Catalytic sector of complex I (NADH:ubiquinone oxidoreductase): subunit stoichiometry and substrate-induced conformation changes.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Belogrudov
G
G
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Hatefi
Y
Y
D001665:Binding Sites; D002384:Catalysis; D003432:Cross-Linking Reagents; D004735:Energy Transfer; D005292:Ferricyanides / Q000494:pharmacology; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D009492:Neurospora crassa / Q000201:enzymology; D011487:Protein Conformation; D011804:Quinolines / Q000494:pharmacology
D003432:Cross-Linking Reagents; D005292:Ferricyanides; D007506:Iron-Sulfur Proteins; D011804:Quinolines; D009243:NAD; D009249:NADP; C000470:EEDQ; D016660:NAD(P)H Dehydrogenase (Quinone); C028033:potassium ferricyanide