Nitrogen is essential for all life processes. Although abundant in the Earth's atmosphere as N2 gas, this gas is not metabolized by most life forms. Consequently, cellular nitrogen is usually obtained from ammonia, nitrate, or as part of an organic molecule. These sources of fixed nitrogen are relatively scarce and represent the limiting nutrient in global food production. Because organic nitrogen is incompletely recycled in the global ecosystem due to the processes of nitrification and denitrification, biological N2 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 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 the genetic regulation of the nitrogen-fixation components. Moreover, an understanding of the molecular mechanism 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. A series of altered molybdenum-iron proteins, which are constructed in, and isolated from, mutant Azotobacter vinelandii strains, will be used. These altered proteins have targeted amino-acid residues, which lie in the vicinity of either of the prosthetic groups, substituted by other residues. They will be used to investigate the molecular origins of catalysis by nitrogenase. The specific objectives of these studies are to determine how nitrogenase is organized to effect biological nitrogen fixation and specifically how each of the prosthetic groups, the iron-molybdenum cofactor and the P cluster, functions in catalysis. The results should aid both in understanding the catalytic mechanism and in generating targets for beneficial modifications of nitrogenase, such that a significant health and nutritional benefit accrues in the future.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Metallobiochemistry Study Section (BMT)
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May, Michael K
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Virginia Polytechnic Institute and State University
Schools of Earth Sciences/Natur
United States
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Fisher, Karl; Lowe, David J; Tavares, Pedro et al. (2007) Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function. J Inorg Biochem 101:1649-56
Xiao, Yuming; Fisher, Karl; Smith, Matt C et al. (2006) How nitrogenase shakes--initial information about P-cluster and FeMo-cofactor normal modes from nuclear resonance vibrational spectroscopy (NRVS). J Am Chem Soc 128:7608-12
Durrant, Marcus C; Francis, Amanda; Lowe, David J et al. (2006) Evidence for a dynamic role for homocitrate during nitrogen fixation: the effect of substitution at the alpha-Lys426 position in MoFe-protein of Azotobacter vinelandii. Biochem J 397:261-70
Fisher, Karl; Dilworth, Michael J; Newton, William E (2006) Azotobacter vinelandii vanadium nitrogenase: formaldehyde is a product of catalyzed HCN reduction, and excess ammonia arises directly from catalyzed azide reduction. Biochemistry 45:4190-8
Maskos, Zofia; Fisher, Karl; Sorlie, Morten et al. (2005) Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover. J Biol Inorg Chem 10:394-406
Fisher, Karl; Newton, William E (2005) Nitrogenase proteins from Gluconacetobacter diazotrophicus, a sugarcane-colonizing bacterium. Biochim Biophys Acta 1750:154-65
Han, Jaehong; Newton, William E (2004) Differentiation of acetylene-reduction sites by stereoselective proton addition during Azotobacter vinelandii nitrogenase-catalyzed C2D2 reduction. Biochemistry 43:2947-56
Fisher, K; Newton, W E; Lowe, D J (2001) Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: evidence for S = 3/2 spin states from reduced MoFe-protein intermediates. Biochemistry 40:3333-9
Fisher, K; Dilworth, M J; Kim, C H et al. (2000) Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene. Biochemistry 39:2970-9
Fisher, K; Dilworth, M J; Kim, C H et al. (2000) Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: effects of acetylene or ethylene on interactions with H+, HCN, and CN-. Biochemistry 39:10855-65

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