Studies on the Electron Transfer System c REDUCTASE*

Shortly after the discovery of coenzyme Q in mitochondria (I), it was shown that the oxidation of externally added reduced coenzyme Q (&Hz)1 was catalyzed by mitochondrial preparations and that this reaction was inhibited in the presence of cyanide (2), also that submitochondrial preparations containing cytochromes b and cl but lacking cytochrome oxidase could not oxidize &Hz unless substrate amounts of cytochrome c were provided in the assay medium as electron acceptors (2). Subsequent studies also showed that catalytic amounts of cytochrome c were required for the oxidation of &Hz by molecular oxygen in the presence of a submitochondrial particle containing cytochrome oxidase (3). This activity was shown to be inhibited by both cyanide and antimycin A. Since the reactions of cytochrome oxidase are clearly defined (4), it was apparent from these studies that there must exist in mitochondria an enzyme system capable of catalyzing electron transfer between QHp and cytochrome c by an antimycin A-sensitive reaction. Studies on a highly active reduced diphosphopyridine nucleotide-cytochrome c reductase (5) isolated in this laboratory from beef heart mitochondria, have shown that this enzyme system could be cleaved into a reduced diphosphopyridine nucleotidecoenzyme Q reductase essentially free from cytochromes (6) and a cytochrome-rich fraction containing cytochromes b and cl and nonheme iron. The properties of the former fraction (DPNHcoenzyme Q reductase) have been described in the accompanying communication (7). From the cytochrome-rich fraction a highly active &Hz-cytochrome c reductase has been prepared. The present communication deals with the purification from mitochondrial DPNH-cytochrome c reductase and the general properties of this enzyme complex. Its activity, as the corresponding activity in mitochondria, is inhibited by antimycin A, 2-alkyl-3hydroxynaphthoquinone, and 2-nonyl-4-hydroxyquinoline-N-