
Facilitated by its unique location, the ΔnifH MoFe protein-as-
sociated P*-cluster takes up a resemblance to a surface-exposed
Fe/S cluster that is capable of substrate reduction when a proper
reducing agent is supplied. Like some of the synthetic Fe/S-based
clusters, which can reduce C2H2 (24), H+ (25), and N2H4 (26) in
the presence of strong reductants, the P*-cluster can catalyze the
simple, two-electron reduction of the same set of substrates in
reactions driven by Eu(II)-DTPA. However, this cluster can also
reduce CN− and CO to hydrocarbons under ambient conditions,
a catalytic property that has been attributed so far only to the
isolated and protein-bound cofactors of nitrogenase (16, 27, 28).
Consistent with its surface location, the P*-cluster of ΔnifH MoFe
protein behaves more like the isolated cofactors in the Eu(II)-
DTPA–driven reactions of CN−- and CO-reduction (16), because
all these clusters (i) produce CH4 as the major product and (ii)
show a clear and consistent preference for the formation of 1-
alkenes over n-alkanes at alkene/alkane ratios of as high as 6.7/1
(Fig. 3). These observations point to a structural-functional anal-
ogy between the P-cluster and cofactor species of nitrogenases;
moreover, they strengthen the link between nitrogenase- and
Fischer–Tropsch-based reactions of hydrocarbon formation, as the
latter reaction also has a well-documented tendency toward excess
methanation and high ratios of alkene-to-alkane formation (29).
The catalytic properties of P*-cluster originate from its unique
structural arrangement. Previous XAS/EXAFS studies of ΔnifH
MoFe protein have shown that the P*-cluster consists of one
standard [Fe4S4] subcubane and one atypical [Fe4S4]-like sub-
cubane (8, 12). Thus, the P*-cluster in ΔnifH MoFe protein may
resemble the catalytic modules in Ni/Fe-containing carbon
monoxide dehydrogenase/acetyl-CoA synthase (30), siroheme-
type sulfite reductase (31), and iron-only hydrogenase (32), all of
Materials and Methods
Cell Growth and Protein Purification. A. vinelandii strains DJ1143, YM6A, and
DJ1165 were grown in 180 L batches in a 200 L New Brunswick fermentor
(New Brunswick Scientific) in Burke’s minimal medium supplemented with
2 mM ammonium acetate. The growth rate was measured by cell density at
436 nm using a Spectronic 20 Genesys (Spectronic Instruments). Cells were
harvested in the late exponential phase using a flow-through centrifugal
harvester (Cepa). The cell paste was washed with a buffer containing 50 mM
Tris-HCl (pH 8.0). Previously published methods were used for the purifica-
tion of His-tagged ΔnifB, ΔnifBΔnifZ, and ΔnifH MoFe proteins (7, 10).
EPR Spectroscopy. All EPR samples were prepared in a Vacuum Atmospheres
dry box at an oxygen level of less than 4 ppm. The ΔnifB, ΔnifBΔnifZ, and
ΔnifH MoFe proteins were incubated with 5 mM Eu(II)-DTPA for 15 min,
followed by the removal of Eu(II)-DTPA with a G25 column. Subsequently,
these proteins were incubated with 2 mM sodium dithionite (Na2S2O4) be-
fore being transferred to and frozen in the EPR sample tubes. All protein
samples contained 25 mM Tris-HCl (pH 8.0), 10% (vol/vol) glycerol, and 2 mM
Na2S2O4. Perpendicular-mode EPR spectra of these samples were recorded
by a Bruker ESP 300 Ez spectrometer (Bruker) interfaced with an Oxford
Instruments ESR-9002 liquid helium continuous-flow cryostat (Oxford
‡The L-cluster is a precursor form of FeMoco, which can be converted to a mature cofactor
on NifEN before it is delivered to its target location in the MoFe protein. XAS/EXAFS and
X-ray crystallographic studies show that the L-cluster is an 8Fe homolog to FeMoco,
which is free of Mo and homocitrate (35, 36). Moreover, biochemical analyses reveal
that the NifEN-bound L-cluster is catalytically active and capable of reducing C2H2 and
−
N3 to C2H4 and NH3, respectively, in a Fe protein/ATP-dependent reaction (37).
