The objective of this project is to study the mechanism by which ATP hydrolysis is linked to electron transfer and the reduction of molecular nitrogen and/or protons in the metalloenzyme complex, nitrogenase. These studies may serve as a more general model of energy transduction for other systems. The approach is to combine recombinant DNA techniques and physicochemical techniques to study the site of ATP binding and the mechanism of ATP hydrolysis in nitrogenase. In particular, oligonucleotide directed mutagenesis will be used to create mutants--initially in the Fe-protein component of nitrogenase since this protein is known to bind nucleotides in the absence of any other protein component. Target residues have been suggested by computer modeling of the likely nucleotide binding domain with references to known nucleotide binding domains. Computer modeling studies of this component will continue. Detailed comparison of purified wild-type and mutant proteins will be carried out. The thrust of the biochemical studies will be to employ ATP analogs, chemical modifying agents and protein crosslinking chemicals to probe the ATP binding site of the iron protein and the protein docking sites on both nitrogenase components. Physical techniques will be used to monitor the integrity of modified proteins and to look for specific interactions of modifying agents with the metal centers of the nitrogenase components. These studies will increase understanding of the site at which ATP hydrolysis occurs, its relationship to the various metal clusters (Fe-S and Mo-Fe-S) in the component proteins, the role of ATP in the system, and provide important structure/function information about the enzyme complex.
