3.3 Assembly of M-Cluster
3.3.1 Formation of the [Fe 8 S 9 C] L-Cluster on NifB
Like in the case of the P-cluster, the assembly of the M-cluster also begins with the
fusion of two [Fe 4 S 4 ]-like modules which occurs on NifB (Fig. 4). The investigation of NifB was historically hindered by the difficulty to express this protein in
the diazotrophic model organism A. vinelandii [51]. Early sequence analysis
revealed a number of conserved cysteine residues, indicating the presence of several
putative Fe-S clusters in NifB. One of these was predicted to be a radical
S-adenosyl-L-methionine (SAM)-binding [Fe 4 S 4 ] cluster because of the occurrence
of a signature CxxxCxxC motif—a sequence pattern commonly associated with
cluster-ligation in radical SAM proteins—and was later referred to as the SAM
cluster [52]. This intriguing [Fe 4 S 4 ] cluster has one open coordination site that
binds the sacrificial co-substrate SAM, which can either serve as methyl donor in
methylation and/or as a source of a 5′ deoxyadenosyl radical (5′-dAÁ) in
radical-mediated reactions [53]. Further analysis led to the identification of a pair of
[Fe 4 S 4 ] clusters, collectively referred to as the K-cluster, which was proposed to be
the building block that makes up the 8Fe core of the M-cluster (Fig. 4) [51, 52].
This was eventually demonstrated by a series of biochemical breakthrough
experiments, in which NifB was expressed as a fusion-protein with NifEN, both, in
A. vinelandii, and, heterologously, in E. coli [51, 52, 54–56]. Remarkably, the
different versions of NifB proteins essentially displayed identical properties and
behavior, despite the different expression systems and source organisms. EPR
analysis revealed a composite S = 1/2 signal at g = 2.02, 1.95, and 1.90, representing the presence of both the K- and the SAM cluster. Significantly, this signal
disappeared upon addition of SAM, indicating that both metal clusters of NifB
reacted with SAM molecules [51, 56]. Concomitantly, a g = 1.94 EPR signal,
previously established to be characteristic of the [Fe 8 S 9 C] L-cluster, emerged and
thus demonstrated a novel radical SAM-mediated biosynthetic route that fuses two
[Fe 4 S 4 ] clusters into an 8Fe cluster [51, 56]. The exact mechanism of this fascinatingly complex reaction is still a topic of ongoing research, although many details
have been elucidated in the last decade. It is currently understood that the action of
NifB can be roughly grouped into three stages: (i) Insertion of the methyl moiety
(Fig. 4, Step 1 & 2); (ii) cluster rearrangement (Fig. 4, Step 3), and (iii) incorporation of the 9th sulfur atom (Fig. 4, Step 4). These events are described in the
following sections.
The insertion of the methyl group from SAM. The involvement of SAM in the
conversion of the K to the L cluster was initially demonstrated by detecting the
cleavage products of SAM [51, 56]. HPLC analysis revealed that cleavage of SAM
by NifB produces 5′-deoxyadenosine (5′-dAH) and S-adenosyl-L-homocysteine
(SAH) (Fig. 4, Step 1) [51, 54]. In parallel, using [methyl-d 3 ] SAM instead of
regular SAM, deuterated 5′-deoxyadenosine (5′-dAD) could be detected together
with undeuterated SAH [54]. Interestingly, previous studies of the SAM-dependent
RNA-methyltransferases RlmN and Cfr also shown a similar product profile, so
Assembly and Function of Nitrogenase
163
3.3.1 Formation of the [Fe 8 S 9 C] L-Cluster on NifB
Like in the case of the P-cluster, the assembly of the M-cluster also begins with the
fusion of two [Fe 4 S 4 ]-like modules which occurs on NifB (Fig. 4). The investigation of NifB was historically hindered by the difficulty to express this protein in
the diazotrophic model organism A. vinelandii [51]. Early sequence analysis
revealed a number of conserved cysteine residues, indicating the presence of several
putative Fe-S clusters in NifB. One of these was predicted to be a radical
S-adenosyl-L-methionine (SAM)-binding [Fe 4 S 4 ] cluster because of the occurrence
of a signature CxxxCxxC motif—a sequence pattern commonly associated with
cluster-ligation in radical SAM proteins—and was later referred to as the SAM
cluster [52]. This intriguing [Fe 4 S 4 ] cluster has one open coordination site that
binds the sacrificial co-substrate SAM, which can either serve as methyl donor in
methylation and/or as a source of a 5′ deoxyadenosyl radical (5′-dAÁ) in
radical-mediated reactions [53]. Further analysis led to the identification of a pair of
[Fe 4 S 4 ] clusters, collectively referred to as the K-cluster, which was proposed to be
the building block that makes up the 8Fe core of the M-cluster (Fig. 4) [51, 52].
This was eventually demonstrated by a series of biochemical breakthrough
experiments, in which NifB was expressed as a fusion-protein with NifEN, both, in
A. vinelandii, and, heterologously, in E. coli [51, 52, 54–56]. Remarkably, the
different versions of NifB proteins essentially displayed identical properties and
behavior, despite the different expression systems and source organisms. EPR
analysis revealed a composite S = 1/2 signal at g = 2.02, 1.95, and 1.90, representing the presence of both the K- and the SAM cluster. Significantly, this signal
disappeared upon addition of SAM, indicating that both metal clusters of NifB
reacted with SAM molecules [51, 56]. Concomitantly, a g = 1.94 EPR signal,
previously established to be characteristic of the [Fe 8 S 9 C] L-cluster, emerged and
thus demonstrated a novel radical SAM-mediated biosynthetic route that fuses two
[Fe 4 S 4 ] clusters into an 8Fe cluster [51, 56]. The exact mechanism of this fascinatingly complex reaction is still a topic of ongoing research, although many details
have been elucidated in the last decade. It is currently understood that the action of
NifB can be roughly grouped into three stages: (i) Insertion of the methyl moiety
(Fig. 4, Step 1 & 2); (ii) cluster rearrangement (Fig. 4, Step 3), and (iii) incorporation of the 9th sulfur atom (Fig. 4, Step 4). These events are described in the
following sections.
The insertion of the methyl group from SAM. The involvement of SAM in the
conversion of the K to the L cluster was initially demonstrated by detecting the
cleavage products of SAM [51, 56]. HPLC analysis revealed that cleavage of SAM
by NifB produces 5′-deoxyadenosine (5′-dAH) and S-adenosyl-L-homocysteine
(SAH) (Fig. 4, Step 1) [51, 54]. In parallel, using [methyl-d 3 ] SAM instead of
regular SAM, deuterated 5′-deoxyadenosine (5′-dAD) could be detected together
with undeuterated SAH [54]. Interestingly, previous studies of the SAM-dependent
RNA-methyltransferases RlmN and Cfr also shown a similar product profile, so
Assembly and Function of Nitrogenase
163