A Comparative Analysis of the CO-Reducing Activities of MoFe Proteins Containing Mo- and V-Nitrogenase Cofactors

Article abstract of DOI:10.1002/cbic.201800035

The Mo and V nitrogenases are structurally homologous yet catalytically distinct in their abilities to reduce CO to hydrocarbons. Here we report a comparative analysis of the CO-reducing activities of the Mo- and V-nitrogenase cofactors (i.e., the M and V clusters) upon insertion of the respective cofactor into the same, cofactor-deficient MoFe protein scaffold. Our data reveal a combined contribution from the protein environment and cofactor properties to the reactivity of nitrogenase toward CO, thus laying a foundation for further mechanistic investigation of the enzymatic CO reduction, while suggesting the potential of targeting both the protein scaffold and the cofactor species for nitrogenase-based applications in the future.

Full text of DOI:10.1002/cbic.201800035

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Accepted Article
Title: A comparative analysis of the CO-reducing activities of MoFe
proteins containing the Mo- and V-nitrogenase cofactors
Authors: Markus Walter Ribbe, Chi Chung Lee, Kazuki Tanifuji, Megan
Newcomb, Jasper Liedtke, and Yilin Hu
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To be cited as: ChemBioChem 10.1002/cbic.201800035
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A comparative analysis of the CO-reducing activities of MoFe
proteins containing the Mo- and V-nitrogenase cofactors
Chi Chung Lee,[a] Kazuki Tanifuji,[a] Megan Newcomb,[a,b] Jasper Liedtke,[a] Yilin Hu,*[a] and Markus W.
Ribbe*[a,b]
Abstract: The Mo- and V-nitrogenases are structurally homologous
yet catalytically distinct in their abilities to reduce CO to hydrocarbons.
Here we report a comparative analysis of the CO-reducing activities
of the Mo- and V-nitrogenase cofactors (i.e., the M- and V-clusters)
upon insertion of the respective cofactor into the same, cofactor-
also closely resemble each other in composition and
architecture,[10,11] particularly with regard to the presence of a
certain sulfide at the ‘belt’ region of the cofactor that is displaced
upon CO binding.[12] The observation of disparate CO-reducing
activities of the two highly homologous nitrogenases, therefore,
raises the question of whether the protein environment or the
cofactor itself is responsible for this reactivity. Clarification of this
point is important, as it is prerequisite to any further mechanistic
investigation of the enzymatic CO reduction by nitrogenase.
Moreover, a better understanding of the structural determinants
of the CO-reducing ability of nitrogenase is the first step towards
future engineering of artificial enzymes that efficiently convert CO
or CO2 to hydrocarbons.
deficient MoFe protein scaffold. Our data reveal
a combined
contribution of protein environment and cofactor properties to the
reactivity of nitrogenase toward CO, laying a foundation for further
mechanistic investigation of the enzymatic CO reduction while
suggesting the potential of targeting both the protein scaffold and the
cofactor species for nitrogenase-based applications in the future.
Nitrogenase is a complex metalloenzyme catalyzing the ambient
reduction of a wide range of substrates. The best-known function
of nitrogenase is the reduction of nitrogen (N2) to ammonia (NH3)
in a process that parallels the industrial Haber-Bosch process,
which supplies the essential nitrogen element for the entire food
chain.[1-3] In recent years, nitrogenase has been shown to reduce
small C1 substrates, such as carbon monoxide (CO), carbon
dioxide (CO2) and cyanide (CN-), to hydrocarbons, in a process
that mirrors the industrial Fischer-Tropsch process, thereby
adding another important function to its catalytic repertoire.[4-7]
The activities of nitrogenase in these reactions are enabled by a
two-component system and, in the cases of the homologous
molybdenum (Mo)- and vanadium (V)-nitrogenases, a reductase
