Nitrogen fixation, the conversion of dinitrogen from the air into ammonia, is catalyzed by the enzyme nitrogenase. Nitrogenase uses two unique metal clusters to split the nitrogen triple bond. The unusual structures of these metal clusters have captured the imagination of biochemists and inorganic chemists. Recently, two X-ray structures of component 1 proteins containing these metal clusters have been determined. Despite the large volume of research that has been done on nitrogenase during the past twenty-five years, the mechanism of nitrogen fixation is not known. The availability of the X-ray structure opens the door to structure-based mechanistic studies of nitrogenase. Our laboratory has the advantage of using two enzyme forms, the conventional Mo-nitrogenase and the alternative V-nitrogenase from the bacterium Azotobacter vinelandii, to study the various roles of the metal clusters and protein components in enzyme function. An innovative experimental approach to the enzyme mechanism is proposed based on the use of hybrid and mutant forms of nitrogenase. The hybrid forms will be produced by incorporation of the Vfe-cofactor into the Mo- nitrogenase protein lacking cofactor, and the reciprocal incorporation of the MoFe-cofactor into the V-nitrogenase protein. Preliminary studies show that the latter hybrid has altered substrate reduction patterns. Mutant proteins will be produced by using site-directed mutagenesis to systematically replace key amino acids surrounding the cofactor in V- nitrogenase with the corresponding amino acid of Mo-nitrogenase. The enzymology of these proteins will be fully characterized (i.e., Km, product distribution and response to varying electron flux) for nitrogen fixation, acetylene reduction and dihydrogen evolution. CO inhibition of exogenous substrate reduction and H2 inhibition of nitrogen fixation will also be characterized. In the latter system, D2 and H2O (or H2 and D2O) will be used to investigate the ability of these new protein to support HD formation during nitrogen fixation. Finally, these proteins will be investigated to determine whether the mutation has produced an uncoupling of MgATP hydrolysis from electron transport. EPR spectroscopy will be used to probe changes in the electronic structure of the cofactor in the new proteins. This technique will also be used to monitor whether the cofactor is reduced by component 2 and whether CO-induced S = 1/2 signals are generated when the proteins are in turnover-competent medium with CO. Finally, some of the proteins (later selected according to their unique phenotype) will be crystallized and their x-ray diffraction spectra taken in order to gain greater structural information. These proposed experiments with hybrid and mutant enzymes will lay the foundation for future work on designed nitrogenases.
