REACTION MECHANISM OF MYOCARDIAC UBIQUINONE-PROTEINS

心肌泛醌蛋白的反应机制

• 批准号: 6882562
• 负责人: CHANG-AN YU
• 金额: $ 2.28万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6882562
• 关键词: NADPH cytochrome c2 reductase; Rhodospirillales; X ray crystallography; active sites; animal tissue; binding sites; cellular respiration; cytochrome b; cytochrome c; electron transport; enzyme complex; enzyme mechanism; iron sulfur protein; mitochondria; myocardium; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein reconstitution; protein structure function; site directed mutagenesis; succinate dehydrogenase; ubiquinone

The objective of this application is to elucidate the structure, function, and mechanism of the ubiquinone (Q)-mediated electron transfer complexes of the mitochondrial respiratory chain. During past support periods the applicant has established protocols for site-directed mutagenesis coupled with in vivo and in vitro reconstitution to study some of the structural and functional relationship in succinate-Q reductase (SQR) and ubiquinol-cytochrome c reductase (QCR). He has crystallized QCR, solved its structure to 2.9 A resolution and indicated that QCR is a functional dimer and that the catalytic cycle of complex involves movement of the head domain of iron-sulfur protein (ISP). Functional evidence for such movement was obtained from characterization of Rhodobacter sphaeroides bc1 complexes with altered ISP necks. In addition to electron and proton transfer, this group has discovered and studied two more functions of QCR, superoxide radical generation and mitochondrial processing peptidase activity (MPP). In the next grant period Dr.Yu will focus on the elucidation of the mechanism of electron transfer and proton translocation in QCR and of electron transfer in SQR. The approaches to be used include kinetic rate determinations, various spectroscopic measurements, organic synthesis, resolution and reconstitution, molecular genetics, and protein crystallography. The specific aims are: (A) to obtain structural data for mechanistic studies, including the Q-binding at the Qo site pocket, the proton transfer path, structure water, hydrogen bonding, and the interaction domain between cytochrome c1 and c. This will be achieved by obtaining high resolution QCR structure and by X-ray analysis of QCR crystals in different redox states, in the presence of non-oxidizable Q derivatives, or with cytochrome c; (B) to elucidate the reaction mechanisms for electron and proton transfer in QCR. This will be achieved by investigating the movement of the head domain of ISP via functional analysis of mutants having an intersubunit cystine bridge, salt bridge, or hydrogen bond at the interface between cytochrome b and ISP, and via the conformational changes of cytochrome b or c1, at the interface with ISP, during the oxidation-reduction cycle or binding with different Qo site inhibitors; by determining the acid induced intra-molecular electron transfer rates between 2Fe2S and heme c1 using proton caged compound activated by pulse laser; and probing the nature of the intra-molecular path of QH2 in QCR by site directed mutagenesis; (C) to synthesize Q-derivatives and inhibitors for protein:Q interaction studies; (D) to elucidate electron transfer mechanisms in SQR by establishing the identity of QPs2; reconstitution of SQR from recombinant QPs subunit and isolated SDH; identification of SDH docking site(s) in QPs subunits; and crystallization and 3-D structural analysis of mitochondrial SQR. Successful elucidation of the reaction mechanisms for electron and/or proton transfer of SQR and QCR will provide information crucial to understanding the mitochondrial bioenergetic process and thus help in drug design for mitochondrial related diseases. Also, since the quinone reactive sites are responsible for superoxide generation and Q can scavenge singlet oxygen, detailed knowledge of the structure-function relationships and reaction mechanisms of Q-mediated electron transfer should provide valuable information for the study of oxidative stress. This information will be useful in pharmaceutical investigations of cytotoxicity and the aging process.

本申请的目的是阐明结构、功能、 泛醌（Q）介导的电子转移及其机制 线粒体呼吸链复合体。在过去的支持期间 申请人已经建立了定点诱变偶联方案 通过体内和体外重建来研究一些结构和 琥珀酸 Q 还原酶 (SQR) 和泛醇细胞色素的功能关系 c还原酶（QCR）。他结晶了 QCR，将其结构解析为 2.9 A 分辨率并表明 QCR 是一种功能性二聚体，并且催化 复合物的循环涉及铁硫蛋白头域的运动 （ISP）。这种运动的功能证据是从表征中获得的 具有改变的 ISP 颈的球形红杆菌 bc1 复合物。 除了电子和质子转移之外，该小组还发现了 研究了 QCR 的另外两个功能，超氧自由基的产生和 线粒体加工肽酶活性（MPP）。在下一个资助期内 于博士将重点阐述电子转移的机理和 QCR 中的质子易位和 SQR 中的电子转移。的方法 可使用的包括动力学速率测定、各种光谱 测量、有机合成、分辨率和重构、分子 遗传学和蛋白质晶体学。 具体目标是：（A）获取用于机械研究的结构数据， 包括 Qo 位点口袋处的 Q 结合、质子转移路径、结构 水、氢键以及细胞色素 c1 和 c.这将通过获得高分辨率 QCR 结构和 X 射线来实现 在存在以下物质的情况下，分析不同氧化还原态的 QCR 晶体 不可氧化的 Q 衍生物，或与细胞色素 c 一起使用； (B) 阐明 QCR 中电子和质子转移的反应机制。这将是 通过功能性调查 ISP 头域的移动来实现 分析具有亚基间胱氨酸桥、盐桥或 细胞色素b和ISP之间的界面上形成氢键，并通过 细胞色素 b 或 c1 在与 ISP 界面处的构象变化 氧化还原循环或与不同Qo位点抑制剂的结合；经过 测定酸诱导的分子内电子转移速率 2Fe2S和血红素c1使用脉冲激光激活的质子笼化合物；和 通过位点定向探索 QCR 中 QH2 分子内路径的性质 诱变； (C) 合成Q-衍生物和蛋白质抑制剂：Q 相互作用研究； (D) 阐明 SQR 中的电子转移机制 确定 QP2 的身份；从重组 QP 重建 SQR 亚基和分离的SDH； QP 中 SDH 对接位点的识别 亚基；线粒体 SQR 的结晶和 3-D 结构分析。 成功阐明电子和/或质子的反应机制 SQR 和 QCR 的传输将为理解 线粒体生物能过程，从而有助于线粒体药物设计 相关疾病。 此外，由于醌反应位点负责 超氧化物生成和Q可以清除单线态氧，详细知识 Q介导的结构-功能关系和反应机制的研究 电子转移应该为氧化研究提供有价值的信息 压力。该信息将有助于药物研究 细胞毒性和衰老过程。