No abstract

The dinitrogen-binding site in the Mo-based nitrogenase is FeMo-cofactor, a metallo-sulfur cluster of composition MoFe7S9·R-homocitrate. The NifV- mutant nitrogenase from Klebsiella pneumoniae contains an FeMo-cofactor in which homocitrate has been replaced by citrate (i.e., MoFe7S9·citrate). Both the wild type and mutant cofactors (in the S = 3/2 spin state) can be extracted into N-methylformamide. The extracted cofactors bind one molecule of PhS- at the tetrahedral Fe, and the rate of this reaction depends on what else is coordinated to the cluster. No differences were observed between the reactivities of wild-type and NifV- cofactors with PhS- when they were complexed with CN-, N3 -, or H+. However, when imidazole is bound, the kinetics of the reactions of PhS- with the two cofactors are very different. Here we propose that R-homocitrate (but not citrate) can hydrogen bond to the imidazole ligand on Mo, and that this perturbs the electron distribution within the cluster core, and hence its reactivity with PhS-. Using the X-ray crystallographic data for the MoFe-protein of nitrogenase and molecular mechanics calculations, we have investigated the implications of these findings on the action of the enzyme. Our model shows that R-homocitrate is uniquely capable of facilitating the binding of dinitrogen by allowing the substrate access to Mo after dissociation of the Mo−carboxylate bond while simultaneously influencing the electron-richness of the cofactor by hydrogen bonding of the pendant −CH2CH2CO2 arm to the imidazole group of Hisα442. The whole process is mediated by hydrogen bonding of amino acid side chains to the carboxylate groups of R-homocitrate.

N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed.

The x-ray crystal structure of NifV ؊ Klebsiella pneumoniae nitrogenase MoFe protein (NifV ؊ Kp1) has been determined and refined to a resolution of 1.9 Å. This is the first structure for a nitrogenase MoFe protein with an altered cofactor. Moreover, it is the first direct evidence that the organic acid citrate is not just present, but replaces homocitrate as a ligand to the molybdenum atom of the iron molybdenum cofactor (FeMoco). Subsequent refinement of the structure revealed that the citrate was present at reduced occupancy.