Lee et al.
PNAS
|
May 1, 2012
|
vol. 109
|
no. 18
|
6925

Instruments). All spectra were recorded at 10 K using a microwave frequency
of 9.62 GHz, a microwave power of 50 mW, a gain of 5 × 104, a modulation
frequency of 100 kHz, and a modulation amplitude of 5 G. A total of 10
scans were collected for each sample.
without the ΔnifH MoFe protein), H2 (under Ar) and C2H4 (under C2H2) were
produced at 27% and 9%, respectively, of those in the complete assays (i.e.,
with the ΔnifH MoFe protein). The specific activities of H2 and C2H4 formation
were reported after subtracting the background activities of the control
assays from those of the complete assays. No activities were observed in the
cases of other substrates in the absence of ΔnifH MoFe protein.
Activity Assays in Eu(II)-DTPA. Eu(II)-DTPA was prepared as described pre-
viously (14). All assays contained, in a total volume of 25 mL, 25 mM Tris-HCl
(pH 7.8), 5 mM Eu-DTPA, and 80 mg of ΔnifH, ΔnifBΔnifZ, or ΔnifB MoFe
protein. The reaction of C2H2-, C2H4-, or CO-reduction was carried out in a gas
atmosphere of 100% (vol/vol) C2H2, C2H4, or CO; whereas the reaction of
CO2-reduction was carried out in a gas atmosphere of 0.05% CO2/99.95%
(vol/vol) Ar and maintained at pH 7.8, with the addition of 0.5 M NaHCO3
to the assay. All other assays were carried out in a gas atmosphere of 100%
(vol/vol) Ar, with the addition of 120 mM NaCN and 100 mM NaN3, re-
spectively, to the reactions of CN−- and N3−-reduction. Production of H2 was
analyzed as described previously (39), and formation of NH3 was determined
by an HPLC fluorescence method (40). Alkene and alkane products CH4, C2H4,
C2H6, C3H6, C3H8, 1-C4H8, n-C4H10, 1-C5H10, n-C5H12, 1-C6H12, n-C6H14, and
n-C7H16 were analyzed by GC-FID on an activated alumina column (Grace),
which was held at 40 °C for 2 min, increased to 200 °C at a rate of 10 °C/min,
and held for an additional 2 min at 200 °C. These 12 hydrocarbons were
quantified as described previously (16) and their detection thresholds were
(in nanomole product/micromole protein): 1.1 (CH4), 1.3 (C2H4), 1.3 (C2H6), 1.5
(C3H6), 1.3 (C3H8), 1.4 (1-C4H8), 1.4 (n-C4H10), 3.1 (1-C5H10), 3.7 (n-C5H12), 6.1 (1-
C6H12), 5.8 (n-C6H14), and 7.0 (n-C7H16), respectively. In control assays (i.e.,
GC-MS Analysis. Hydrocarbon products were identified by GC-MS using
a Hewlett-Packard 5890 GC and a 5972 MSD. The identities of CH4, C2H4, C2H6,
C3H6, C3H8, 1-C4H8, n-C4H10, 1-C5H10, n-C5H12, 1-C6H12, n-C6H14, and n-C7H16
were confirmed by using a Scott standard gas mixture of n-alkanes and 1-
alkenes. A total of 50 μL of gas was injected into a split/splitless injector op-
erated at 125 °C in splitless mode. A 1-mm ID liner was used to optimize the
sensitivity of gas detection. The separation of gaseous products was achieved
by using a Restek PLOT-QS capillary column (0.320 mm ID × 30 m length),
which was held at 40 °C for one min, heated to 220 °C at a rate of 10 °C/min,
and held for another 3 min at 220 °C. Carrier gas (He) was passed through the
column at a rate of 1.0 mL/min. The mass spectrometer was operated in
electron impact ionization (EII) and selected ion monitoring mode.
ACKNOWLEDGMENTS. We thank Prof. Douglas Rees [California Institute of
Technology(CIT)] and Dr. Nathan Dalleska (CIT) for their kind help with GC-
MS analysis. This work was supported by Herman Frasch Foundation Grant
617-HF07 and National Institutes of Health Grant GM-67626 (to M.W.R.).
