Abstract

The research proposed herein seeks to determine the molecular level details of nitrogenase turnover, substrate binding, substrate reduction and metal cluster synthesis and insertion. The major approach is to construct, express and purify site-directed mutant and other variants of the Fe and MoFe proteins of nitrogenase. These purified proteins will then be characterized by X-ray crystallography, by biochemical and kinetic studies and by a variety of spectroscopic techniques. These experiments are designed to determine the chemical, redox and physical properties of each component of the Fe and MoFe proteins and how they interact with each other. The information obtained will be invaluable in attempts to enhance biological nitrogen fixation or to duplicate it synthetically. Increased fixed nitrogen equates to increased protein supply, which is a significant factor in good nutrition and health. In addition, the knowledge gained here is expected to greatly increase our understanding of a variety of fundamentally important processes like nucleotide controlled energy transduction, long range electron and proton transfer, and metal cluster insertion.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043144-09
Application #
2608904
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1989-12-01
Project End
1999-03-31
Budget Start
1997-12-01
Budget End
1999-03-31
Support Year
9
Fiscal Year
1998
Total Cost
Indirect Cost

  Institution

Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697

  Publications

Ribbe, Markus W; Hu, Yilin; Guo, Maolin et al. (2002) The FeMoco-deficient MoFe protein produced by a nifH deletion strain of Azotobacter vinelandii shows unusual P-cluster features. J Biol Chem 277:23469-76
Guo, Maolin; Sulc, Filip; Ribbe, Markus W et al. (2002) Direct assessment of the reduction potential of the [4Fe-4S](1+/0) couple of the Fe protein from Azotobacter vinelandii. J Am Chem Soc 124:12100-1
Ribbe, M W; Burgess, B K (2001) The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase. Proc Natl Acad Sci U S A 98:5521-5
Strop, P; Takahara, P M; Chiu, H et al. (2001) Crystal structure of the all-ferrous [4Fe-4S]0 form of the nitrogenase iron protein from Azotobacter vinelandii. Biochemistry 40:651-6
Frank, P; Angove, H C; Burgess, B K et al. (2001) Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior. J Biol Inorg Chem 6:683-97
Ribbe, M W; Bursey, E H; Burgess, B K (2000) Identification of an Fe protein residue (Glu146) of Azotobacter vinelandii nitrogenase that is specifically involved in FeMo cofactor insertion. J Biol Chem 275:17631-8
Kim, S; Burgess, B K (1996) Evidence for the direct interaction of the nifW gene product with the MoFe protein. J Biol Chem 271:9764-70
Ma, L; Brosius, M A; Burgess, B K (1996) Construction of a form of the MoFe protein of nitrogenase that accepts electrons from the Fe protein but does not reduce substrate. J Biol Chem 271:10528-32
Conradson, S D; Burgess, B K; Newton, W E et al. (1994) Selenol binds to iron in nitrogenase iron-molybdenum cofactor: an extended x-ray absorption fine structure study. Proc Natl Acad Sci U S A 91:1290-3
Gavini, N; Ma, L; Watt, G et al. (1994) Purification and characterization of a FeMo cofactor-deficient MoFe protein. Biochemistry 33:11842-9

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