The objective remains to elucidate high resolution crystal structures of electron transport proteins and enzymes which contain Fe-S clusters, heme groups and flavins. Methods of protein crystallography will be used. The results are relevant to studies of electron transfer nitrogen fixation and aerobic metabolism. Primary interests are the structure and function of 3Fe clusters, the protein environmental factors which determine the properties of Fe-S clusters, and structural aspects of electron transfer reactions in proteins. The structure of the Fe-S enzyme aconitase in the [3Fe-4S] state will be solved at 2.7 A resolution. The primary sequence will be determined through collaboration. Data will be collected on the activated [4Fe-4S] state of the aconitase crystals. The structure will be interpreted in relation to existing data on the enzyme mechanism and chemical states of the Fe-S cluster. The structure of Azotobacter ferredoxin will be refined beyond 2.0 A. The structure of the oxidized protein will be refined against data sets collected at pH 8 and pH 5. Data will be collected on reduced crystals as a function of pH. The structures will be analyzed in relation to data on Fe-S cluster interconversions and conformational changes in other ferredoxins and enzymes. Data will be collected to 1.7 A on oxidized and reduced Azotobacter cytochrome c5 crystals, and the structures will be refined. The structures will be compared to the refined structures of oxidized and reduced cytochrome c551.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036325-06
Application #
3290060
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1985-08-01
Project End
1991-07-31
Budget Start
1990-08-01
Budget End
1991-07-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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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
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
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
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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