MOLECULAR AND CONTROL MECHANISM OF ENERGY COUPLING SITE

描述：复合体I(NADH-泛醌氧化还原酶)是 厌氧阶段产生的再氧化NADH的主要入口点 新陈代谢；它是最复杂也是最少被研究的能量 线粒体呼吸链的转换装置。私家侦探 建议继续研究其分子机制。 哺乳动物复合体中的电子/矢量质子转移I. 参与“主动”(脉冲)的结构/功能重排- 还将研究络合物I的“非活动”(休眠)状态转变。 这项工作构成了生理学上最重要的研究之一 由于高能量需求组织中的许多疾病是 与复合体中的遗传性或诱导性缺陷相关的I.特定的新的 拟开展的工作包括：(1)衬底研究 (吡啶核苷酸结合位点(S)，将由 NADH脱氢利用动力学参数的测定 竞争性NAD+和NADH类似物。结合部位的依赖性 催化惰性核苷酸与酶的氧化还原亲和力 国家(S)也将被调查；(2)空间的确定 EPR可检测的氧化还原组分相对于彼此的组织 并在能量传导膜的表面涂有特殊的 强调两种不同的依赖于Delta-Mu(H+)的复合体 与I相关的乌双喹酮类化合物，最近在 P.I.和Co-P.I.S的合作努力；(3)识别 多肽(S)参与的慢性非活性/活性转换 络合物I利用荧光和光谱的特定标记- 可检测的SH试剂；(4)开发一种用于 活性稳定的酶的增溶和纯化 构象通过使用特定的抑制物来唯一结合或 优先于活性络合物I。 美国P.I.在低温EPR方面拥有丰富的经验(唯一 目前可用于研究络合物I氧化还原的技术 原位成分)和呼吸的热力学分析中 链式元件。私家侦探在这方面做出了重大贡献 铁硫蛋白、黄素和半喹酮自由基的分析。 该公司在制备生物化学方面具有丰富的经验。 氧化还原酶和酶动力学分析。协作项目 这些调查人员之间的合作将极大地促进 理解络合物中能量传递的分子机制 I及其与生理相关的调节。

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.