Resolution and Reconstitution of the Mitochondrial Electron Transport System

A procedure is described for separating from preparations of NADH-ubiquinone reductase (Complex I), an NADH dehydrogenase which retains full activity with ferricyanide as acceptor, but which is devoid of rotenone-sensitive NADH-ubiquinone reductase activity. About 50 to 90% of the original NADH-ubiquinone reductase activity is restored by recombination with purified phospholipids in the presence of cholate, and dialysis for at least 10 hours. Reduction of ubiquinone-1 by this preparation is 80 to 90% inhibited by rotenone. 2. After reconstitution with phospholipids, the preparation reacts with ubiquinone-cytochrome c reductase (Complex III) to give rotenone- and antimycin-sensitive NADH-cytochrome c reductase activity. No activity with Complex III is observed with the preparation either before reconstitution or after dialysis without phospholipids. 3. A modification of the separation procedure leads to a preparation of NADH dehydrogenase exhibiting activity with ferricyanide as acceptor. However, after reconstitution with phospholipids, the rates of rotenone-sensitive NADH-ubiquinone reductase obtained are less than 3% of those in Complex I. The properties of the reconstitutively active and inactive NADH dehydrogenase are compared.