the SAM-cluster (coordinated by Cys
50 , Cys
54 , and Cys
57 in the CxxCxxxC motif),
the K1-cluster (part of the K-cluster and coordinated by Cys
30 , Cys
63 , and Cys
129
towards the N-terminus) and the K2-cluster (part of the K-cluster and coordinated
by Cys
264 , Cys
274 , and Cys
277 towards the C-terminus). Deletion of each set of
cysteines, which led to the selective loss of the three [Fe 4 S 4 ] modules, confirmed
that all three modules were necessary for formation of L-cluster. More importantly,
it also revealed that only the NifB mutant that misses the K1 cluster, but retains the
K2- and SAM clusters, can maintain a normal SAM-cleavage-product profile and
the ability to generate methanethiol upon acid quenching. Meanwhile, deletion of
either the K2 or the SAM cluster led to obliteration of these activities [59]. This
observation suggests that the presence of both clusters is the prerequisite for methyl
transfer and the subsequent hydrogen abstraction and thus strongly points to a
K2-associated sulfur atom as the target of the methyl transfer step.
Cluster rearrangement and formation of the L-cluster. The immediate events that
follow the methyl transfer and hydrogen abstraction remain largely unclear at this
point due to the concomitant nature of the insertion of the center carbide, the
rearrangement of the K1- and K2-clusters, and the formation of the L-cluster. The
following steps would need to occur in synchrony: One or both K cluster modules
(K1 and K2) need to open up by breaking off the Fe-S bonds. The clusters then
need to move closer to each other for the coupling reaction to occur. The methylene
moiety which was originated from the methyl group transferred from SAM to the
K2 cluster, and was then generated by hydrogen abstraction, needs to be deprotonated (Fig. 4, Step 3) [36]. These events likely involve nearby residues to either
guide the rearrangement of K1 and K2 rearrangement and their fusion, or act as a
base to deprotonate the K2-bound methylene moiety. One likely candidate that
might simultaneously fulfill both roles could be histidine-43 of MaNifB [60]. Using
mutagenesis and pulse EPR, this residue was shown to coordinate to the K1 cluster,
likely via an N-atom of an imidazole group on the side chain [59, 60]. Significantly,
this ligation disappears as the K1- and K2-cluster is fused into the L-cluster, which
highlights the importance of histidine-43 for this crucial process. Indeed, NifB with
a H43A mutation (designated MaNifB
H43A ) was shown to be unable to form an
intact L-cluster upon incubation with SAM. Moreover, the ability to MaNifB
H43A to
catalyze methyl-transfer and hydrogen-abstraction was unaffected by the mutation,
which was demonstrated by the detection of SAH and 5′-dA by HPLC analysis, as
well as methanethiol upon acid quenching. The cluster species of the SAM-treated
MaNifB
H43A could therefore be considered as an intermediate K-cluster with a
methylene attachment which is on route towards its transformation to the L-cluster
[59, 60]. XAS/EXAFS characterization of this supposed intermediate revealed that
K1- and K2-modules become more aligned with respect to each other when
compared to as original state (i.e. MaNifB
H43A without SAM treatment), while still
lacks the 8Fe core arrangement which is characteristic of the L- and the M-cluster
[60]. Taken together, these observations suggest the following interesting proposals: (i) the presence of histidine-43 might act as a guide to assist the structural
rearrangement during the coupling of K1 and K2 cluster, (ii) histidine-43 might be
166
C.-C. Lee et al.
50 , Cys
54 , and Cys
57 in the CxxCxxxC motif),
the K1-cluster (part of the K-cluster and coordinated by Cys
30 , Cys
63 , and Cys
129
towards the N-terminus) and the K2-cluster (part of the K-cluster and coordinated
by Cys
264 , Cys
274 , and Cys
277 towards the C-terminus). Deletion of each set of
cysteines, which led to the selective loss of the three [Fe 4 S 4 ] modules, confirmed
that all three modules were necessary for formation of L-cluster. More importantly,
it also revealed that only the NifB mutant that misses the K1 cluster, but retains the
K2- and SAM clusters, can maintain a normal SAM-cleavage-product profile and
the ability to generate methanethiol upon acid quenching. Meanwhile, deletion of
either the K2 or the SAM cluster led to obliteration of these activities [59]. This
observation suggests that the presence of both clusters is the prerequisite for methyl
transfer and the subsequent hydrogen abstraction and thus strongly points to a
K2-associated sulfur atom as the target of the methyl transfer step.
Cluster rearrangement and formation of the L-cluster. The immediate events that
follow the methyl transfer and hydrogen abstraction remain largely unclear at this
point due to the concomitant nature of the insertion of the center carbide, the
rearrangement of the K1- and K2-clusters, and the formation of the L-cluster. The
following steps would need to occur in synchrony: One or both K cluster modules
(K1 and K2) need to open up by breaking off the Fe-S bonds. The clusters then
need to move closer to each other for the coupling reaction to occur. The methylene
moiety which was originated from the methyl group transferred from SAM to the
K2 cluster, and was then generated by hydrogen abstraction, needs to be deprotonated (Fig. 4, Step 3) [36]. These events likely involve nearby residues to either
guide the rearrangement of K1 and K2 rearrangement and their fusion, or act as a
base to deprotonate the K2-bound methylene moiety. One likely candidate that
might simultaneously fulfill both roles could be histidine-43 of MaNifB [60]. Using
mutagenesis and pulse EPR, this residue was shown to coordinate to the K1 cluster,
likely via an N-atom of an imidazole group on the side chain [59, 60]. Significantly,
this ligation disappears as the K1- and K2-cluster is fused into the L-cluster, which
highlights the importance of histidine-43 for this crucial process. Indeed, NifB with
a H43A mutation (designated MaNifB
H43A ) was shown to be unable to form an
intact L-cluster upon incubation with SAM. Moreover, the ability to MaNifB
H43A to
catalyze methyl-transfer and hydrogen-abstraction was unaffected by the mutation,
which was demonstrated by the detection of SAH and 5′-dA by HPLC analysis, as
well as methanethiol upon acid quenching. The cluster species of the SAM-treated
MaNifB
H43A could therefore be considered as an intermediate K-cluster with a
methylene attachment which is on route towards its transformation to the L-cluster
[59, 60]. XAS/EXAFS characterization of this supposed intermediate revealed that
K1- and K2-modules become more aligned with respect to each other when
compared to as original state (i.e. MaNifB
H43A without SAM treatment), while still
lacks the 8Fe core arrangement which is characteristic of the L- and the M-cluster
[60]. Taken together, these observations suggest the following interesting proposals: (i) the presence of histidine-43 might act as a guide to assist the structural
rearrangement during the coupling of K1 and K2 cluster, (ii) histidine-43 might be
166
C.-C. Lee et al.