Studies on the Electron Transfer System: LXVIII. FORMATION OF MEMBRANES AS THE BASIS OF THE RECONSTITUTION OF THE MITOCHONDRIAL ELECTRON TRANSFER SYSTEM

Abstract

Cytochrome oxidase (Complex IV), reduced diphosphopyridine nucleotide-cytochrome c reductase (a mixture of Complexes I and III), and succinic-cytochrome c reductase (a mixture of Complexes II and III) can be induced to form membranes by reducing the bile salt level of the suspending medium (in concentrated solutions of the complexes as prepared, the level of bile salt is sufficient to achieve molecular dispersion of the repeating units). When the membranes formed separately from the individual complex or sets of complexes are mixed prior to or during assay, the mixture of membranes fails to show DPNH oxidase activity (mixture of I + III and IV) or succinic oxidase activity (mixture of II + HI and IV). In a sucrose gradient these mixtures are separable by centrifugation into two distinct bands corresponding to the component membranes. However, the membranes formed by mixing either I + III or II + III with IV in concentrated solution, and then diluting, will catalyze the oxidation of DPNH or succinate by molecular oxygen (the test of reconstitution). Each of these membranes shows only a single band when centrifuged in a sucrose gradient. On the basis of these observations, reconstitution is interpreted in terms of the incorporation within a single membrane of the complete set of complexes necessary for a reconstituted activity. Reconstitution of electron transfer activity does not require selective interactions between, or alignments of, the complexes of the electron transfer chain. A membrane can be formed by any one complex with or without any of the other complexes; any proportions of two or more complexes can be achieved. These observations exclude the notion of stoichiometry among complexes. Cytochrome c, one of the electron transfer links between complexes, is incapable of moving from one membrane to another. Thus, reconstitution requires that not only the complexes but also the mobile components must be in the same membrane system.