9513512 Howard Nitrogenase is a two-protein component enzyme which mediates both electron transfer between the two proteins (Fe-protein and Mo-Fe protein) and ATP hydrolysis, which provides the energy for the electron transfer. Fe-protein has a strong secondary structural motif similar to G-proteins and Ras which suggests that Fe-protein functions as a nucleotide-dependent switch. The important distinction is that Fe-protein is competent for electron transfer to MoFe-protein only in the complex and during ATP hydrolysis. The objective of this proposal is to develop methods to stabilize the complex by trapping the transition from ATP to ADP states. Mutagenesis and kinetic analyses with nucleotide analogues provide a rational approach based upon the nucleotide switch model. Specific questions to be addressed are: l. What is the location of the electron in the AlF4- ADP complex of Avl and Av2? 2. Can electron transfer be trapped in one of the redox clusters of the complex? 3. What are the amino acid residues that stabilize the transition state during the electron transfer step? %%% Nitrogen fixation is the process whereby atmospheric nitrogen gas is converted to the biologically important form, ammonia. The latter is incorporated into all major classes of biomolecules and, hence, is a required component of all living systems. How this process is performed by the nitrogenase enzyme has both practical and general biological importance. For example, nitrogenase is able to catalyze the reaction at 30 C and normal atmospheric pressure while the industrial process requires a hundred times the normal atmospheric pressure and elevated temperature. Understanding details of the enzyme catalysis portends more efficient and therefore more cheaply abundant ammonia for fertilizer. The nitrogenase reaction involves the transfer, one at a time, of electrons between the component proteins. Concomitant with the electron transfer, 2 ATP are hydrolyzed (broken down) to provide the energy for the transfer. It is the coupling of the two events that controls the overall chemical reaction. Based upon the elegant x-ray crystal structures of the two nitrogenase proteins, we noted that the electron donor component (Fe-protein) had a striking structural similarity to other nucleotide-dependent switch proteins such as those involved in muscle contraction, metabolic regulation associated with tumor initiation and others. In all of these molecules, the role of the nucleotide is to stabilize specific protein conformations; the conformation switches as the nucleotide is hydrolyzed. The hypothesis that a conformational "gate" in nitrogenase is opened during ATP hydrolysis that allows the electron to move from one site to another will be tested by trapping the proteins in the conformation where the gate is momentarily open. Because of the similarity between the nitrogenase protein and the proteins involved in the other biological processes, these results should help to elucidate the mechanisms of these other systems as well. ***
