Factor B and energy coupling in mitochondria

线粒体中的因子 B 和能量耦合

• 批准号: 6678756
• 负责人: GRIGORY I BELOGRUDOV
• 金额: $ 20.59万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678756
• 关键词: NAD(P)H dehydrogenase; adenosine triphosphate; adenosinetriphosphatase; bioenergetics; cadmium; cell component structure /function; crosslink; dithiol; electron transport; enzyme complex; free radical oxygen; membrane activity; mitochondria; oxidative phosphorylation; oxidoreductase inhibitor; protein protein interaction; site directed mutagenesis

DESCRIPTION (provided by applicant): In mitochondria, substrate oxidation by multisubunit complexes of the electron transport chain is coupled to ATP synthesis by the ATP synthase complex via an electrochemical proton gradient, Delta-mu-H+, across the mitochondrial inner membrane. The ability to synthesize ATP, establish Delta-mu-H+ and catalyze the energy-linked reactions is lost in bovine heart submitochondrial particles sonicated at pH 8.8 in the presence of 0.6 mM EDTA, due to proton leakiness of the mitochondrial inner membrane. Recently, we cloned and expressed in bacteria a human mitochondrial polypeptide, factor B, that efficiently restored all the energy-linked reactions, including the ATP synthesis, in the pH 8.8/EDTA treated submitochondrial particles. Because factor B coupling activity is similar to that of low amounts of oligomycin, a specific inhibitor of proton translocation through the membrane sector F-0 of ATP synthase, we propose that factor B is a regulatory component of mitochondrial ATP synthase, loosely associated with F-0 sector. We hypothesize that in mitochondria, factor B is involved in regulation of proton flux through F-0 sector coupled to ATP synthesis in the active site of the enzyme. We further hypothesize that via redox status of its essential thiols, specifically a vicinal dithiol, factor B acts as a "pressure valve" for maintaining proton-motive force below the threshold that favors production of reactive oxygen species in mitochondria, by inducing a mild uncoupling of the organelle. The following specific aims are proposed: 1) To characterize the interaction of factor B with the membrane sector F-0 in the membrane bound and detergent solubilized ATP synthase complex; and 2) To elucidate the structure-function relationship of the vicinal dithiol motif found in the amino acid sequence of the mature factor B, and the role it plays in the factor B sensitivity toward Cd 2+ and phenylarsine oxide, both of which uncouple oxidative phosphorylation in mitochondria. The proposed research is expected to advance the knowledge of the molecular mechanisms of energy transfer and conservation in mitochondria, and shed light on the role of mitochondria in a number of human disorders.

描述（由申请人提供）：在线粒体中，电子传递链的多亚基复合物的底物氧化与 ATP 合酶复合物通过电化学质子梯度 Delta-mu-H+ 穿过线粒体内膜耦合 ATP 合成。由于线粒体内膜的质子泄漏，在 pH 8.8、0.6 mM EDTA 存在下超声处理的牛心脏软骨下颗粒中，合成 ATP、建立 Delta-mu-H+ 和催化能量相关反应的能力丧失。最近，我们在细菌中克隆并表达了一种人类线粒体多肽（B 因子），它可以在 pH 8.8/EDTA 处理的线粒体颗粒中有效恢复所有能量相关反应，包括 ATP 合成。由于 B 因子偶联活性与低量寡霉素（一种通过 ATP 合酶膜 F-0 区质子易位的特异性抑制剂）相似，因此我们推测 B 因子是线粒体 ATP 合酶的调节成分，与 F 松散相关。 -0 扇区。我们假设在线粒体中，因子 B 参与调节通过 F-0 区域的质子通量，并与酶活性位点中的 ATP 合成相结合。我们进一步假设，通过其必需硫醇（特别是邻位二硫醇）的氧化还原状态，因子 B 充当“压力阀”，通过诱导温和的解偶联，将质子动力维持在有利于线粒体中活性氧生成的阈值以下的细胞器。提出以下具体目标： 1) 表征因子 B 与膜结合且去污剂溶解的 ATP 合酶复合物中膜扇区 F-0 的相互作用； 2) 阐明成熟因子 B 氨基酸序列中邻位二硫醇基序的结构-功能关系，以及它在因子 B 对 Cd 2+ 和氧化苯胂的敏感性中所起的作用，这两种物质都解偶联氧化线粒体中的磷酸化。拟议的研究预计将增进对线粒体能量转移和保存的分子机制的了解，并阐明线粒体在许多人类疾病中的作用。