The overall goal of this research project is to investigate the proton releasing reactions that accompany electron transfer in the cytochrome bc1, region of the mitochondrial respiratory chain. Recently, we reported that dicyclohexylcarbodiimide (DCCD), the well-characterized carboxyl modifying reagent, blocked the electrogenic release of protons in the bc, complex isolated from yeast mitochondria and in a bf complex isolated from spinach chloroplasts. Radiolabeled DCCD was preferentially bound to cytochrome b in the bc1 complex and to cytochrome b6 in the bf complex suggesting that cytochrome b (b6) plays a role in proton translocation. Moreover, DCCD is covalently bound to aspartate-160 in yeast cytochrome b and to either aspartate-152 or glutamate-165 in the chloroplast cytochrome b6. Recent topographical projections for the b-cytochromes based on hydropathy plots have predicted that these acidic residues are localized in an amphipathic helix (initially proposed as membrane-spanning helix 4) bound to the outer surface of the model membrane. This implies that protons released during the oxidation of the quinol substrate are translocated through this extra-membranous helix to the cytoplasmic side of the membrane, This grant application proposes to address the role of helix 4 of cytochrome b in proton-translocation by the following specific aims: 1) To determine the topographical orientation of cytochrome b in the inner mitochondrial membrane and cytochrome b6 in the thylakoid membrane using specific antibodies against regions of the protein and selective proteolytic digestion, 2) To use fluorescent derivatives of DCCD to characterize the environment of the carboxyl group to which DCCD binds, 3) To study enzymatic activity and proton movements in mutants of cytochrome b to establish the amino acids involved in these processes, and 4) To establish the role of the 14 kDa protein, subunit 7, of the bc1 complex in electron transfer and proton translocation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038433-09
Application #
2179329
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-09-01
Project End
1995-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
9
Fiscal Year
1994
Total Cost
Indirect Cost
Name
West Virginia University
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
191510239
City
Morgantown
State
WV
Country
United States
Zip Code
26506
Wang, Y; Bruel, C; Yan, L et al. (1998) Exogenous ubiquinol analogues affect the fluorescence of NCD-4 bound to aspartate-160 of yeast cytochrome b. J Bioenerg Biomembr 30:455-64
Obungu, V H; Wang, Y; Beattie, D S (1998) The role of charged amino acids in the alpha1-beta4 loop of the iron-sulfur protein of the cytochrome bc1 complex of yeast mitochondria. J Biol Chem 273:11917-22
Wang, Y; Obungu, V; Beattie, D S (1998) Dicyclohexylcarbodiimide inhibits proton pumping in ubiquinol:cytochrome c oxidoreductase of Rhodobacter sphaeroides and binds to aspartate-187 of cytochrome b. Arch Biochem Biophys 352:193-8
Ramabadran, R S; Japa, S; Beattie, D S (1997) Assembly of deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc1 complex of yeast mitochondria. J Bioenerg Biomembr 29:45-54
Obungu, V; Yu, L P; Japa, S et al. (1997) The role of the membrane-spanning and extra-membranous regions of the iron-sulfur protein in its assembly into the cytochrome bc1 complex of yeast mitochondria. Biochim Biophys Acta 1321:229-37
Beattie, D S; Howton, M M (1996) The presence of rotenone-sensitive NADH dehydrogenase in the long slender bloodstream and the procyclic forms of Trypanosoma brucei brucei. Eur J Biochem 241:888-94
Wang, Y; Howton, M M; Beattie, D S (1995) Topographical organization of cytochrome b in the yeast mitochondrial membrane determined by fluorescence studies with N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]carbodiimide. Biochemistry 34:7476-82
Japa, S; Sun, J Z; Beattie, D S (1995) Import of subunit VII of the cytochrome bc1 complex into yeast mitochondrial. Arch Biochem Biophys 319:250-6
Japa, S; Beattie, D S (1994) Sequences of the iron-sulfur protein precursor necessary for its import and two-step processing in yeast mitochondria. Arch Biochem Biophys 312:414-20
Beattie, D S; Obungu, V H; Kiaira, J K (1994) Oxidation of NADH by a rotenone and antimycin-sensitive pathway in the mitochondrion of procyclic Trypanosoma brucei brucei. Mol Biochem Parasitol 64:87-94

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