The molecular morphology of bovine heart mitochondrial NADH----ubiquinone reductase. Native disulfide-linked subunits and rotenone-induced conformational changes.
Bovine heart mitochondrial NADH----ubiquinone reductase (complex I), contains two disulfide-linked subunits of 75 and 33 kDa as revealed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with beta-mercaptoethanol omitted from preparation of the sample for the first dimension. Two unidentified polypeptides (110-115 and 69 kDa) are also found in disulfide linkage with the two complex I subunits. The 110-115-kDa polypeptide appears to be pyridine dinucleotide transhydrogenase by several criteria including selective precipitation with an antibody raised to the purified transhydrogenase. The two disulfide-linked subunits were also found in a product cross-linked for 2 min with dithiobis (succinimidyl propionate) (DSP) along with five other complex I subunits of 53-57, 42, 24-27, 17-18, and 12.5-15.5 kDa (Gondal, J.A., and Anderson, W.M. (1985) J. Biol. Chem. 260, 5931-5935) indicating that these seven subunits lie within 11-12 A of each other at one or more points in space in the enzyme's interior. Cross-linking of complex I with DSP for 2 min in the presence of 1 microM rotenone yielded a cross-linked product consisting of the two natural disulfide-linked subunits and the 110-115- and 69-kDa polypeptides. This suggests that rotenone induces a conformational change in the enzyme that moves the seven DSP cross-linked subunits away from each other and outside the 11-12 A bridging distance of DSP. This alteration in conformation may be communicated to iron-sulfur center N-2 within the hydrophobic outer shell of the enzyme to prevent electron transfer to its natural electron acceptor, ubiquinone. A model of rotenone action based upon these observations is presented.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008623:Mercaptoethanol / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D016660:NAD(P)H Dehydrogenase (Quinone); D011487:Protein Conformation / Q000187:drug effects; D011808:Quinone Reductases; D012402:Rotenone / Q000494:pharmacology*; D013388:Succinimides / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D013388:Succinimides; D012402:Rotenone; D008623:Mercaptoethanol; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; D014357:Trypsin; C011240:dithiobis(succinimidylpropionate)