Humankind’s tremendous industrial and technological progress over the last two centuries has been driven by the natural abundance and availability of fossil fuels. As those reserves deplete, the prudent course of action would be to develop other readily available fuel sources. Some research efforts using biomolecules involve the hydrogenases and nitrogenases with the goal of evolving dihydrogen. At the nitrogenase active site, the iron-molybdenum cofactor (FeMo-co) catalyzes the reduction of dinitrogen and protons to form ammonia and dihydrogen. Toward the goal of producing dihydrogen passively as an alternative fuel, a novel advanced material has been developed. CdSe nanoparticles complexed with FeMo-co, in both aqueous and organic solvent systems showed complex formation. When the system was interrogated by EPR spectroscopy, evidence of electron transfer was observed. The CdSeMSA●NafY●FeMo-co system when illuminated with visible light evolved dihydrogen consistently in four different experimental sets under the same reaction conditions. NifEN protein plays an important role in the biosynthesis of FeMo-co in addition to the involvement of NifU, NifS, NifB, NifX, NifH and NafY. After NifB synthesizes a FeMo-co precursor, 6-Fe NifB-co, NifEN further incorporates additional Fe, S, Mo, and (R)-homocitrate to complete the synthesis of FeMo-co. Molybdenum is provided to NifEN as its oxoanion, Mo(VI)O4 2- ; however, in FeMo-co molybdenum is in the oxidation state of Mo(IV). EPR spectroscopic investigation of NifEN turnover samples showed a signal at g = 2.00 that was dependent on molybdate concentration. Power and temperature profiles gave evidence that the g iv = 2.00 EPR signal was distinct from the Fe-S clusters in NifEN. The species observed at g = 2.00 was assigned to the reduction of Mo(VI) to Mo(V). How to utilize the effectiveness of FeMo-co and complex it to photoactive materials for the purpose of evolving dihyrogen upon illumination, thus providing a sustainable alternative energy source is one subject of this dissertation. A related subject is to gain an understanding of the biosynthetic pathway of FeMo-co by investigation of NifEN turnover experiments. This understanding should contribute towards the development of improved catalysts for meeting future energy demands.

The Mo-nitrogenase complex is composed of two components, Fe-protein and MoFe-protein. This complex is able to catalyze the reduction of N2 through the MgATP dependent transfer of electrons from the Fe-protein Fe4S4 cluster to the MoFe-protein P-cluster and, subsequently, to the iron-molybdenum cofactor (FeMoco). FeMo-co is a Fe7S9MoC-(R)-homocitrate cluster and has two biosynthetic precursors, NifB-co and L-cluster, of unknown structure and composition. The biosynthesis of FeMo-co is an enigmatic process that minimally requires NifB, NifEN, Fe-protein, MoO4 2- , (R)-homocitrate and S-adenolsylmethionine. A means to isolate the NifB enzyme for characterization has been developed through use of a GST-fusion tag. Double recombination of A. vinelandii strains with a constructed vector has yielded strains capable of nif promoter regulated expression of GST-NifB. Extracts of strains containing GST-NifB were shown to activate the Monitrogenase complex in biochemical complementation assays. Mass spectroscopy was then used to verify successful isolation of GST-NifB by GSH-Sepharose affinity purification. The number of NifB-co ligand binding sites and ligand types were examined by EXAFS analysis of samples containing selenol and thiol ligands. A Fe6S9C model for NifB-co was optimized to best fit the EXAFS data, where a 2-fold discrepancy in binding sites implied by thiol or selenol only ligand samples suggests Fe-(μ2S)-Fe binding in the absence of Se. Samples containing heterogeneous ligand types indicated that NifX bound NifB-co ligates to four cysteine residues and one molecule of DTT.