1. Burgess BK, Lowe DJ (1996) Mechanism of molybdenum nitrogenase. Chem Rev 96:
2983–3012.
2. Einsle O, et al. (2002) Nitrogenase MoFe-protein at 1.16 A resolution: A central ligand
in the FeMo-cofactor. Science 297:1696–1700.
3. Lancaster KM, et al. (2011) X-ray emission spectroscopy evidences a central carbon in
the nitrogenase iron-molybdenum cofactor. Science 334:974–977.
4. Spatzal T, et al. (2011) Evidence for interstitial carbon in nitrogenase FeMo cofactor.
Science 334:940.
5. Schindelin H, Kisker C, Schlessman JL, Howard JB, Rees DC (1997) Structure of ADP x
AIF4(-)-stabilized nitrogenase complex and its implications for signal transduction.
Nature 387:370–376.
6. Schmid B, et al. (2002) Structure of a cofactor-deficient nitrogenase MoFe protein.
Science 296:352–356.
7. Ribbe MW, Hu Y, Guo M, Schmid B, Burgess BK (2002) The FeMoco-deficient MoFe
protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-
cluster features. J Biol Chem 277:23469–23476.
8. Corbett MC, et al. (2004) Comparison of iron-molybdenum cofactor-deficient nitro-
genase MoFe proteins by X-ray absorption spectroscopy: implications for P-cluster
biosynthesis. J Biol Chem 279:28276–28282.
9. Broach RB, et al. (2006) Variable-temperature, variable-field magnetic circular di-
chroism spectroscopic study of the metal clusters in the DeltanifB and DeltanifH mofe
proteins of nitrogenase from Azotobacter vinelandii. Biochemistry 45:15039–15048.
10. Hu Y, Fay AW, Dos Santos PC, Naderi F, Ribbe MW (2004) Characterization of Azo-
tobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein as-
sembly. J Biol Chem 279:54963–54971.
21. Seefeldt LC, Rasche ME, Ensign SA (1995) Carbonyl sulfide and carbon dioxide as new
substrates, and carbon disulfide as a new inhibitor, of nitrogenase. Biochemistry 34:
5382–5389.
22. Corbett MC, et al. (2007) Conformational differences between Azotobacter vinelandii
nitrogenase MoFe proteins as studied by small-angle X-ray scattering. Biochemistry
46:8066–8074.
23. Torres RA, Lovell T, Noodleman L, Case DA (2003) Density functional and reduction
potential calculations of Fe4S4 clusters. J Am Chem Soc 125:1923–1936.
24. McMillan RS, Renaud J, Reynolds JG, Holm RH (1979) Biologically related iron-sulfur
clusters as reaction centers. Reduction of acetylene to ethylene in systems based on
[Fe4S4(SR)4]3-. J Inorg Biochem 11:213–227.
25. Ghosh S, et al. (2011) Bio-inspired hydrogenase models: Mixed-valence triion com-
plexes as proton reduction catalysts. Chem Commun (Camb) 47:11222–11224.
26. Malinak SM, Demadis KD, Coucouvanis D (1995) Catalytic reduction of hydrazine of
ammonia by the VFe3S4 cubanes—Further evidence for the direct involvement of the
heterometal in the reduction of nitrogenase substrates and possible relevance of the
vanadium nitrogenase. J Am Chem Soc 117:3126–3133.
27. Lee CC, Hu Y, Ribbe MW (2010) Vanadium nitrogenase reduces CO. Science 329:642.
28. Hu Y, Lee CC, Ribbe MW (2011) Extending the carbon chain: Hydrocarbon formation
catalyzed by vanadium/molybdenum nitrogenases. Science 333:753–755.
29. Santilli DS, Castner DG (1989) Mechanism of chain growth and product formation for
the Fischer–Tropsch reaction over iron catalysts. Energy Fuels 3:8–15.
30. Doukov TI, Iverson TM, Seravalli J, Ragsdale SW, Drennan CL (2002) A Ni-Fe-Cu center
in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase. Science 298:
567–572.
