.
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whereas the P-cluster of the V nitrogenase consists of a pair of
[Fe4S4]-like clusters (Figure 1B).[5,7–9] Likewise, despite a strik-
ing homology in structure, the cofactors of the Mo and
V nitrogenases are distinguishable not only by the incorpo-
rated heterometals, but also by their electronic properties
(Figure 1C).[10] The differences between the metal clusters in
the Mo and V nitrogenases underline the differences in the
catalytic behavior of these homologous enzymes. It has been
documented that the V nitrogenase is less efficient than its
Mo counterpart in terms of N2 reduction; yet, this nitrogenase
can reduce C2H2 to ethane (C2H6), a catalytic activity not
observed in the case of the Mo nitrogenase.[6,8] Perhaps the
biggest discrepancy between the catalytic properties of the
two nitrogenases is their ability to reduce CO to hydro-
carbons, with the V nitrogenase showing an overall activity
that is nearly 700 times higher than that of its Mo counter-
part.[11,12] This observation has prompted us to conduct
a comparative study with the Mo and V nitrogenases to
address 1) whether the two nitrogenases can also reduce CO2
to hydrocarbons, and 2) whether they have the same discrep-
ancy in their activities to generate hydrocarbons from this
substrate.
CO2 upon substitution of D2O for H2O, reaching a maximum
increase of activity at 120 minutes (Figure 2A). Apart from
CO, CH4, which is a further reduced C1 product, could be
detected in reaction mixtures in the presence of the Mo and
V nitrogenases when CO2 was supplied as a substrate (Fig-
ure 2B). However, when H2O was replaced by D2O, the
activity of CH4 formation by the V nitrogenase increased
from 0 to a maximum of 22.2 nmol per mmol of protein
*
*
(Figure 2B, vs. ), whereas the activity of CH4 formation by
the Mo nitrogenase decreased from a maximum of 7.3 nmol
!
!
per mmol of protein to 0 (Figure 2B, vs. ). Such a disparate
D2O effect implies a difference in the routes to CH4
formation taken by the two nitrogenases.
The difference between the Vand Mo nitrogenases in CO2
reduction is further illustrated by the difference in their
À
abilities to use CO2 as a substrate to form C C bonds. In the
presence of H2O, little or no C2 product was detected during
CO2 reduction by either the Mo or the V nitrogenase
!
*
(Figure 2C and D,
and ). In the presence of D2O,
*
*
however, C2D4 (Figure 2C, ) and C2D6 (Figure 2D, ) were
detected as products of CO2 reduction by the V nitrogenase,
whereas these C2 products were hardly detectable in the same
reaction catalyzed by the Mo nitrogenase (Figure 2C and D,
Consistent with an earlier report,[13] the Mo nitrogenase
can reduce CO2 to CO (Figure 2A, triangles). Like its
Mo counterpart, the V nitrogenase can also catalyze the
reduction of CO2 to CO (Figure 2A, circles) in an ATP-
!
). Thus, as was observed in the case of CH4 formation, there
was a clear increase in the activities of C2D4 and C2D6
formation by the V nitrogenase upon substitution of D2O
for H2O, whereas these activities remained marginal in the
reaction catalyzed by the Mo nitrogenase following such
a substitution. Moreover, like the formation of CH4, the
formation of C2 products by the V nitrogenase was ATP-
dependent, as C2D4 and C2D6 could not be detected in the
absence of ATP (Supporting Information, Figure S1).
GC–MS analysis supplied further evidence for the differ-
ences between the Mo and V nitrogenases in hydrocarbon
formation from CO2. When 12CO2 was replaced by 13CO2,
13CD4 could be detected in the V nitrogenase-catalyzed
reaction in D2O (Figure 3B); however, 13CH4 was absent
from the Mo nitrogenase-catalyzed reaction in H2O (Fig-
ure 3A). This observation confirmed CO2 as the carbon
source for CD4 generated by the V nitrogenase while
suggesting a different carbon source for the same C1 product
generated by the Mo nitrogenase. Aside from CD4, CO2 also
gave rise to the C2 products in the V nitrogenase-catalyzed
reaction, as 13C2D4 (Figure 3C) and 13C2D6 (Figure 3D) could
be detected in the presence of D2O upon substitution of 13CO2
for 12CO2. Together, the GC–MS and activity data highlight
the difference between the reactions of CO2 reduction by the
V and Mo nitrogenases, showing the ability of the V nitroge-
nase to form C1 and C2 hydrocarbons along with CO and the
inability of its Mo counterpart to generate products other
than CO under these experimental conditions. Given the
previous observation that the V nitrogenase can reduce CO to
hydrocarbons,[11,12] the co-production of CO and hydrocar-
bons by this enzyme as products of CO2 reduction raises the
relevant question of whether it is the CO2-derived CO that
gives rise to the hydrocarbon products.
Figure 2. Product formation by the Mo and V nitrogenases in the
presence of CO2. Time-dependent formation of CO (A), CH4 (B), C2H4
!
!
,
(C), and C2H6 (D) by the Mo nitrogenase in H2O ( , b) or D2O (
*
*
c) and by the V nitrogenase in H2O ( , b) or D2O ( , c).
Data are presented as meanÆSD (N=3) after background correction.
dependent reaction (Figure S1) using dithionite (20 mm) at
pH 8.5. The two nitrogenases displayed comparable efficien-
cies in H2O-based reactions, forming approximately the same
amount of CO from CO2 over a time period of 180 minutes
(Figure 2A). Moreover, both nitrogenases exhibited roughly
the same increase in activity for the formation of CO from
This question can be addressed by directly supplying CO
to the V nitrogenase in a concentration simulating the
maximum concentration of CO achieved in the “equilibrated
2
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
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