Mo center, and Cysa275, which coordinates to the terminal Fe atom at the opposite
end of the cluster (Fig. 1c) [18]. The fact that the M-cluster only requires two
protein ligands is indicative of the integrity of the core structure, which likely is
upheld by the central carbide and the three belt sulfur atoms. The limited ligation
that tethers the M-cluster to the NifDK polypeptide also allows the metal cofactor to
be chemically extracted from the protein into organic solvents such as N-methylformamide (NMF) [33, 34]. The extracted M-cluster (also called isolated cofactor)
can reconstitute the M-cluster-deficient, but P-cluster-containing variant of NifDK
(sometimes known as apo-NifDK), thereby fully restoring the substrate-reduction
activity and spectroscopic features of holo-NifDK [33].
3 Assembly of Nitrogenase
The assembly of nitrogenase is a broad topic with many interesting facets: the
synthesis of the catalytic and reductase components, the maturation of the unique
metal clusters, the involvement of accessory proteins and the genetic regulation of
all these processes [6, 7, 35–37]. This chapter focuses on the biosynthesis of the Pand M-cluster of NifDK and discusses the latest developments in this area of active
research.
3.1 Overview: General Scheme and Major Players Involved
The biosynthesis of both the P- and the M-cluster starts with the assembly of a
common building block: the [Fe 4 S 4 ] cluster (Fig. 2). Its synthesis is achieved by the
NifUS complex, which mobilizes cellular Fe and S to first generate [Fe 2 S 2 ] subunits
and then fuses two such subunits together into a [Fe 4 S 4 ] cluster (Fig. 2, top left)
[38–40]. From there, the biosynthetic path diverges. For the formation of the
P-cluster, a pair of [Fe 4 S 4 ] clusters are transferred directly to NifDK to form the
P-cluster in-situ with the help of NifH and NifZ (Fig. 2, bottom left). In contrast, the
assembly of the M-cluster begins with a pair of [Fe 4 S 4 ] clusters on NifB, on which
a Fe 8 S 9 C intermediate, known as the L-cluster, is formed (Fig. 2, top right). The
L-cluster is subsequently transferred to NifEN, the site where Mo and homocitrate
are installed—again with the participation of NifH (Fig. 2, top right). Finally, the
matured M-cluster is inserted into the P-cluster-containing, but M-cluster-deficient
“apo-NifDK” to form the competent holo-NifDK (Fig. 2, bottom right) [36, 37].
3.2 Assembly of P-Cluster
The identification of the P-cluster’s biosynthetic precursor was made possible by
two NifDK variants: one isolated in a nifH deletion background (which is referred
to as DnifH NifDK) and one isolated in a nifB and nifZ double deletion background
Assembly and Function of Nitrogenase
159
end of the cluster (Fig. 1c) [18]. The fact that the M-cluster only requires two
protein ligands is indicative of the integrity of the core structure, which likely is
upheld by the central carbide and the three belt sulfur atoms. The limited ligation
that tethers the M-cluster to the NifDK polypeptide also allows the metal cofactor to
be chemically extracted from the protein into organic solvents such as N-methylformamide (NMF) [33, 34]. The extracted M-cluster (also called isolated cofactor)
can reconstitute the M-cluster-deficient, but P-cluster-containing variant of NifDK
(sometimes known as apo-NifDK), thereby fully restoring the substrate-reduction
activity and spectroscopic features of holo-NifDK [33].
3 Assembly of Nitrogenase
The assembly of nitrogenase is a broad topic with many interesting facets: the
synthesis of the catalytic and reductase components, the maturation of the unique
metal clusters, the involvement of accessory proteins and the genetic regulation of
all these processes [6, 7, 35–37]. This chapter focuses on the biosynthesis of the Pand M-cluster of NifDK and discusses the latest developments in this area of active
research.
3.1 Overview: General Scheme and Major Players Involved
The biosynthesis of both the P- and the M-cluster starts with the assembly of a
common building block: the [Fe 4 S 4 ] cluster (Fig. 2). Its synthesis is achieved by the
NifUS complex, which mobilizes cellular Fe and S to first generate [Fe 2 S 2 ] subunits
and then fuses two such subunits together into a [Fe 4 S 4 ] cluster (Fig. 2, top left)
[38–40]. From there, the biosynthetic path diverges. For the formation of the
P-cluster, a pair of [Fe 4 S 4 ] clusters are transferred directly to NifDK to form the
P-cluster in-situ with the help of NifH and NifZ (Fig. 2, bottom left). In contrast, the
assembly of the M-cluster begins with a pair of [Fe 4 S 4 ] clusters on NifB, on which
a Fe 8 S 9 C intermediate, known as the L-cluster, is formed (Fig. 2, top right). The
L-cluster is subsequently transferred to NifEN, the site where Mo and homocitrate
are installed—again with the participation of NifH (Fig. 2, top right). Finally, the
matured M-cluster is inserted into the P-cluster-containing, but M-cluster-deficient
“apo-NifDK” to form the competent holo-NifDK (Fig. 2, bottom right) [36, 37].
3.2 Assembly of P-Cluster
The identification of the P-cluster’s biosynthetic precursor was made possible by
two NifDK variants: one isolated in a nifH deletion background (which is referred
to as DnifH NifDK) and one isolated in a nifB and nifZ double deletion background
Assembly and Function of Nitrogenase
159