Abstract

The research proposed herein seeks to determine the structure and function of the iron-molybdenum cofactor (FeMoco) from the MoFe protein of Azotobacter vinelandii nitrogenase and how nitrogenase is organized to deliver the required reducing equivalents to substrate. Experiments include: purification and crystallization of FeMoco to determine its chemical composition and ultimately its structure by x-ray crystallography; x-ray absorption spectroscopy at Mo and S edges of FeMoco to gain structural insights and changes therein on redox and chemical modification; electrochemical, chemical and spectroscopic studies to elucidate FeMoco's redox properties and potential for catalysis; and the purification, characterization and reconstitution with FeMoco of the cofactor-deficient MoFe protein from mutant cells.
This research aims to determine the individual responsibilities of FeMoco and its complementary protein in catalysis. Such information will be invaluable in attempts to enhance biological nitrogen fixation and to duplicate it synthetically.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK037255-04
Application #
3236079
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1985-08-01
Project End
1990-03-31
Budget Start
1988-12-01
Budget End
1990-03-31
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
University of California Davis
Department
Type
Earth Sciences/Resources
DUNS #
094878337
City
Davis
State
CA
Country
United States
Zip Code
95618

  Publications

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 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
Fisher, K; Dilworth, M J; Newton, W E (2000) Differential effects on N(2) binding and reduction, HD formation, and azide reduction with alpha-195(His)- and alpha-191(Gln)-substituted MoFe proteins of Azotobacter vinelandii nitrogenase. Biochemistry 39:15570-7

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