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.