The objective of this project is to study in depth the structure and function of two Fe-S proteins: aconitase and a 7Fe ferredoxin. Aconitase is a large enzyme which employs a [4Fe-4S] cluster to catalyse stereospecific dehydration/rehydration of citrate in the second and third steps of the Krebs cycle. Aconitase is the best studied example of a Fe-S enzyme catalysing a non-redox reaction. The goal is to deduce specific steps in the mechanism and the role of the protein and cluster in each. The 7Fe ferredoxin from Azotobacter vinelandii functions in electron transfer reactions and contains [3Fe-4S] and [4Fe-4S] clusters in 106 amino-acids. This protein provides an ideal system for understanding the inter-relationship of polypeptide and Fe-S cluster. Both aconitase and ferredoxin will be studied using x-ray crystallography coupled with -molecular biology, biochemistry and spectroscopy. Each part of the project involves a close collaboration. The P.I. (Stout) will solve, refine and analyze crystal structures of aconitase complexed with substrates,inhibitors and reaction intermediate analogs, and structures of aconitase mutants prepared by the Co-P.I. (Zalkin). Zalkin will carry out mutational analysis of porcine aconitase directed toward active site residues, Fe-S cluster ligands,the hinge/linker region and the leader sequence. Aconitase mutants will be expressed in yeast to study in vivo the effect of mutations, and the mechanisms of mitochondrial import and Fe-S cluster assembly. Samples of bovine aconitase and its substrates will be provided by, and mutant enzyme activity and spectroscopic analyses will be done by collaborators H. Beinert and M. C. Kennedy as part of their continuing study of the enzyme. The P.I. will solve, refine and analyze crystal structures of site-directed mutants of the 7Fe ferredoxin. These proteins will be prepared by B. K. Burgess as part of her on-going mutational analysis of structure and function of this ferredoxin.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036325-09
Application #
3290061
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1985-08-01
Project End
1995-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Jung, Y S; Bonagura, C A; Tilley, G J et al. (2000) Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster. J Biol Chem 275:36974-83
Lloyd, S J; Lauble, H; Prasad, G S et al. (1999) The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex. Protein Sci 8:2655-62
Schipke, C G; Goodin, D B; McRee, D E et al. (1999) Oxidized and reduced Azotobacter vinelandii ferredoxin I at 1.4 A resolution: conformational change of surface residues without significant change in the [3Fe-4S]+/0 cluster. Biochemistry 38:8228-39
Chen, K; Tilley, G J; Sridhar, V et al. (1999) Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I. J Biol Chem 274:36479-87
Gao-Sheridan, H S; Kemper, M A; Khayat, R et al. (1998) A T14C variant of Azotobacter vinelandii ferredoxin I undergoes facile [3Fe-4S]0 to [4Fe-4S]2+ conversion in vitro but not in vivo. J Biol Chem 273:33692-701
Sridhar Prasad, G; Kresge, N; Muhlberg, A B et al. (1998) The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii. Protein Sci 7:2541-9
Stout, C D; Stura, E A; McRee, D E (1998) Structure of Azotobacter vinelandii 7Fe ferredoxin at 1.35 A resolution and determination of the [Fe-S] bonds with 0.01 A accuracy. J Mol Biol 278:629-39
Kemper, M A; Stout, C D; Lloyd, S J et al. (1997) Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide. J Biol Chem 272:15620-7
Lauble, H; Kennedy, M C; Emptage, M H et al. (1996) The reaction of fluorocitrate with aconitase and the crystal structure of the enzyme-inhibitor complex. Proc Natl Acad Sci U S A 93:13699-703
Lauble, H; Stout, C D (1995) Steric and conformational features of the aconitase mechanism. Proteins 22:1-11

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