MoFe proteins biosynthesis

Fe protein polypeptide NifH is also involved in FeMoco and MoFe protein biosynthesis MoFe protein polypeptides... 

The VFe protein also has the equivalent of P-cluster pairs which have similar properties to those found in the MoFe protein (159). No information is available on whether P-cluster pairs exist in the FeFe protein, but because of the relatively high sequence identity and the similar genetic basis of its biosynthesis, the occurrence seems highly likely. The catalytic role assigned to the P-cluster pair involves accepting electrons from the Fe protein for storage and future deUvery to the substrate via the FeMo-cofactor centers. As of this writing (ca early 1995), this role has yet to be proved. 

As well as donating electrons to the MoFe protein, the Fe protein has at least two and possibly three other functions (see Section IV,C) It is involved in the biosynthesis of the iron molybdenum cofactor, FeMoco it is required for insertion of the FeMoco into the MoFe protein polypeptides and it has been implicated in the regulation of the biosynthesis of the alternative nitrogenases. 

The significance of the observed interactions between MoFe proteins and nucleotides is not easy to determine. However, as noted in Section IV, ATP is involved in the maturation of the MoFe protein and the biosynthesis of FeMoco. It is therefore possible that the nucleotide interactions noted here are associated with the maturation of the MoFe protein and not with its catalytic activity. 

The biosynthesis of the MoFe protein is extremely complex. Here we will first describe the biosynthesis of FeMoco, then that of the apo-MoFe protein, encoded by the nifD and nifK genes and containing the P clusters, and finally we will summarize what is known about the combination of FeMoco with the apo-MoFe protein to form active MoFe protein. 

Fe-protein, the unique, highly specific electron donor to MoFe-protein, mediates coupling between ATP hydrolysis and electron transfer to MoFe-protein and also participates in the biosynthesis and insertion of FeMoco into MoFe-protein. Fe-protein contains one ferredoxin-like [Fe4S 4 2 /1+ cluster as its redox center. There is now evidence for an [Fe4S4]° super-reduced state in which four high-spin iron(II) (S= 2) sites are postulated. These were previously discussed in Section 6.3 and illustrated in Table 6.1.16 The [Fe4S4] cluster in this state bridges a dimer of... 

The assembly of the MoFe protein involves the biosynthesis of the FeMo cofactor, and in a separate pathway, the biosynthesis of a P cluster containing yet FeMo cofactor deficient species of the MoFe protein and the insertion of the completed FeMo cofactor into the FeMo cofactor deficient MoFe protein. ... 

Figure 12 illustrates the current model of FeMo cofactor biosynthesis, a process independent of the production of MoFe protein polypeptides, which starts with the mobihzation of iron and sulfur and the assembly of Fe/S fragments by NifS and NifU. These Fe/S fragments are transferred to NifB nifB gene product), which is subsequently involved in the production of the Fe/S core of the FeMo cofactor (designated NifB cofactor) that probably contains all of... 

Biosynthesis of aP Cluster-containing, FeMo Cofactor-deficient MoFe Protein... 

Like the biosynthesis of the FeMo cofactor (Section 4.2.1), the insertion of the FeMo cofactor into the FeMo cofactor-deficient form of the MoFe protein also requires the presence of the Fe protein. This final maturation process of the MoFe protein most likely occurs in a series of steps, which are shown in Figure 12. Initially, the FeMo cofactor site is not accessible to FeMo cofactor insertion ( closed conformation). The conversion of this site to another form with the FeMo cofactor site accessible to... 

Figure 12 Assembly of the molybdenum nitrogenase. Pathways of FeMo cofactor biosynthesis, Fe protein maturation, and MoFe protein assembly are indicated in dotted, dashed, and solid arrows, respectively. The nif gene products that are also required for the vanadium and iron-only nitrogenase systems are indicated by ...

The assembly of the molybdenum nitrogenase Fe protein involves the nifH gene and at least the nifS, nifU, and niJM gene prodncts that are implicated in the [4Fe 4S] cluster assembly, while the assembly of MoFe protein requires at least 15 nif gene products and involves the biosynthesis of FeMo cofactor and FeMo cofactor deficient MoFe protein in separate pathways and the insertion of FeMo cofactor into the MoFe protein. 

In addition to the mechanistic role in the nitrogenase enzymatic function, Fe-protein also participates at several stages in the biosynthesis of the nitrogenase proteins. Fe-protein is essential for the production of active MoFe-protein and is involved in both the synthesis of FeMo-cofactor and its insertion into cofactor-deficient MoFe-protein (40-42). Fe-protein may also function as an activator for the expression of alternative nitrogenases (43). In turn, formation of active Fe-protein requires the nifM gene product (44, 45), which perhaps functions either in cluster insertion or in promoting the correct subunit-subunit and subunit-cofactor interactions in the Fe-protein dimer (i.e., a chaperone-type role). The significant sequence conservation observed in the Fe-protein family may reflect the structural constraints associated with these diverse aspects of Fe-protein function. 

The component proteins of Mo nitrogenase are MoFe protein, an 2)32 tetramer encoded by the nifDK genes, and an Fe protein, a y2 dimer encoded by nifH. The formation of an active enzyme requires, in addition to these structural genes, the functions of several other nif genes. The nif M in some unknown way activates the Fe protein polypeptide and is essential for its function. A number of nif genes are involved in FeMoco biosynthesis (nifHBENVQ) and are therefore essential for the synthesis of an active MoFe protein. 

At each cycle, MgATP complexed Fe-protein associates with MoFe-protein, 2ATP are hydrolyzed with the transfer of one electron to MoFe-protein and the complex dissociates. Turnover is extremely slow, 6.4 s 1 (estimated from in vitro rapid kinetic measurements), necessitating the biosynthesis of enormous quantities of the two proteins. Electrons are presumably accumulated in the P-center before transfer to the FeMo cofactor where substrate reduction is thought to occur. 

Genes nif and vnf) involved in the biosynthesis of the VFe-protein. FeS is a low molecnlar weight iron-snlfur cluster used in both the synthesis of the VFe- and MoFe-protein. The gene for homocitrate synthase (nifV) is equally used in both systems. Modified from ref. 102. For the M cluster and the complete protein, see Figure 4.26. 

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