11. Cotton MS, et al. (2009) VTVH-MCD study of the Δ nifB Δ nifZ MoFe protein from
Azotobacter vinelandii. J Am Chem Soc 131:4558–4559.
12. Lee CC, et al. (2009) Stepwise formation of P-cluster in nitrogenase MoFe protein.
Proc Natl Acad Sci USA 106:18474–18478.
31. Oliveira TF, et al. (2008) The crystal structure of Desulfovibrio vulgaris dissimilatory
sulfite reductase bound to DsrC provides novel insights into the mechanism of sulfate
respiration. J Biol Chem 283:34141–34149.
13. Hu Y, Fay AW, Lee CC, Ribbe MW (2007) P-cluster maturation on nitrogenase MoFe
protein. Proc Natl Acad Sci USA 104:10424–10429.
14. Vincent KA, et al. (2003) Instantaneous, stoichiometric generation of powerfully re-
ducing states of protein active sites using Eu(II) and polyaminocarboxylate ligands.
Chem Commun (Camb) 21:2590–2591.
15. Danyal K, et al. (2010) Uncoupling nitrogenase: Catalytic reduction of hydrazine to
ammonia by a MoFe protein in the absence of Fe protein-ATP. J Am Chem Soc 132:
13197–13199.
16. Lee CC, Hu Y, Ribbe MW (2012) ATP-independent formation of hydrocarbons cata-
lyzed by isolated nitrogenase cofactors. Angew Chem Int Ed Engl 51:1947–1949.
17. Zhang Y, Holm RH (2004) Structural conversions of molybdenum-iron-sulfur edge-
bridged double cubanes and P(n)-type clusters topologically related to the nitroge-
nase P-cluster. Inorg Chem 43:674–682.
18. Ohki Y, Ikagawa Y, Tatsumi K (2007) Synthesis of new [8Fe-7S] clusters: A topological
link between the core structures of P-cluster, FeMo-co, and FeFe-co of nitrogenases. J
Am Chem Soc 129:10457–10465.
19. Pickett CJ, et al. (2004) Synergic binding of carbon monoxide and cyanide to the FeMo
cofactor of nitrogenase: Relic chemistry of an ancient enzyme? Chemistry 10:
4770–4776.
32. Peters JW, Lanzilotta WN, Lemon BJ, Seefeldt LC (1998) X-ray crystal structure of the
Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution.
Science 282:1853–1858.
33. Beinert H (2000) Iron-sulfur proteins: Ancient structures, still full of surprises. J Biol
Inorg Chem 5:2–15.
34. Beinert H, Holm RH, Münck E (1997) Iron-sulfur clusters: Nature’s modular, multi-
purpose structures. Science 277:653–659.
35. Corbett MC, et al. (2006) Structural insights into a protein-bound iron-molybdenum
cofactor precursor. Proc Natl Acad Sci USA 103:1238–1243.
36. Kaiser JT, Hu Y, Wiig JA, Rees DC, Ribbe MW (2011) Structure of precursor-bound
NifEN: A nitrogenase FeMo cofactor maturase/insertase. Science 331:91–94.
37. Hu Y, et al. (2009) Catalytic activities of NifEN: Implications for nitrogenase evolution
and mechanism. Proc Natl Acad Sci USA 106:16962–16966.
38. Zhang Y, Zuo JL, Zhou HC, Holm RH (2002) Rearrangement of symmetrical dicubane
clusters into topological analogues of the P cluster of nitrogenase: Nature’s choice? J
Am Chem Soc 124:14292–14293.
39. Gavini N, Burgess BK (1992) FeMo cofactor synthesis by a nifH mutant with altered
MgATP reactivity. J Biol Chem 267:21179–21186.
40. Corbin JL (1984) Liquid chromatographic-fluorescence determination of ammonia
from nitrogenase reactions: A 2-min assay. Appl Environ Microbiol 47:1027–1030.
20. Carnahan EM, Protasiewicz JD, Lippard SJ (1993) 15 years of reductive coupling: What
have we learned? Acc Chem Res 26:90–97.
6926
|
www.pnas.org/cgi/doi/10.1073/pnas.1202429109
Lee et al.