hydride accumulation in the early reactive states (i.e. E 1 to E 3 ) would force the
sulfur ligand out of the cofactor, possibly through protonation of the sulfide, and
into the nearby sulfur-holding site. It was further suggested that in VnfDGK the
sulfur atom might resume its original S2B position upon after the release of the final
product, a process which might be facilitated by Gln176, in order to regenerate the
E 0 ground state (Fig. 7b) [107]. Based on the assumption that the assigned HN
−
ligand in VnfDGK is mechanistically relevant, Einsle hypothesized that it might
represent the E 6 state of the distal pathway introduced in Sect. 4.2. In this scenario,
N 2 might bind symmetrically in bridging mode between Fe2 and Fe6 as a l 1 , 1 -
N − NH 2 species.
Finally, to complicate matters further, Rees and coworkers showed that all three
of the sulfur atoms, not just S2B, can be lost during catalysis and thus might be
mechanistically relevant [109]. First, by turnover with selenocyanate (SeCN
−
), an
alternative substrate and inhibitor of nitrogenase, it was first demonstrated in a
1.60 Å resolution crystal structure that a Se atom can be selectively incorporated
into the S2B position of M-cluster [109]. Subsequently, this Se2B-containingNifDK was then subjected to further turnover with C 2 H 2 in a time-dependent
structural study in order to monitor the eventual fate of the Se atom. This was
achieved by terminating the reaction by freeze quenching at different time points
and followed by crystallographic characterization. Remarkably, while the Se
occupancy in the 2B position decreased during catalytic turnover, while increasing
levels of Se were detected at the other two belt positions (3A and 5A) [109]. The
observation that the Se atom can migrate from one position to another strongly
suggests the structural lability of the belt sulfur atoms, although it remains unclear
whether substrates or other ligands would be able to access the 3A and 5A sites. In
addition, it was also shown that the total Se occupancy disappeared after 5000
turnover cycles, indicating the replenishment of S on the M-cluster [109]. The
source of this sulfur has not been identified, although dithionite from the solution
would be the major suspect. Taken together, these results suggest the existence of a
channel within the matrix of NifDK that allows S and Se (or derivatives thereof) to
migrate from the surface of the protein to the active site and vice versa.
5 Conclusion and Future Prospects
In the last decade or so, exciting advances have been made in the field of nitrogenase research which greatly extended our understanding of its assembly and
function. On the biosynthesis side, we now have a complete picture of the components and conditions necessary for the synthesis of the P-cluster. Moreover, we
have unraveled the once mysterious action of NifB and now have the ability to trace
the individual atoms that make up the M-cluster. On the catalysis side, details of the
catalytic mechanism are being filled in as an increasing number of reaction intermediates are trapped and studied spectroscopically, while the ligand-bound nitrogenase structures offer incredible insights that were unimaginable a decade ago. All
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C.-C. Lee et al.
sulfur ligand out of the cofactor, possibly through protonation of the sulfide, and
into the nearby sulfur-holding site. It was further suggested that in VnfDGK the
sulfur atom might resume its original S2B position upon after the release of the final
product, a process which might be facilitated by Gln176, in order to regenerate the
E 0 ground state (Fig. 7b) [107]. Based on the assumption that the assigned HN
−
ligand in VnfDGK is mechanistically relevant, Einsle hypothesized that it might
represent the E 6 state of the distal pathway introduced in Sect. 4.2. In this scenario,
N 2 might bind symmetrically in bridging mode between Fe2 and Fe6 as a l 1 , 1 -
N − NH 2 species.
Finally, to complicate matters further, Rees and coworkers showed that all three
of the sulfur atoms, not just S2B, can be lost during catalysis and thus might be
mechanistically relevant [109]. First, by turnover with selenocyanate (SeCN
−
), an
alternative substrate and inhibitor of nitrogenase, it was first demonstrated in a
1.60 Å resolution crystal structure that a Se atom can be selectively incorporated
into the S2B position of M-cluster [109]. Subsequently, this Se2B-containingNifDK was then subjected to further turnover with C 2 H 2 in a time-dependent
structural study in order to monitor the eventual fate of the Se atom. This was
achieved by terminating the reaction by freeze quenching at different time points
and followed by crystallographic characterization. Remarkably, while the Se
occupancy in the 2B position decreased during catalytic turnover, while increasing
levels of Se were detected at the other two belt positions (3A and 5A) [109]. The
observation that the Se atom can migrate from one position to another strongly
suggests the structural lability of the belt sulfur atoms, although it remains unclear
whether substrates or other ligands would be able to access the 3A and 5A sites. In
addition, it was also shown that the total Se occupancy disappeared after 5000
turnover cycles, indicating the replenishment of S on the M-cluster [109]. The
source of this sulfur has not been identified, although dithionite from the solution
would be the major suspect. Taken together, these results suggest the existence of a
channel within the matrix of NifDK that allows S and Se (or derivatives thereof) to
migrate from the surface of the protein to the active site and vice versa.
5 Conclusion and Future Prospects
In the last decade or so, exciting advances have been made in the field of nitrogenase research which greatly extended our understanding of its assembly and
function. On the biosynthesis side, we now have a complete picture of the components and conditions necessary for the synthesis of the P-cluster. Moreover, we
have unraveled the once mysterious action of NifB and now have the ability to trace
the individual atoms that make up the M-cluster. On the catalysis side, details of the
catalytic mechanism are being filled in as an increasing number of reaction intermediates are trapped and studied spectroscopically, while the ligand-bound nitrogenase structures offer incredible insights that were unimaginable a decade ago. All
178
C.-C. Lee et al.