component (collectively termed the Fe protein) is used to donate
electrons in an ATP-dependent manner to the cofactor of the
catalytic component (designated the MoFe and VFe protein,
respectively), where substrate reduction occurs.[8-10] Interestingly,
the two nitrogenases display highly differential reactivities toward
CO, with the Mo-nitrogenase generating hydrocarbons at ~0.02
nmol/nmol protein/min, and the V-nitrogenase at 16 nmol/nmol
protein/min—approximately 680-fold higher than its Mo-
counterpart.[5] Such a discrepancy in activity is surprising, as not
only are the protein structures of the catalytic components of the
two systems highly homologous to each other, their respective
cofactor centers (designated the M- and V-cluster, respectively)
One approach to address this question is to insert the M- and V-
clusters into the same, cofactor-deficient MoFe protein scaffold
and compare the activities of these reconstituted proteins with
each other, and with those of the native MoFe protein (designated
NifDK) and VFe protein (designated VnfDGK). As observed in the
cases of the native NifDK and VnfDGK, NifDK reconstituted with
the M-cluster (designated NifDKM) or the V-cluster (designated
NifDKV) is capable of reducing CO to hydrocarbons in a H2O-
based buffer (Figure 1A, B). Compared to the native NifDK, both
NifDKM and NifDKV generate the same C2 (C2H4, C2H6) and C3
(C3H6, C3H8) hydrocarbons at comparable rates (Figure 1A, B, ,
, ). Compared to the native VnfDGK, however, NifDKM and
NifDKV do not generate detectable amounts of C1 (CH4) and C4
(C4H8, C4H10) hydrocarbons as VnfDGK does under the same
experimental conditions, and the overall rates of hydrocarbon
production by both reconstituted NifDK proteins are 640-fold
lower than that by VnfDGK (Figure 1A, B, , , ). Upon
substitution of D2O for H2O in the reaction buffer, NifDKM and
NifDKV appear to follow the pattern of the native NifDK again in
terms of changes in product range and reaction rate (Figure 1C,
D).[5,13] Like NifDK, NifDKM and NifDKV display the ability to form
detectable amounts of C4 hydrocarbons (C4H8, C4H10), as well as
a substantial increase of the reaction rate by 24-fold in the
presence of D2O (Figure 1C, D, , , ). The rate increases in
the cases of NifDKM and NifDKV (Figure 1D vs. B, , ) are
similar to that of the native NifDK (Figure 1D, ) but contrast that
of the native VnfDGK (Figure 1D vs. B, ), which is only
increased by 1.1-fold upon substitution of D2O for H2O. Taken
[a]
[b]
Dr. C. C. Lee, Dr. K. Tanifuji, Megan Newcomb, Jasper Liedtke,
Prof. Y. Hu, Prof. M. W. Ribbe
Department of Molecular Biology and Biochemistry
University of California, Irvine
2230/2236 McGaugh Hall, Irvine, CA 92697-3900 (USA)
E-mail: yilinh@uci.edu; mribbe@uci.edu
Megan Newcomb, Prof. M. W. Ribbe
Department of Chemistry
University of California, Irvine
2236 McGaugh Hall, Irvine, CA 92697-2025 (USA)
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Figure 1. Specific activities of hydrocarbon formation from CO reduction by  NifDK,  VnfDGK,  NifDKM, and  NifDKV in the presence of H2O (A, B) and D2O
(C, D). Shown are bar charts of the individual activities of CH4-, C2H4-, C2H6-, C3H6-, C3H8-, C4H8-, and C4H10-formation (A, C) and stacked bar charts of the sum of
the individual activities (B, D) of each protein species. The colors of the stacked bars (B, D) correspond to those of the individual bars (A, C). Specific activities are
expressed as nmol hydrocarbon (HC)/nmol protein/min and were determined as described in Materials in Methods. Data are presented as mean ± SD (N = 4
repetitions of each experiment).
together, these observations suggest that the protein environment
plays a key role on the CO-reducing activity of nitrogenase, as the
M- and V-clusters seem to be ‘normalized’ in terms of their
reactivities toward CO upon insertion into the same protein
scaffold, NifDK.
A closer examination of the C2 product distributions of NifDKM and
NifDKV in D2O and H2O provides further support for this argument.
