the two pathways diverge between the states E 4 and E 6 , and then converge back into
the same route for the reduction and the release of the final product ammonia (steps
E 7 and E 8 ) (Fig. 6a) [9, 81, 82]. In the first step of the distal pathway, N 2 binds to an
Fe center of the M-cluster in the E 4 (4H) state and the subsequent reduction occurs
by the preferential addition of protons and electrons to the N atom that is distal to
the coordinated metal center. This allows for the formation of a bound hydrazido
(Fe=N–NH 2 ) species at the E 4 state [4, 81]. In the next step (E 5 ), the addition of a
proton and an electron, again at the distal N atom, results in the release of one
equivalent of NH 3 , and leaves a terminal iron nitride (FeN) species behind. The
reduction/protonation of this species in the E 6 state leads to the formation of an iron
imido (Fe=NH) moiety which undergoes further electron/proton transfer and, via an
iron amido (M-NH 2 ) intermediate (state E 7 ), is finally transformed into free NH 3
(state E 8 ) (Fig. 6a). In contrast, the alternating pathway starts at the N 2 -bound E 4
state with the addition of one proton/electron per nitrogen atom. This generates a
diazene-bound species (Fe–HN=NH) as the E 4 (2N2H) intermediate. The following
two electron/proton addition steps (E 5 and E 6 ) are also proposed to occur alternatingly on both N atoms, thus producing a hydrazine species (M–H 2 N–NH 2 ) as the
E 6 state [9, 82]. Further reduction/protonation releases NH 3 , generating the same
amido-bound species as described in the distal pathway in the E 7 state, followed by
the reduction and release of the second equivalent of NH 3 in the final step in E 8
(Fig. 6a).
There is currently no evidence that would conclusively prove the validity of
either pathway, although more recent biochemical and spectroscopic findings favor
the alternating pathway. Support for the distal N 2 reduction pathway mainly stem
from the early work on the synthetic, N 2 -reducing, mononuclear Mo complexes by
Chatt and Schrock [98, 99]. In addition, the detection of hydrazine formation upon
acid- and base-quenching during the pre-steady-state turnover of N 2 , was initially
viewed as support for the distal pathway by Lowe and others, as this observation
was interpreted as the release of the two-electron-reduced hydrazido intermediate in
stage E 4 proposed in the distal pathway [100]. However, it was later realized that
the same result can also be interpreted as evidence for the presence of the hydrazine
bound intermediate which is proposed to be present in the E 6 state of the alternating
pathway [9, 82]. On the other hand, biochemical studies revealed that hydrazine
(N 2 H 4 ) and diazene (N 2 H 2 ) can be reduced to ammonia by nitrogenase, indicating
that these nitrogenous species could be on-route intermediates of N 2 reduction
along the alternating pathway [101, 102]. Subsequent spectroscopic studies identified the presence of an intermediate that is generated in the reduction of N 2 H 4 ,
N 2 H 2 and N 2 [103]. This suggests the reduction of these species might share the
same route, hence strongly favoring the alternating pathway. However, such
intermediate was also found to only consist of one nitrogen, thus likely representing
E 7 or E 8 states, which are shared by both the distal and the alternating pathway
[104, 105]. In this light, future attempts to accumulate and characterize
nitrogen-bound E 4 , E 5 or E 6 will likely provide the definitive answer for this
longstanding question in the N 2 reduction mechanism.
Assembly and Function of Nitrogenase
175
the same route for the reduction and the release of the final product ammonia (steps
E 7 and E 8 ) (Fig. 6a) [9, 81, 82]. In the first step of the distal pathway, N 2 binds to an
Fe center of the M-cluster in the E 4 (4H) state and the subsequent reduction occurs
by the preferential addition of protons and electrons to the N atom that is distal to
the coordinated metal center. This allows for the formation of a bound hydrazido
(Fe=N–NH 2 ) species at the E 4 state [4, 81]. In the next step (E 5 ), the addition of a
proton and an electron, again at the distal N atom, results in the release of one
equivalent of NH 3 , and leaves a terminal iron nitride (FeN) species behind. The
reduction/protonation of this species in the E 6 state leads to the formation of an iron
imido (Fe=NH) moiety which undergoes further electron/proton transfer and, via an
iron amido (M-NH 2 ) intermediate (state E 7 ), is finally transformed into free NH 3
(state E 8 ) (Fig. 6a). In contrast, the alternating pathway starts at the N 2 -bound E 4
state with the addition of one proton/electron per nitrogen atom. This generates a
diazene-bound species (Fe–HN=NH) as the E 4 (2N2H) intermediate. The following
two electron/proton addition steps (E 5 and E 6 ) are also proposed to occur alternatingly on both N atoms, thus producing a hydrazine species (M–H 2 N–NH 2 ) as the
E 6 state [9, 82]. Further reduction/protonation releases NH 3 , generating the same
amido-bound species as described in the distal pathway in the E 7 state, followed by
the reduction and release of the second equivalent of NH 3 in the final step in E 8
(Fig. 6a).
There is currently no evidence that would conclusively prove the validity of
either pathway, although more recent biochemical and spectroscopic findings favor
the alternating pathway. Support for the distal N 2 reduction pathway mainly stem
from the early work on the synthetic, N 2 -reducing, mononuclear Mo complexes by
Chatt and Schrock [98, 99]. In addition, the detection of hydrazine formation upon
acid- and base-quenching during the pre-steady-state turnover of N 2 , was initially
viewed as support for the distal pathway by Lowe and others, as this observation
was interpreted as the release of the two-electron-reduced hydrazido intermediate in
stage E 4 proposed in the distal pathway [100]. However, it was later realized that
the same result can also be interpreted as evidence for the presence of the hydrazine
bound intermediate which is proposed to be present in the E 6 state of the alternating
pathway [9, 82]. On the other hand, biochemical studies revealed that hydrazine
(N 2 H 4 ) and diazene (N 2 H 2 ) can be reduced to ammonia by nitrogenase, indicating
that these nitrogenous species could be on-route intermediates of N 2 reduction
along the alternating pathway [101, 102]. Subsequent spectroscopic studies identified the presence of an intermediate that is generated in the reduction of N 2 H 4 ,
N 2 H 2 and N 2 [103]. This suggests the reduction of these species might share the
same route, hence strongly favoring the alternating pathway. However, such
intermediate was also found to only consist of one nitrogen, thus likely representing
E 7 or E 8 states, which are shared by both the distal and the alternating pathway
[104, 105]. In this light, future attempts to accumulate and characterize
nitrogen-bound E 4 , E 5 or E 6 will likely provide the definitive answer for this
longstanding question in the N 2 reduction mechanism.
Assembly and Function of Nitrogenase
175