monitor the progression of P-cluster maturation at different time points. The data
showed that 50% of P-cluster are formed within the first 5 min, as indicated by the
appearance of the g * 12 EPR signal, the characteristic Fe–Fe backscattering
distances and substrate reducing activities; while the rest of the activity and
P-cluster-related spectroscopic features gradually emerged in the subsequent 2 h
(Fig. 3) [47]. Based on these observations, a stepwise P-cluster maturation model
was proposed, in which one P-cluster is formed first in a fast step, followed by the
slow maturation of the second P-cluster. In support of this model, DnifBnifZ NifDK
displayed the P* cluster-specific S = 1/2 EPR signal (perpendicular mode) with
roughly 50% intensity as compared to DnifH NifDK (which has two P* clusters)
while at the same time also showed the P-cluster-specific g * 12 signal with
approximately 50% intensity as compared to DnifB NifDK (which has two
P-clusters). In addition, when compared to DnifB, DnifBnifZ NifDK could only be
activated to a level of 50% in the presence of isolated M-cluster. Taken together,
these results suggest that DnifBnifZ NifDK contains one P*-cluster in one half of
NifDK and one P-cluster in the other half and could therefore represent an in
vivo-intermediate in the stepwise P-cluster maturation model. Indeed, the P*-cluster
on DnifBnifZ NifDK can also be matured into a P-cluster upon incubation with
NifZ, NifH, MgATP, and dithionite. Interestingly, it was shown that the action of
NifZ has to occur before the action of NifH/MgATP, thereby suggesting a potential
role of the former as a chaperone that facilitates the action of the NifH in the
maturation of the second P-cluster (Fig. 3) [47, 48].
While the molecular mechanism of P-cluster formation remains unclear, the
consistent requirement of NifH, MgATP, and high concentrations of reductant (i.e.,
dithionite) indicates involvement of reduction of P*cluster. Reduction of P*cluster
as a prerequisite of P-cluster maturation is supported by a study by Rupnik et al., in
which the conversion P*- to P-cluster was achieved chemically [49]. By reducing
DnifH NifDK with the strong reductant Ti(III) citrate, followed by oxidation with
either dithionite or IDS, a small portion of P* cluster was shown to be matured even
without NifH and MgATP. MCD characterization of the Ti(III)citrate-reduced
DnifH NifDK revealed the presence of the [Fe 4 S 4 ]
0 cluster, suggesting the [Fe 4 S 4 ]like sub-clusters of the P* cluster might undergo the “super reduced” all ferrous
state before being fused to form the P cluster. Beyond that, the need for conformational rearrangement in NifDK in P-cluster maturation is also indicated by a
small angle X-ray scattering (SAXS) [50]. In this study, comparison between DnifH
NifDK and DnifB NifDK revealed a much larger gap between the ab interface in
the NifDK variant compared to the later. This may suggest that, as the P* cluster is
matured, the a and b subunits of the NifDK have to move towards each other to
bring the two [Fe 4 S 4 ]-type modules closer to each other for a purported reductive
coupling of the metal clusters to occur (Fig. 3).
162
C.-C. Lee et al.
showed that 50% of P-cluster are formed within the first 5 min, as indicated by the
appearance of the g * 12 EPR signal, the characteristic Fe–Fe backscattering
distances and substrate reducing activities; while the rest of the activity and
P-cluster-related spectroscopic features gradually emerged in the subsequent 2 h
(Fig. 3) [47]. Based on these observations, a stepwise P-cluster maturation model
was proposed, in which one P-cluster is formed first in a fast step, followed by the
slow maturation of the second P-cluster. In support of this model, DnifBnifZ NifDK
displayed the P* cluster-specific S = 1/2 EPR signal (perpendicular mode) with
roughly 50% intensity as compared to DnifH NifDK (which has two P* clusters)
while at the same time also showed the P-cluster-specific g * 12 signal with
approximately 50% intensity as compared to DnifB NifDK (which has two
P-clusters). In addition, when compared to DnifB, DnifBnifZ NifDK could only be
activated to a level of 50% in the presence of isolated M-cluster. Taken together,
these results suggest that DnifBnifZ NifDK contains one P*-cluster in one half of
NifDK and one P-cluster in the other half and could therefore represent an in
vivo-intermediate in the stepwise P-cluster maturation model. Indeed, the P*-cluster
on DnifBnifZ NifDK can also be matured into a P-cluster upon incubation with
NifZ, NifH, MgATP, and dithionite. Interestingly, it was shown that the action of
NifZ has to occur before the action of NifH/MgATP, thereby suggesting a potential
role of the former as a chaperone that facilitates the action of the NifH in the
maturation of the second P-cluster (Fig. 3) [47, 48].
While the molecular mechanism of P-cluster formation remains unclear, the
consistent requirement of NifH, MgATP, and high concentrations of reductant (i.e.,
dithionite) indicates involvement of reduction of P*cluster. Reduction of P*cluster
as a prerequisite of P-cluster maturation is supported by a study by Rupnik et al., in
which the conversion P*- to P-cluster was achieved chemically [49]. By reducing
DnifH NifDK with the strong reductant Ti(III) citrate, followed by oxidation with
either dithionite or IDS, a small portion of P* cluster was shown to be matured even
without NifH and MgATP. MCD characterization of the Ti(III)citrate-reduced
DnifH NifDK revealed the presence of the [Fe 4 S 4 ]
0 cluster, suggesting the [Fe 4 S 4 ]like sub-clusters of the P* cluster might undergo the “super reduced” all ferrous
state before being fused to form the P cluster. Beyond that, the need for conformational rearrangement in NifDK in P-cluster maturation is also indicated by a
small angle X-ray scattering (SAXS) [50]. In this study, comparison between DnifH
NifDK and DnifB NifDK revealed a much larger gap between the ab interface in
the NifDK variant compared to the later. This may suggest that, as the P* cluster is
matured, the a and b subunits of the NifDK have to move towards each other to
bring the two [Fe 4 S 4 ]-type modules closer to each other for a purported reductive
coupling of the metal clusters to occur (Fig. 3).
162
C.-C. Lee et al.