The ratio between the percentages of the unsaturated C2 product
(C2H4) formed by VnfDGK in D2O and H2O is lower than that by
NifDK (Figure 3A,  vs. ), whereas the ratio between the
percentages of the saturated C2 product (C2H6) formed by
VnfDGK in D2O and H2O is much higher than that by NifDK
(Figure 3B,  vs. ), suggesting a shift of C2 product formation
toward the saturated products in the case of VnfDGK upon
substitution of D2O for H2O. The same trend is observed when
NifDKV and NifDKM are compared for the formation of unsaturated
(Figure 3A,  vs. ) and saturated (Figure 3B,  vs. ) C2
products in D2O and H2O, highlighting the discrete catalytic
characteristic of the V-cluster that renders the product pattern of
NifDKV similar to that of VnfDGK in this reaction. Interestingly, the
V-cluster seems to be specifically tailored for the reaction of CO
reduction. It has been reported that, compared to the native
NifDK, the native VnfDGK displays decreased activities of C2H2-,
H+- and N2-reduction, but a considerably higher activity of CO
reduction.[4,5,8] In analogy to this discrepancy, NifDKV reduces
C2H2, H+, and N2 at 18%, 19% and 27%, respectively, of the
activities of NifDK (Figure 3C, gray bars); yet, it generates
hydrocarbons at approximately the same level as NifDKM in the
presence of both H2O (Figure 3C, blue bar) and D2O (Figure 3D,
blue bar). This observation points to CO as a preferred substrate
for the V-cluster; only in the case of NifDKV, the ability of the V-
cluster to reduce CO is ‘capped’ by the relatively inefficient protein
scaffold, NifDK. These results suggest a combined impact of
There are, however, similarities between the reconstituted
proteins and the native VnfDGK. In the presence of D2O, the C1
product (CH4) remains undetectable in the reaction catalyzed by
NifDK (Figure 1C, ), yet it appears in the product profiles of
NifDKM (Figure 1C, ) and NifDKV (Figure 1C, ), consistent with
an increased formation of this product by VnfDGK upon
substitution of D2O for H2O (Figure 1C, ). Of the two
reconstituted NifDK proteins, NifDKV displays
a
closer
resemblance than NifDKM to the native VnfDGK. Notably, in
analogy to the ability of VnfDGK to generate considerably more
C2H4 than NifDK (Figure 2A,  vs. ), NifDKV generates ~12%
more C2H4 than NifDKM from CO reduction (Figure 2A,  vs. ).
Moreover, upon substitution of D2O for H2O, the ~5% reduction of
C2H4 formation by VnfDGK (Figure 2A vs. B, ) is paralleled by
the ~6% reduction of C2H4 formation by NifDKV (Figure 2A vs. B,
); whereas the ~19% increase of C2H4 formation by NifDK
(Figure 2A vs. B, ) is paralleled by the ~10% increase of C2H4
formation by NifDKM (Figure 2A vs. B, ). These results point to
a contribution of the distinct characteristics the cofactor species
(i.e., the M- or V-cluster) to the reactivity of nitrogenase toward
CO.
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Figure 2. Distribution of hydrocarbons formed from CO reduction by  NifDK,  VnfDGK,  NifDKM, and  NifDKV in the presence of H2O (A) and D2O (B). The
total amount of hydrocarbons detected in the case of each protein was set as 100%. The color code for hydrocarbons is shown on the right.
cofactor properties and protein environment on the CO reactivity
of nitrogenase.
different heterometals (i.e., V or Mo) in these clusters, as it has
been reported that synthetic V-compounds have higher capacities
than their Mo-counterparts in reductively coupling two CO
moieties into functionalized acetylene ligands.[17] Other than the
difference in heterometals, the recently reported crystal structure
of VnfDGK reveals the displacement of a ‘belt’ sulfide of the V-
cluster by a carbonate moiety,[18] a feature that is absent from the
structure of the M-cluster in NifDK.[19] Such a difference could very
well account for the difference between the catalytic properties of
the two cofactors. However, the origin and catalytic relevance of
carbonate appear unclear as of yet, particularly given the earlier
report of the XAS/EXAFS-derived solution structure of the V-
cluster that is free of carbonate.[11] While the underlying reason
behind the different catalytic properties of the cofactors is yet to
Efforts in understanding nitrogenase catalysis have been focused
primarily on the properties of the cofactors.[14-16] which underscore
the central roles of M- and V- clusters in nitrogenase function and
the argument that the observed catalytic differences between Mo-
and V nitrogenases, particularly their differential CO-reducing
activities, originate from the different properties of these two
metallocofactors. In the current study, the apparent preference of
NifDKV for CO over other substrates (C2H2, H+ and N2), as
compared to that of NifDKM (see Figure 3C, D), showcases a
better efficiency of the V-cluster in CO reduction than the M-
cluster. Such a discrepancy could originate from the presence of
Figure 3. Ratios between percentages of C2H4- (A) and C2H6-formation (B) in the presence of D2O and H2O in reactions catalyzed by  NifDK,  VnfDGK, 
NifDKM, and  NifDKV; and activities of NifDKV relative to those of NifDKM in H2O (C) and D2O (D). The ratios were calculated based on the activities shown in Figure
1 (A, B). Activities of  C2H4 formation under C2H2,  H2 formation under Ar,  NH3 formation under N2,  H2 formation under CO), and  hydrocarbon formation
under CO are shown in C. Activities  H2 formation under CO and  hydrocarbon formation under CO are shown in D. Data in C and D are presented as mean ±
SD (N = 4 repetitions of each experiment). HCs, hydrocarbons.
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