Nitrogen is essential for all life processes. It is found abundantly in the atmosphere as nitrogen gas. However, nitrogen gas cannot be metabolized by most organisms and, consequently, cellular nitrogen is usually obtained from nitrate, ammonia or part of an organic molecule. These sources of fixed nitrogen are relatively scarce and represent the limiting factor in Man's continuing efforts to feed the World population. Because fixed nitrogen is incompletely recycled in the global ecosystem due to nitrification and denitrification, biological nitrogen fixation occupies a pivotal position in the nitrogen cycle. It is performed only by certain prokaryotic microorganisms and is catalyzed by the enzyme nitrogenase. The significance of symbiotic microorganisms that fix N2 and deliver it to their plant hosts is well established and there are real prospects that the economy of nitrogen fixation and can be improved through the genetic manipulation of N2-fixing species. Such improvements, however, are dependent upon a fundamental understanding of the biochemical action and genetic regulation of the nitrogen-fixation components. Moreover, an understanding of the molecular mechanism of nitrogen fixation should prove invaluable in duplicating the activity of nitrogenase in chemical systems, which might lead to viable commercial processes. The proposed research centers on the molybdenum-iron protein of nitrogenase and specifically on the roles of its two prosthetic group, FeMoco and the P clusters, in catalysis. Using a series of altered MoFe proteins isolated from mutant strains of Azotobacter vinelandii, constructed by substituting residues in the immediate vicinity of either FeMoco or the P clusters, we propose to investigate the molecular origins of the catalytic modifications that these substitutions cause. Specifically, we plan to: (i) map the reaction surface of FeMoco to determine where substrates and inhibitors bind; (ii) determine the changes that occur in FeMoco when it becomes involved in enzyme turnover; and (iii) probe how the electron- accepting and substrate-reducing properties of the MoFe protein are affected by changes in the polypeptide environment of the P clusters to determine their function(s). The results should give significant insight into both understanding the catalytic mechanism and in generating targets for beneficial modifications of nitrogenase, such that a significant health and nutritional benefit should accrue in the future.
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