Abstract

The goal of our laboratory is to determine the role of protein structure in redox reactions, especially those involving Fe:S centers for electron transfer. We have chosen nitrogen reduction as a model. Although elegant spectroscopic studies have identified new Fe:S and Mo:Fe:S centers in nitrogenase proteins, we have only a limited picture of the protein structure. Yet, it is the latter which undoubtedly influences the properties of the metallo centers. For nitrogenase, no specific amino acids have been identified as part of the electron transfer processes. Only the Fe:S center ligands for the Fe-protein have been determined. It is our intention to use solution chemical methods to study changes in protein structure during catalysis and electron transfer. We will correlate the structural changes and chemical reactivity with the spectral properties of Fe:S proteins. For the proposed studies, the Fe-protein (Av2) and MoFe-protein (Av1) from Azotobacter vinelandii nitrogenase will be used. The specific objectives for the next five years are: 1. To study the interconversion of the Fe:S center in Av2 using spectral and chemical modification methods. 2. To reconstitute enzymatically active Av2 from inactive forms. 3. To identify amino acid residues in the ATP/ADP binding site and their relationship to the Fe:S center. 4. To identify potential thiol ligands of Fe:S and Mo:Fe:S centers in Avl. 5. To identify the binding regions on Av1 and Av2 for complex formation. 6. To identify potential catalytically important residues in the substrate reduction site. 7. To study the thiol reactivity in """"""""simple"""""""", spectrally well-defined Fe:S proteins.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034321-02
Application #
3285094
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1986-08-01
Project End
1991-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455

  Publications

Eccleston, E D; White, T W; Howard, J B et al. (1994) Characterization of a cell surface glycoprotein associated with maturation of rat spermatozoa. Mol Reprod Dev 37:110-9
Wolle, D; Kim, C; Dean, D et al. (1992) Ionic interactions in the nitrogenase complex. Properties of Fe-protein containing substitutions for Arg-100. J Biol Chem 267:3667-73
Busse, S C; La Mar, G N; Howard, J B (1991) Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation. J Biol Chem 266:23714-23
Howard, J B; Rees, D C (1991) Perspectives on non-heme iron protein chemistry. Adv Protein Chem 42:199-280
Deits, T L; Howard, J B (1990) Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction. J Biol Chem 265:3859-67
Willing, A; Howard, J B (1990) Cross-linking site in Azotobacter vinelandii complex. J Biol Chem 265:6596-9
Adams, M W; Eccleston, E; Howard, J B (1989) Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum. Proc Natl Acad Sci U S A 86:4932-6
Louis, C F; Hur, K C; Galvan, A C et al. (1989) Identification of an 18,000-dalton protein in mammalian lens fiber cell membranes. J Biol Chem 264:19967-73
Willing, A H; Georgiadis, M M; Rees, D C et al. (1989) Cross-linking of nitrogenase components. Structure and activity of the covalent complex. J Biol Chem 264:8499-503
Plank, D W; Kennedy, M C; Beinert, H et al. (1989) Cysteine labeling studies of beef heart aconitase containing a 4Fe, a cubane 3Fe, or a linear 3Fe cluster. J Biol Chem 264:20385-93

Showing the most recent 10 out of 13 publications