Nitrogen is essential for all life processes. It is found abundantly in the atmosphere as nitrogen gas (N2). However, nitrogen gas cannot be metabolized by most organisms and, consequently, cellular nitrogen is usually obtained from nitrate, ammonia, or as 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, 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 impact of symbiotic microorganisms that fix N2 for delivery 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 depend 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 developing environmentally-sound fertilizer systems.

We are applying a biochemical-genetical approach to determine how nitrogenase converts N2 into ammonia. We focus mainly on the larger of the two nitrogenase component proteins, called the molybdenum-iron (MoFe) protein, and specifically on the catalytic roles of its two biologically unique catalytic groups, the iron-molybdenum cofactor (FeMo-cofactor) and the iron-sulfur-containing P cluster. After constructing a series of altered MoFe proteins, which are isolated from mutant strains of Azotobacter vinelandii, with amino acid substitutions in the immediate vicinity of either FeMo-cofactor or the P cluster, we investigate the molecular origins of the catalytic modifications that these substitutions cause and so gain insight into the mechanism of the enzyme. Through this protocol, we have determined that FeMo-cofactor either contains or is part of the N2-fixing site and the P cluster acts as an electron storage and transfer agent. Currently we are: (1) mapping the reaction surface of FeMo-cofactor to determine where the various substrates and inhibitors bind; (2) determining electronic and structural changes that occur in FeMo-cofactor when it engages in enzyme turnover; and (3) probing how the electron-accepting and substrate-reducing properties of the MoFe protein are affected by changes in the polypeptide environment of the P cluster to determine its electron-storage capacity and to confirm its mechanistic role(s). The results of our investigations should both increase our understanding of the catalytic mechanism and aid in generating targets for beneficial targets modifications of nitrogenase, such that a significant health and nutritional benefit accrues in the future.

Han, J. and Newton, W. E. (2004) Differentiation of Acetylene-Reduction Sites by Stereoselective Proton Addition during Azotobacter vinelandii Nitrogenase-Catalyzed C2D2 Reduction. Biochem. 43:2947-2956.   [Abstract]

Vichitphan, K. and W. E. Newton (2002) Acetylene reduction with Azotobacter vinelandii Mo-nitrogenase: Role of glutamine-191 of the MoFe protein, in Nitrogen Fixation: Global Perspectives (T. Finan, M. R. O?Brian, D. B. Layzell, J. K. Vessey and W. E. Newton, eds.) CABI Publishing, NY, p. 365.   [Abstract]

Fisher, K., Newton, W. E., and 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. Biochem. 40:3333-3339.   [Abstract]

Fisher, K., Dilworth, M. J., Kim, C.-H., and Newton, W. E. (2000) Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on Catalyzed Reduction of Acetylene and Ethylene. Biochem. 39:2970-2979.   [Abstract]

Fisher, K., Dilworth, M. J., Kim, C.-H., and Newton, W. E. (2000) Azotobacter vinelandii Nitrogenases with Substitutions in the FeMo-Cofactor Environment of the MoFe Protein: Effects of Acetylene and Ethylene on Interactions with H+, HCN, and CN-. Biochem. 39:10855-10865.   [Abstract]

Fisher, K., Dilworth, M. J., and Newton, W. E. (2000) Differential Effects on N2 Binding, HD Formation, and Azide Reduction with alpha-195-Histidine and alpha-191-Glutamine-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase. Biochem. 39:15570-15577.   [Abstract]

Maskos, Z., Sorlie, M., Fisher, K., Newton, W. E., and Hales, B. J. (1999) EPR Studies of the MoFe Proteins of Nitrogenase during Enzymatic Turnover in the Presence of Carbon Monoxide. J. Inorg. Biochem. 74:224.

Dilworth, M. J., Fisher, K., Kim, C.-H., and Newton, W. E. (1998) Effects on substrate reduction of substitution of histidine-195 by glutamine in the -subunit of the MoFe protein of Azotobacter vinelandii nitrogenase. Biochemistry 37:17495.   [Abstract]

Lee, H.-I., Thrasher, K. S., Dean, D. R., Newton, W. E., and Hoffman, B. M. (1998) 14N electron spin-echo envelope modulation of the S=3/2 spin system of the Azotobacter vinelandii nitrogenase iron-molybdenum cofactor. Biochemistry 37:13370.   [Abstract]

Shen, J., Dean, D. R., and Newton, W. E. (1997) Evidence for multiple redox states, multiple substrate-binding sites and distinct inhibitor sites from an altered Azotobacter vinelandii nitrogenase MoFe protein. Biochemistry 36:4884.   [Abstract]