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.

Previous studies revealed that β-95-Cys provides an essential ligand to one of the Fe atoms on the P cluster within the MoFe protein of nitrogenase, and a limited number of substitutions at this position resulted in inactive nitrogenase. It was also found that the counterpart of β-95-Cys, α-88-Cys, which also acts as a cysteinyl ligand to the P cluster, is replaceable without a complete loss of activity. In order to study the structure-function relationship of the protein environment in this region with respect to the P-cluster, subtle changes were introduced at β-95-Cys in <i>Azotobacter vinelandii</I>nitrogenase through site-directed mutagenesis and gene replacement method. Some crude extracts from the mutants with substitutions at β-Cys contain typical FeMo cofactor EPR signal. The β-95^Asp MoFe protein also has significant nitrogenase activity, but lower, suggesting that β-Cys is not absolutely required for both FeMo cofactor insertion and nitrogenase activity. In order to characterize its catalytic features, the β-95^Asp MoFe protein was purified from mutant strain DJ1096. It has significantly reduced H⁺ reduction, C₂H₂-reduction and N₂-reduction activity. It was found that a higher percentage of electron flux goes to H⁺ compared to the wild type MoFe protein. It was also found that reductant independent ATP hydrolysis occurs during H⁺ reduction, suggesting that the altered MoFe protein has an increased affinity for Fe protein-ADP complex. Surprisingly, CO has a significant enhancement effect on H⁺ reduction at low electron flux, but not at high electron flux, and highly couples the electron transfer to ATP hydrolysis. These results indicate that the binding of CO to the MoFe protein may either decrease the affinity of Fe-ADP complex for the β-95^Asp MoFe protein or facilitate electron acceptance by the P cluster, thus improving the electron transfer to substrate. / Master of Science