BIOENERGETICS OF THE CYTOCHROME BC1 COMPLEX

细胞色素 BC1 复合物的生物能量学

• 批准号: 2179329
• 负责人: DIANA S. BEATTIE
• 金额: $ 17.2万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179329
• 关键词: antibody; bioenergetics; cytochrome b; cytochrome c; cytochrome oxidase; electron transport; enzyme complex; enzyme mechanism; fluorescent dye /probe; hemoprotein structure; hydrogen transport; laboratory rabbit; liposomes; mitochondria; mitochondrial membrane; molecular cloning; proteolysis; radioimmunoassay; radiotracer; site directed mutagenesis; yeasts

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.

这个研究项目的总体目标是研究质子