MOLECULAR AND CONTROL MECHANISM OF ENERGY COUPLING SITE

能量耦合位点的分子及控制机制

基本信息

批准号：
2736201
负责人：
Tomoko None Ohnishi
金额：
$ 2.43万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1993
资助国家：
美国
起止时间：
1993-07-01 至 1999-12-31
项目状态：
已结题

项目摘要

DESCRIPTION: Complex I (NADH-ubiquinone oxidoreductase) serves as the main entry point for reoxidation NADH produced by the anaerobic phase of metabolism; it is the most complicated and least studied energy transducing device of the mitochondrial respiratory chain. The P.I.s propose to continue studying the molecular mechanism of electron/vectorial proton transfer in mammalian Complex I. The structure/function rearrangements involved in the "active" (pulsed) - "inactive" (resting) state transition of Complex I will also be studied. This work constitutes one of the physiologically most important research areas, since many diseases in highly energy-demanding tissues are associated with inherited or induced defects in Complex I. Specific new directions of the proposed work include: (1) studies on the substrate (pyridine nucleotide binding site(s), which will be conducted by measuring kinetic parameters of NADH dehydrogenation utilizing competitive NAD+ and NADH analogues. The dependence of the binding site affinities for catalytically inert nucleotides on the enzyme redox state(s) will also be investigated; (2) determination of the spatial organization of EPR-detectable redox components relative to each other and to the surfaces of the energy transducing membrane, with a special emphasis on the two distinct species of delta-mu(H+)-dependent, complex I-associated ubisemiquinones, which have recently been discovered in the P.I. and Co-P.I.'s collaborative endeavor; (3) identification of the polypeptide(s) involved in the slow inactive/active transition of Complex I utilizing specific labelling with fluorescent and spectrally- detectable SH-reagents; (4) development of a procedure for solubilization and purification of the enzyme stabilized in the active conformation by using specific inhibitors which bind exclusively or preferentially to active Complex I. The U.S. P.I. has extensive experience in cryogenic EPR (the only technique which is presently available for studies of Complex I redox components in situ) and in thermodynamic analysis of the respiratory chain components. The P.I. has made significant contributions in the analysis of iron-sulfur proteins, flavin, and semiquinone free radicals. The Co-P.I. has extensive experience in preparative biochemistry of redox enzymes and in enzyme kinetic analysis. The collaborative project between these investigators will greatly enhance the progress in understanding the molecular mechanisms of energy-transduction in Complex I and its physiologically relevant regulation.

描述：复杂的I（NADH-泛酮氧化还原酶）用作通过厌氧产生的重新氧化NADH的主要切入点代谢；它是最复杂，研究最少的能量线粒体呼吸链的转导装置。 P.I.S 建议继续研究哺乳动物复合物中的电子/矢量质子转移I。 “主动”（脉冲） - 结构/功能重排 - 还将研究“不活动”（静止）状态过渡I。这项工作构成了生理上最重要的研究之一区域，由于高能量组织中的许多疾病是与复合物中的遗传或诱导缺陷有关拟议工作的方向包括：（1）底物的研究（吡啶核苷酸结合位点，将通过测量使用NADH脱氢的动力学参数竞争性NAD+和NADH类似物。结合位点的依赖性催化惰性核苷酸酶上的亲和力州也将进行调查；（2）确定空间 EPR可检测的氧化还原组件相对于彼此的组织并带有特殊的能量转导膜的表面强调三角洲（H+）的两种不同种类 - 依赖，复杂 I相关的泛氨基喹酮，最近在 P.I. 和Co-P.I。的合作努力；（3）识别多肽参与缓慢的非活性/主动过渡复杂的我利用特定的标记与荧光和光谱可检测的sh-reangents; （4）制定程序稳定在活动中的酶的溶解和纯化通过使用专门结合的特定抑制剂或优先于主动复合物I。美国P.I.在低温EPR方面拥有丰富的经验（唯一目前可用于研究复杂I氧化还原的技术原位成分）和呼吸的热力学分析链组件。 P.I.在铁硫蛋白，黄素和半喹酮自由基的分析。 Co-p.i。在制备生物化学方面拥有丰富的经验氧化还原酶和酶动力学分析。协作项目在这些调查人员之间将大大提高了解复合物中能量转移的分子机制 I及其生理相关的调节。