Site-directed mutagenesis and gene replacement procedures were used to isolate mutant strains of <i>Azotobacter vinelandii</i> that produce altered MoFe proteins where the α-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. The present study investigates the role of the α-histidine-195 residue in nitrogenase catalysis by examining the altered MoFe proteins. Comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by the <i>Azotobacter vinelandii</i> mutant strains suggest that the α-histidine-195 residue has a structural role which serves to keep the FeMo-cofactor attached to the MoFe protein and to correctly position the FeMo-cofactor within the polypeptide matrix such that N₂ binding is accommodated. Substitution of the α-His-195 residue by a glutamine residue results in an altered MoFe protein that binds but does not reduce N₂, the physiological substrate. Stopped-flow spectroscopic analyses indicate that the α-195^gln MoFe protein is unable to reduce N₂ even though the altered MoFe protein can reach the redox state necessary for N₂ reduction. Although, N₂ is not a substrate for the altered MoFe protein, it is an 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 the active site. The α-195^gln MoFe protein catalyzes HD formation in the presence of N₂ and D₂. Moreover, N₂ binding at the active site of the altered MoFe protein is inhibited by the addition of D₂. These observations indicate that binding of nitrogen to the enzyme is necessary but its reduction is not required for the formation of HD. N₂ uncouples MgATP from proton reduction catalyzed by the α-195gln MoFe protein, but does so without lowering the overall rate of MgA TP 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. N₂-induced uncoupling of ATP hydrolysis from substrate reduction by the α-195^gln MoFe protein is reversed by the addition of H₂ (D₂) in the assay atmosphere. This observation can successfully be explained if it-is assumed that the altered MoFe protein has a much greater binding affinity for H₂ (D₂) than for N₂. Substitution of the α-histidie-195 residue by glutamine also imparts hypersensitivity of acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the α-histidine- 195 residue might protect an Fe contained within FeMo-cofactor from attack by CO. / Ph. D.