The objective of this project is to study the mechanism by which ATP hydrolysis is linked to electron transfer and the reduction of molecular nitrogen and/or protons in the metalloenzyme complex, nitrogenase. These studies may serve as a more general model of energy transduction for other systems. The approach is to combine recombinant DNA techniques and physicochemical techniques to study the site of ATP binding and the mechanism of ATP hydrolysis in nitrogenase. In particular, oligonucleotide directed mutagenesis will be used to create mutants--initially in the Fe-protein component of nitrogenase since this protein is known to bind nucleotides in the absence of any other protein component. Target residues have been suggested by computer modeling of the likely nucleotide binding domain with references to known nucleotide binding domains. Computer modeling studies of this component will continue. Detailed comparison of purified wild-type and mutant proteins will be carried out. The thrust of the biochemical studies will be to employ ATP analogs, chemical modifying agents and protein crosslinking chemicals to probe the ATP binding site of the iron protein and the protein docking sites on both nitrogenase components. Physical techniques will be used to monitor the integrity of modified proteins and to look for specific interactions of modifying agents with the metal centers of the nitrogenase components. These studies will increase understanding of the site at which ATP hydrolysis occurs, its relationship to the various metal clusters (Fe-S and Mo-Fe-S) in the component proteins, the role of ATP in the system, and provide important structure/function information about the enzyme complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM040067-02
Application #
3297376
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Bolin, J T; Ronco, A E; Morgan, T V et al. (1993) The unusual metal clusters of nitrogenase: structural features revealed by x-ray anomalous diffraction studies of the MoFe protein from Clostridium pasteurianum. Proc Natl Acad Sci U S A 90:1078-82
Seefeldt, L C; Mortenson, L E (1993) Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+. Protein Sci 2:93-102
Mortenson, L E; Seefeldt, L C; Morgan, T V et al. (1993) The role of metal clusters and MgATP in nitrogenase catalysis. Adv Enzymol Relat Areas Mol Biol 67:299-374
Jones, R; Woodley, P; Birkmann-Zinoni, A et al. (1993) The nifH gene encoding the Fe protein component of the molybdenum nitrogenase from Azotobacter chroococcum. Gene 123:145-6
Hyman, M R; Seefeldt, L C; Morgan, T V et al. (1992) Kinetic and spectroscopic analysis of the inactivating effects of nitric oxide on the individual components of Azotobacter vinelandii nitrogenase. Biochemistry 31:2947-55
Seefeldt, L C; Morgan, T V; Dean, D R et al. (1992) Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction. J Biol Chem 267:6680-8
Fu, W G; Morgan, T V; Mortenson, L E et al. (1991) Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase. FEBS Lett 284:165-8
Morgan, T V; McCracken, J; Orme-Johnson, W H et al. (1990) Pulsed electron paramagnetic resonance studies of the interaction of Mg-ATP and D2O with the iron protein of nitrogenase. Biochemistry 29:3077-82