Site-directed mutagenesis and gene-replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe proteins in which the alpha-subunit residue-195 position, normally occupied by a histidine residue, was individually substituted by a variety of other amino acids. Structural studies have revealed that this histidine residue is associated with the FeMo-cofactor binding domain and probably provides an NH-->S hydrogen bond to a central bridging sulfide located within FeMo-cofactor. Substitution by a glutamine residue results in an altered MoFe protein that binds but does not reduce N2, the physiological substrate. Although N2 is not a substrate for the altered MoFe protein, it is a potent inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. This result provides evidence that N2 inhibits proton and acetylene reduction by simple occupancy of a common active site. N2 also uncouples MgATP from proton reduction catalyzed by the altered MoFe protein but does so without lowering the overall rate of MgATP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe protein to the MoFe protein that occurs during nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. Substitution of the alpha-histidine-195 residue by glutamine also imparts to the altered MoFe protein hypersensitivity of both its acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the alpha-histidine-195 residue might protect an Fe contained within the FeMo-cofactor from attack by CO. Finally, comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by various mutant strains suggest that the alpha-histidine-195 residue has a structural role, which serves to keep FeMo-cofactor attached to the MoFe protein and to correctly position FeMo-cofactor within the polypeptide matrix, such that N2 binding is accommodated.
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http://dx.doi.org/10.1021/bi00009a008 | DOI Listing |
Acta Crystallogr D Struct Biol
February 2025
State Key Laboratory for Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China.
P-clusters have been statistically analysed using the bond-valence sum (BVS) method together with weighting schemes. The crystallographic data come from the VFe proteins deposited in the Protein Data Bank (PDB) with high resolutions of better than 1.35 Å.
View Article and Find Full Text PDFNat Commun
December 2024
Division of Chemistry and Chemical Engineering 147-75 California Institute of Technology, Pasadena, CA, USA.
Biological nitrogen fixation, performed by the enzyme nitrogenase, supplies nearly 50% of the bioavailable nitrogen pool on Earth, yet the structural nature of the enzyme intermediates involved in this cycle remains ambiguous. Here we present four high resolution cryoEM structures of the nitrogenase MoFe-protein, sampled along a time course of alkaline reaction mixtures under an acetylene atmosphere. This series of structures reveals a sequence of salient changes including perturbations to the inorganic framework of the FeMo-cofactor; depletion of the homocitrate moiety; diminished density around the S2B belt sulfur of the FeMo-cofactor; rearrangements of cluster-adjacent side chains; and the asymmetric displacement of the FeMo-cofactor.
View Article and Find Full Text PDFChembiochem
November 2024
Department of Inorganic Spectroscopy, Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34-36, Mülheim an der Ruhr, 45470, Germany.
Proc Natl Acad Sci U S A
November 2024
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid e Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria/Consejo Superior de Investigaciones Científicas, Madrid 28223, Spain.
The maturation and installation of the active site metal cluster (FeMo-co, FeSCMo--homocitrate) in Mo-dependent nitrogenase requires the protein product of the gene for production of the FeS cluster precursor (NifB-co, [FeSC]) and the action of the maturase complex composed of the protein products from the and genes. However, some putative diazotrophic bacteria, like sp. RS-1, lack the genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN.
View Article and Find Full Text PDFJ Biol Chem
November 2024
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain; Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain. Electronic address:
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