ELECTRON TRIBUTARIES OF THE RESPIRATORY CHAIN

呼吸链的电子支流

• 批准号: 3272615
• 负责人: YOUSSEF HATEFI
• 金额: $ 24.16万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3272615
• 关键词: Escherichia coli; NAD(P) transhydrogenase; active sites; bioenergetics; conformation; electron transport; enzyme inhibitors; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; laboratory rabbit; liposomes; membrane potentials; mitochondrial membrane; monoclonal antibody; nicotinamide adenine dinucleotide; protein sequence; protonation; ubiquinone

Mitochondrial nicotinamide nucleotide transhydrogenase (TH) is a homodimer of monomer M(r)=109228, is located in the inner membrane, and catalyzes hydride ion transfer between the 4A position of NAD(H) and the 4B position of NADP(H) in a reaction that is coupled to transmembrane proton translocation with a H+/H- stoichiometry close to unity. Among other things, the matrix NADPH so produced is utilized by the combined actions of glutathione reductase and glutathione peroxidase to consume H2 0 2, which results from dismutation of O2- generated by the respiratory chain. H2O2 and hydroxyl radicals produced via the Haber-Weiss and Fenton reactions can initiate lipid peroxidation and cause extensive damage to mitochondrial membranes and mtDNA. During the past grant period, we have deduced the amino acid sequences of the mature TH (1043 residues) and its signal peptide (43 residues); identified the NAD and NADP binding domains and the DCCD and N-ethylmaleimide (NEM) binding sites; investigated the membrane topography of TH; shown that NADP and NADPH, but not NAD and NADH, binding alters TH conformation in different ways; and arrived from these and previous studies at a working hypothesis regarding the mechanism of energy transduction and H+ translocation by TH. This mechanism involves utilization of substrate binding energy for proton translocation via TH conformation change. Studies contemplated for the next grant period include: I. Structural Studies: (a) Further study of the topography of the central hydrophobic domain of TH. (b) Study of the spatial relationship between the NAD- and NADP-binding domains, which are, respectively, the N- and C-terminal extramembranous segments of TH. (c) Further elucidation of the NAD(H) binding site. II. Mechanistic Studies: (a) Study of the effect of membrane potential on Kd for NADP and NADPH, and on TH conformation change as monitored by the rate of TH modification by various proteases and NEM. (b) Reconstitution of bovine TH lacking the N-terminal NAD(H)-binding extramembranous segment (43 kDa) and mutated E. coli membranes lacking the TH alpha subunit (corresponds to bovine 43 kDa plus a short hydrophobic tail) with purified, soluble 43 kDa and possibly other 4A-specific NAD-binding proteins. (c) Proteinase K treatment of mitoplasts splits TH into a 72 kDa N-terminal and a 37 kDa C-terminal fragment. The bisected TH has been purified and shown to have high TH activity and H+ translocation capability when embedded in liposomes. Attempt will be made to separate the 37 kDa fragment and reconstitute it with the soluble 43 kDa piece and demonstrate H+ translocation. If successful, these studies will confine the TH machinery for utilization of substrate binding energy for H+ translocation to the C-terminal 37 kDa fragment, which contains a short hydrophobic head capable of forming only 5 membrane-intercalating amino acid clusters. III. E. Coli TH will be isolated, its NAD and NADP binding sites will be determined, its possible substrate-promoted conformation change will be investigated, and attempts will be made to crystallize it.

线粒体烟酰胺核苷酸转氢酶（TH）是一种 单体M(r)=109228的同二聚体，位于内膜，并且 催化 NAD(H) 的 4A 位和 跨膜偶联反应中 NADP(H) 的 4B 位置 H+/H- 化学计量接近统一的质子易位。 之中 除此之外，如此产生的矩阵 NADPH 被组合利用 谷胱甘肽还原酶和谷胱甘肽过氧化物酶消耗H2的作用 0 2，这是呼吸系统产生的 O2- 歧化的结果 链。 H2O2 和羟基自由基通过 Haber-Weiss 和 芬顿反应可以引发脂质过氧化并导致广泛的 线粒体膜和 mtDNA 损伤。 在过去的补助期间 在此期间，我们推导出了成熟TH的氨基酸序列（1043） 残基）及其信号肽（43 个残基）；确定了 NAD 和 NADP 结合域以及 DCCD 和 N-乙基马来酰亚胺 (NEM) 结合 网站；研究了 TH 的膜形貌；表明 NADP 和 NADPH，但不是 NAD 和 NADH，结合会改变不同的 TH 构象 方式；并从这些和之前的研究中得出了一个可行的假设 关于能量转换和H+易位的机制 TH。 该机制涉及利用底物结合能 通过 TH 构象变化的质子易位。 下一个资助期预期的研究包括： I. 结构 研究：（a）进一步研究中心疏水性的形貌 TH 域。 (b) NAD-和NAD-之间的空间关系研究 NADP 结合域，分别是 N 端和 C 端 TH 的膜外节段。 (c) NAD(H)的进一步阐明 结合位点。 二.机制研究：(a) 研究 NADP 和 NADPH 的 Kd 膜电位以及 TH 构象 通过各种蛋白酶的 TH 修饰率监测变化 和新经济法。 (b) 缺乏 N 末端的牛 TH 的重建 NAD(H) 结合膜外片段 (43 kDa) 和突变的大肠杆菌 缺乏 TH α 亚基的膜（相当于牛 43 kDa plus 短的疏水尾）具有纯化的、可溶的 43 kDa，并且可能 其他 4A 特异性 NAD 结合蛋白。 (c) 蛋白酶K处理 有丝分裂体将 TH 分裂为 72 kDa N 端和 37 kDa C 端 分段。 平分的 TH 已被纯化并显示出具有高 TH 包埋在脂质体中时的活性和 H+ 易位能力。 将尝试分离 37 kDa 片段并重建它 与可溶性 43 kDa 片段并证明 H+ 易位。 如果 成功的话，这些研究将限制 TH 机器的利用 H+ 转位至 C 末端的底物结合能 37 kDa 片段，其中包含一个短的疏水头，只能形成 5 个膜嵌入氨基酸簇。三． 大肠杆菌 TH 将是 分离后，其 NAD 和 NADP 结合位点将被确定，其可能 将研究底物促进的构象变化，并尝试 将使其结晶。