Nitrogen fixation, the conversion of dinitrogen to ammonia, is directly related to world-wide crop production and, as such, is one of the most important enzymatic reactions in nature. However, inspite of the large volume of research that has been done on this system, the mechanism of nitrogen fixation is still unclear. The purpose of this research is to gain a better understanding of this mechanism by studying the nitrogen-fixing enzyme, nitrogenase, in a mutationally altered system. Since nitrogenase is a molybdenum-containing enzyme and since it generally is felt that Mo is at the active site of the enzyme serving both for reduction and binding of dinitrogen, the mutants to be studied are those that fix dinitrogen in the absence of Mo but the presence of tungsten (W), a proven inhibitor of wild-type nitrogen fixation. A preliminary study of the enzyme in these mutants suggest that it contains equal amounts of Mo and W. In this proposal, this enzyme will be isolated and purified to homogeneity. It will be characterized in terms of metal (i.e. Fe, Mo and W) content, molecular weight, polypeptide composition and substrate specificity relative to the wild-type enzyme. Spectroscopic characterization will be accomplished by recording the enzyme's esr, optical and low-temperature MCD spectra. The active-site cofactor will also be isolated from this mutant and its metal composition determined. The second stage of the research proposed is the search for a possible alternate nitrogen-fixing system in azotobacter. It has been proposed by others that this alternate system functions during conditions of Mo deprivation. Differences in the relative rates of C2H2 and N2 reduction in the mutant will be measured to determine whether there are two different nitrogen-fixing systems. Antibodies against component I of normal nitrogenase will be used to inhibit in vitro fixation by the ordinary system thus allowing the detection of the possible alternate system. Finally, wild-type azotobacter will be used to deplete W-containing medium of Mo to allow the study of the growth of the mutant in W-medium without any Mo present to see if nitrogen fixation can occur in the absence of Mo.
