No description available.

The FeMo cofactor is one of two types of prosthetic group found in the larger of the two nitrogenase component proteins, called the MoFe protein, and it is strongly implicated as the substrate binding and reduction site. The glutamine-191 residue in the Alpha-subunit of the MoFe protein of A. vinelandii nitrogenase was targeted for substitution because its side chain is involved in a hydrogen-bond network from one of the terminal carboxylates of the homocitrate component of FeMo cofactor through to the backbone NH of Alpha Gly-61, which is adjacent to Alpha Cys-62, which ligates to the P cluster (the second type of prosthetic group in the MoFe protein). A variety of altered MoFe proteins produced by the A. vinelandii mutant strains, namely the Alpha Pro-191, Alpha Ser-191, Alpha Thr-191, Alpha His-191, Alpha Glu-191, and Alpha Arg-191 altered MoFe proteins, have been purified to homogeneity and the catalytic properties of these altered MoFe proteins have been compared to those of wild type MoFe protein. Unlike wild type, the six altered MoFe proteins have decreased catalytic activity on substrate reduction and exhibited H2 evolution that was partially inhibited by added CO. Moreover, some of altered MoFe proteins with lower specific activity for the C2H4 production can produce C2H6 from C2H2. The results from the pH and activity studies indicate that the substitutions on the MoFe protein have an effect on the contribution of the responsible acid-base group(s) involved in proton transfer for H+- and C2H2-reduction. Furthermore, the inhibition by CO of hydrogen evolution by these altered MoFe proteins is likely from a lowering of the rate of both electron and proton transfer to the H+- reduction site(s). Some altered MoFe proteins but not wild type MoFe protein can produce C2H6 from C2H2. This observation suggested a lower apparent binding affinity for C2H2 and a slower proton transfer to C2H2 reduction with these altered MoFe proteins, which allow the intermediate to stay at the site longer and be further reduced by two electrons and two protons to give C2H6. These changes in the biochemical properties of these altered MoFe proteins indicate that the Alpha Gln-191 residue is intimately involved in substrate binding and reduction including proton delivery to substrate. / Ph. D.

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.

Master of Science

Thesis (Ph.D.)--Mississippi State University. Department of Physics and Astronomy. / Title from title screen. Includes bibliographical references.

Sorghum plants excrete phenolic acids which reduce subsequent crop yields. These acids accumulate in field soil by combining with soil and clay particles to form stable complexes which remain until degraded by bacterial metabolism. The amount of phenolic acids in soil samples were obtained by gas chromatography measurements, while Azotobacter populations were obtained by plate counts in 40 sorghum field samples from Denton County, Texas. One can conclude that increasing the Azotobacter population in the soil increased the degradation rate of phenolic acids proportionally. It is proposed that seed inoculation will introduce selected strains of Azotobacter into the soil. The presence of Azotobacter should increase crop size in subsequent plantings.