CATALYTIC SECTOR OF COMPLEX-I (NADH-UBIQUINONE OXIDOREDUCTASE) - SUBUNIT STOICHIOMETRY AND SUBSTRATE-INDUCED CONFORMATION CHANGES
CATALYTIC SECTOR OF COMPLEX-I (NADH-UBIQUINONE OXIDOREDUCTASE) - SUBUNIT STOICHIOMETRY AND SUBSTRATE-INDUCED CONFORMATION CHANGES
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DOI：
10.1021/bi00181a018
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发表时间：
1994-04-19
期刊：
影响因子：
2.9
通讯作者：
HATEFI, Y
中科院分区：
文献类型：
--
作者：
BELOGRUDOV, G;HATEFI, Y
线粒体NADH的电子载体：泛醌氧化还原酶（复合物I）主要包含在两个膜外亚复合物，黄素蛋白（FP）和铁硫蛋白（IP）。FP含有分子量为51、24和9 kDa的三个亚基。51-kDa亚基携带NADH结合位点，并含有FMN和一个四核铁硫簇。24-kDa亚基含有双核铁硫簇。IP含有7个亚基，分子量为75、49、30、18、15、13和11 kDa。在75 kDa亚基中含有一个四核和很可能是一个双核铁硫簇。FP和IP通过51-和75-kDa亚基进行接触。复合物I（疏水蛋白（HP），31个亚基）的其余部分主要是膜插入，并包含两个铁硫簇显然在23 kDa的亚基，并可能在20 kDa的亚基。在这项研究中，化学计量的FP和IP的复合物I的亚基的放射免疫测定。每摩尔复合物I，有2摩尔的15-kDa亚基和1摩尔的FP和四个最大的IP亚基。13-和11-kDa亚基的化学计量不能分别测定，因为它们在凝胶电泳时共迁移。此外，使用三种不同分子长度的交联剂，研究了底物（NADH、NADPH、NAD和NADH加铁氰化钾以通过FP快速氧化NADH）对FP和IP亚基的交联模式的影响。结果表明，在交联之前，用NADH或NADPH处理复合物I，而不是用NAD或NADH + K3 Fe（CN）（6），导致FP亚基之间、75-和51-kDa亚基之间、IP亚基之间以及IP和HP亚基之间交联程度的变化（减少或增加）。换句话说，NAD（P）H对复合物I的还原似乎引起了涉及FP、IP和HP亚基之间的邻近性的构象变化。有人提出，通过类比最近的证据，关于ATP合酶复合物中的能量转移模式，在复合物I的底物还原后观察到的广泛的亚基邻近变化可能是这种酶复合物中发生能量偶联和转移的方式，即，通过从FP和IP到HP的构象能量转移，在HP中实现质子移位。
The electron carriers of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) are contained predominately in two extramembranous subcomplexes, a flavoprotein (FP) and an iron-sulfur protein (IP). FP contains three subunits with molecular masses of 51, 24, and 9 kDa. The 51-kDa subunit carries the NADH binding site and contains FMN and a tetranuclear iron-sulfur cluster. The 24-kDa subunit contains a binuclear iron-sulfur cluster. IP contains seven subunits with molecular masses of 75, 49, 30, 18, 15, 13, and 11 kDa. It contains a tetranuclear and very likely a binuclear iron-sulfur cluster in the 75-kDa subunit. FP and IP make contact through the 51- and the 75-kDa subunits. The remainder of complex I (hydrophobic protein (HP), 31 subunits) is largely membrane-intercalated and contains two iron-sulfur clusters apparently in a 23-kDa subunit and possibly another in a 20-kDa subunit. In this study, the stoichiometries of the FP and IP subunits in complex I were determined by radioimmunoassay. Per mole of complex I, there are 2 mol of the 15-kDa subunit and 1 mol each of the FP and the four largest IP subunits. The stoichiometries of the 13- and the 11-kDa subunits could not be determined separately, because they comigrate upon gel electrophoresis. In addition, the effect of substrates (NADH, NADPH, NAD, and NADH plus potassium ferricyanide to rapidly oxidize NADH via FP) on the cross-linking patterns of FP and IP subunits was investigated, using three different cross-linking reagents of different molecular lengths. Results showed that treatment of complex I with NADH or NADPH, but not with NAD or NADH + K3Fe(CN)(6), prior to cross-linking resulted in changes in the extent (decrease or increase) of cross-linking among the FP subunits, between the 75- and the 51-kDa subunits, among the IP subunits, and between the IP and the HP subunits. In other words, reduction of complex I by NAD(P)H appeared to cause conformational changes involving proximities among and between the FP, IP, and HP subunits. It is proposed that, by analogy to recent evidence regarding the mode of energy transfer in the ATP synthase complex, the extensive subunit proximity changes observed upon substrate reduction of complex I may be the manner in which energy coupling and transfer take place within this enzyme complex, i.e., via conformational energy transfer from FP and IP to HP, where proton translocation is effected.