FeMoco biosynthesis

Subunits of NifN2E2 possibly a scaffold for FeMoco biosynthesis... 

The NifN2E2 complex has been isolated from both a nifB mutant (72) and a nifH mutant (75), that is, mutants blocked at different stages in FeMoco biosynthesis, with the aim of determining whether FeMoco precursors might accumulate on NifN2E2. 

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. 

These genetic data support the suggestion of a parallel route for the synthesis of cofactors of Mo-independent nitrogenase function involving some early steps in common with FeMoco biosynthesis. 

The detailed characterization of the FeMoco site has involved parallel studies of the site within the protein and in its extracted form. The authentication of the extracted FeMoco involves the production and use of mutant organisms that make an inactive FeMo protein that contains all subunits and P clusters, but lacks the FeMoco sites.A mutant of Azotobacter vinelandii called UW-45 (UW = C/niversity of Wisconsin) was first used to assay for isolated FeMoco.Since several genes are involved in specifying FeMoco biosynthesis, mutants lacking these genes produce FeMo protein either lacking FeMoco or having a defective version of FeMoco. Mutants such as NifB of Klebsiella pneumoniae lack cofactor, and an inactive apo protein can be isolated from them. 

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. 

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

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. 

A great deal has been learned about the biosynthesis of nitrogenases, but at the moment the process is understood only in broad outline. The detailed roles of the individual gene products require much further investigation, which may once more indicate fresh approaches to some of the problems identified herein. In particular, if the biosynthetic steps can be emulated chemically, then it may be possible to synthesize FeMoco in large quantities in order to allow its detailed analysis at the atomic level. 

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... 

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