intermediate led to the release of two equivalents of H 2 , thereby demonstrating the
accumulation of four electrons/protons on M-cluster [85–87, 89].
1,2 H,
57 Fe, and
95 Mo ENDOR experiments suggested that the spectroscopic features of this intermediate can be interpreted as an Fe-bound hydride fragment, plausibly in a l 2 -
bridging conformation (Fe–H–Fe) [85–87, 89, 90]. This finding led to the proposal
that the E 4 state of NifDK, which is represented by the freeze-trapped intermediate
mentioned above, is an M-cluster having two l 2 -hydride ligands bridged between
two different pairs of Fe atoms with two protons bound elsewhere on the cluster.
This proposed E 4 state, also termed E 4 (4H), then formed the basis for the reductive
elimination mechanism of N 2 binding. Its structure was further explored in recent
theoretical study, which suggests that the cleavage of an Fe-S bond, if not the entire
removal of the belt sulfur atom, is an energetically feasible reaction for the
M-cluster in the E 4 state [91, 92].
The two hydrides loaded on the M-cluster are thought to be the driving force for
N 2 binding. Studies on synthetic compounds suggested, the binding of N 2 can be
promoted by the reductive elimination of H 2 [93]. Therefore, the M-cluster in the
E 4 (4H) state is similarly thought to reductively eliminate one equivalent of H 2 in
order to enable the transient binding of N 2. This is followed by immediate reduction, resulting in a proposed bound N 2 H 2 species (known as the E 4 (2N2H) state) [9,
82]. It should be mentioned that the experimentally observed stoichiometry of one
H 2 per reduced N 2 can then be easily explained by the reductive elimination
mechanism because in this scenario the elimination of H 2 is a prerequisite for the
binding of N 2 . In addition, based on the micro-reversibility principle, this proposal
also predicts that exogenous H 2 can be oxidatively added to E 4 (2N2H) NifDK,
thereby replacing the bound N 2 on the M-cluster and generating the E 4 (4H)
intermediate (Fig. 6b) [9, 82]. This prediction is consistent with two previous
biochemical findings. First, it was observed that NifDK produces HD gas under a
deuterium D 2 /N 2 atmosphere, thus suggesting the direct uptake of H 2 (or D 2 ) in the
presence of N 2 (Fig. 6b) [94, 95]. Second, addition of H 2 was found to alleviate the
inhibitory effect of N 2 on acetylene (C 2 H 2 ) reduction observed in NifDK
point-mutants, implying that H 2 might compete with N 2 for a specific binding site
on the M-cluster [97]. The reversibility of the reductive elimination of H 2 was
specifically tested biochemically in a recent study [98]. In this experiment, D 2 was
added to the NifDK upon normal N 2 turnover in order to achieve the backward
conversion of E 4 (2N2H) to E 4 (2H2D) as analog to E 4 (4H). Subsequently, acetylene
(C 2 H 2 ) was added to this turnover mixture to outcompete N 2 for the
M-cluster-bound hydrides or deuterides. Incorporation of deuterium into the product ethylene was then observed as C 2 H 2 D 2 and C 2 H 3 D and detected by GC–MS
(Fig. 6b) [98]. This finding supports the transient presence of the E 4 (2H2D)
intermediate, and hence the reductive elimination proposal, although it has not
directly been observed and characterized via spectroscopy.
The distal pathway versus the alternating pathway. In both the proposed distal and
the alternating pathway, the steps E 1 to E 4 are thought to proceed by accumulation
of reducing equivalents on M-clusters followed by the binding of N 2 . Subsequently,
174
C.-C. Lee et al.
accumulation of four electrons/protons on M-cluster [85–87, 89].
1,2 H,
57 Fe, and
95 Mo ENDOR experiments suggested that the spectroscopic features of this intermediate can be interpreted as an Fe-bound hydride fragment, plausibly in a l 2 -
bridging conformation (Fe–H–Fe) [85–87, 89, 90]. This finding led to the proposal
that the E 4 state of NifDK, which is represented by the freeze-trapped intermediate
mentioned above, is an M-cluster having two l 2 -hydride ligands bridged between
two different pairs of Fe atoms with two protons bound elsewhere on the cluster.
This proposed E 4 state, also termed E 4 (4H), then formed the basis for the reductive
elimination mechanism of N 2 binding. Its structure was further explored in recent
theoretical study, which suggests that the cleavage of an Fe-S bond, if not the entire
removal of the belt sulfur atom, is an energetically feasible reaction for the
M-cluster in the E 4 state [91, 92].
The two hydrides loaded on the M-cluster are thought to be the driving force for
N 2 binding. Studies on synthetic compounds suggested, the binding of N 2 can be
promoted by the reductive elimination of H 2 [93]. Therefore, the M-cluster in the
E 4 (4H) state is similarly thought to reductively eliminate one equivalent of H 2 in
order to enable the transient binding of N 2. This is followed by immediate reduction, resulting in a proposed bound N 2 H 2 species (known as the E 4 (2N2H) state) [9,
82]. It should be mentioned that the experimentally observed stoichiometry of one
H 2 per reduced N 2 can then be easily explained by the reductive elimination
mechanism because in this scenario the elimination of H 2 is a prerequisite for the
binding of N 2 . In addition, based on the micro-reversibility principle, this proposal
also predicts that exogenous H 2 can be oxidatively added to E 4 (2N2H) NifDK,
thereby replacing the bound N 2 on the M-cluster and generating the E 4 (4H)
intermediate (Fig. 6b) [9, 82]. This prediction is consistent with two previous
biochemical findings. First, it was observed that NifDK produces HD gas under a
deuterium D 2 /N 2 atmosphere, thus suggesting the direct uptake of H 2 (or D 2 ) in the
presence of N 2 (Fig. 6b) [94, 95]. Second, addition of H 2 was found to alleviate the
inhibitory effect of N 2 on acetylene (C 2 H 2 ) reduction observed in NifDK
point-mutants, implying that H 2 might compete with N 2 for a specific binding site
on the M-cluster [97]. The reversibility of the reductive elimination of H 2 was
specifically tested biochemically in a recent study [98]. In this experiment, D 2 was
added to the NifDK upon normal N 2 turnover in order to achieve the backward
conversion of E 4 (2N2H) to E 4 (2H2D) as analog to E 4 (4H). Subsequently, acetylene
(C 2 H 2 ) was added to this turnover mixture to outcompete N 2 for the
M-cluster-bound hydrides or deuterides. Incorporation of deuterium into the product ethylene was then observed as C 2 H 2 D 2 and C 2 H 3 D and detected by GC–MS
(Fig. 6b) [98]. This finding supports the transient presence of the E 4 (2H2D)
intermediate, and hence the reductive elimination proposal, although it has not
directly been observed and characterized via spectroscopy.
The distal pathway versus the alternating pathway. In both the proposed distal and
the alternating pathway, the steps E 1 to E 4 are thought to proceed by accumulation
of reducing equivalents on M-clusters followed by the binding of N 2 . Subsequently,
174
C.-C. Lee et al.