responsible for the deprotonation of the methylene moiety on the K2 cluster as the
loss of histindine-43 ligation during L-cluster formation could be mediated through
protonation of its side chain, and (iii) histindine-43 might thereby serve as a
functional liaison that orchestrates these two events (Fig. 4, Step 3).
The incorporation of the 9th sulfur atom. Following the rearrangement and fusion
of the two K cluster modules, a [Fe 8 S 8 C] core is formed. This core is referred to as
the L*-cluster because of its structural similarity to the L-cluster that was deduced
from spectroscopic data [61]. EPR analysis revealed that both the L*- and the
L-cluster display almost identical g = 1.94 signals. XAS/EXAFS analysis also
revealed an overall resemblance of the Fe distances between the L* and L-cluster,
although detected the lack of a strong Fe–Fe interaction in L*-cluster which is
present in the L-cluster [62]. This is indicative that the L*-cluster adopts a more
open conformation and thereby inferring that the missing 9th sulfur atom would be
located on one of the three belt positions. Activities analysis revealed the L*-cluster
is incapable of undergoing the same maturation events that turn the fully matured
L-cluster into the M-cluster [61]. This highlights the significance of the incorporation of the so-called “9th-sulfur” atom and raises the question of what the source
of the sulfur might be. Surprisingly, the 9th-sulfur atom does not come from SAM
or NifS as previously thought, but rather from inorganic sulfite (SO 3
2− ) (Fig. 4,
Step 4) [61–64]. In a reaction free from any S contaminations, featuring the absence
of dithionite and the use a synthetic [Fe 4 S 4 ] cluster reconstituted NifB, it could be
shown that the L*-cluster can be activated (in terms of its ability to mature into the
M cluster) by adding sulfite as a sulfur source (Fig. 4, Step 4) [61]. In stark contrast,
the same could not be achieved with sulfate (SO 4
2− ), sulfide (S
2− ) or other common
sulfur metabolites [61]. A
35 S-tracing experiment, finally, provided the definite
proof that S from
35 SO 3
2− was incorporated into the L*-cluster as the radiolabel
was detected on the extracted L-cluster after
35 SO 3
2− had reacted with NifB and
SAM [61]. There is currently no direct evidence for the utilization of sulfite in the
9
th S incorporation step inside the cell, although sulfite along with sulfate and
sulfide are all essential of cellular sulfur metabolism hubs. The absence of the l 2 -
belt sulfide from the L*-cluster is consistent with the labile nature of the belt region
of the cofactor, as demonstrated in recent MCD studies of L cluster as well as
several structures in which the belt sulfur of either M- or V-cluster are missing (see
Sect. 4.3) [65].
3.3.2 Maturation of the L-Cluster on NifEN
After its formation on NifB, the L-cluster is transferred to NifEN to be further
matured into the M-cluster (Fig. 5a). NifEN is a homolog of NifDK and thus
contains analogous metal-cluster binding sites. Indeed, spectroscopic and structural
characterization of A. vinelandii NifEN revealed the presence of a [Fe 4 S 4 ] cluster in
the analogous P-cluster binding site of NifDK at the interface of the a- and
b-subunits [66]. When expressed in a nifHDK-deletion background, EPR, Fe edge
XAS/EXAFS and Fe Kb XES studies showed the accumulation of the [Fe 8 S 9 C]
L-cluster on NifEN [67–69]. A crystal structure of this L-cluster-containing NifEN
Assembly and Function of Nitrogenase
167
loss of histindine-43 ligation during L-cluster formation could be mediated through
protonation of its side chain, and (iii) histindine-43 might thereby serve as a
functional liaison that orchestrates these two events (Fig. 4, Step 3).
The incorporation of the 9th sulfur atom. Following the rearrangement and fusion
of the two K cluster modules, a [Fe 8 S 8 C] core is formed. This core is referred to as
the L*-cluster because of its structural similarity to the L-cluster that was deduced
from spectroscopic data [61]. EPR analysis revealed that both the L*- and the
L-cluster display almost identical g = 1.94 signals. XAS/EXAFS analysis also
revealed an overall resemblance of the Fe distances between the L* and L-cluster,
although detected the lack of a strong Fe–Fe interaction in L*-cluster which is
present in the L-cluster [62]. This is indicative that the L*-cluster adopts a more
open conformation and thereby inferring that the missing 9th sulfur atom would be
located on one of the three belt positions. Activities analysis revealed the L*-cluster
is incapable of undergoing the same maturation events that turn the fully matured
L-cluster into the M-cluster [61]. This highlights the significance of the incorporation of the so-called “9th-sulfur” atom and raises the question of what the source
of the sulfur might be. Surprisingly, the 9th-sulfur atom does not come from SAM
or NifS as previously thought, but rather from inorganic sulfite (SO 3
2− ) (Fig. 4,
Step 4) [61–64]. In a reaction free from any S contaminations, featuring the absence
of dithionite and the use a synthetic [Fe 4 S 4 ] cluster reconstituted NifB, it could be
shown that the L*-cluster can be activated (in terms of its ability to mature into the
M cluster) by adding sulfite as a sulfur source (Fig. 4, Step 4) [61]. In stark contrast,
the same could not be achieved with sulfate (SO 4
2− ), sulfide (S
2− ) or other common
sulfur metabolites [61]. A
35 S-tracing experiment, finally, provided the definite
proof that S from
35 SO 3
2− was incorporated into the L*-cluster as the radiolabel
was detected on the extracted L-cluster after
35 SO 3
2− had reacted with NifB and
SAM [61]. There is currently no direct evidence for the utilization of sulfite in the
9
th S incorporation step inside the cell, although sulfite along with sulfate and
sulfide are all essential of cellular sulfur metabolism hubs. The absence of the l 2 -
belt sulfide from the L*-cluster is consistent with the labile nature of the belt region
of the cofactor, as demonstrated in recent MCD studies of L cluster as well as
several structures in which the belt sulfur of either M- or V-cluster are missing (see
Sect. 4.3) [65].
3.3.2 Maturation of the L-Cluster on NifEN
After its formation on NifB, the L-cluster is transferred to NifEN to be further
matured into the M-cluster (Fig. 5a). NifEN is a homolog of NifDK and thus
contains analogous metal-cluster binding sites. Indeed, spectroscopic and structural
characterization of A. vinelandii NifEN revealed the presence of a [Fe 4 S 4 ] cluster in
the analogous P-cluster binding site of NifDK at the interface of the a- and
b-subunits [66]. When expressed in a nifHDK-deletion background, EPR, Fe edge
XAS/EXAFS and Fe Kb XES studies showed the accumulation of the [Fe 8 S 9 C]
L-cluster on NifEN [67–69]. A crystal structure of this L-cluster-containing NifEN
Assembly and Function of Nitrogenase
167