Topology of beef heart cytochrome c oxidase from studies on reconstituted membranes.
The orientation of purified beef heart cytochrome c oxidase, incorporated into vesicles by the cholate dialysis procedure [Carroll, R.C., & Racker, E. (1977) J. Biol. Chem. 252, 6981], has been investigated by functional and structural approaches. The level of heme reduction obtained by using cytochrome c along with the membrane-impermeant electron donor ascorbate was 78 +/- 2% of that obtained with cytochrome c and the membrane-permeant reagent N,N,N',N'-tetramethyl-p-phenylenediamine. Electron transfer from cytochrome c is known to occur exclusively from the outer surface of the mitochondrial inner membrane (C side), implying that at least 78% of the oxidase molecules are oriented in the same way in these vesicles as in the intact mitochondria. Trypsin, which cleaves subunit IV near its N terminus, modifies only 5-7% of this subunit in intact vesicles. This removal of the N-terminal residues has been shown to occur only in mitochondrial membranes with their inner side (M side) exposed. Diazobenzene [35S]sulfonate [( 35S]DABS) likewise modifies subunit IV only in submitochondrial particles. Labeling of intact membranes with [35S]DABS resulted in incorporation of only 4-8% of the total counts that could be incorporated into this subunit in membranes made leaky to the reagent by addition of 2% Triton X-100. Therefore, both the functional and structural data show that at least 80% and probably more of the cytochrome c oxidase molecules are oriented with their C domain outermost and M domains in the lumen of vesicles prepared by the cholate dialysis method.(ABSTRACT TRUNCATED AT 250 WORDS)
D000218:Adamantane / Q000031:analogs & derivatives; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D003979:Diazonium Compounds; D004024:Dicyclohexylcarbodiimide; D003576:Electron Transport Complex IV / Q000378:metabolism; D006418:Heme; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008929:Mitochondria, Heart / Q000201:enzymology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D013425:Sulfanilic Acids; D014357:Trypsin / Q000378:metabolism
D003979:Diazonium Compounds; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D013425:Sulfanilic Acids; C024080:adamantane diazirine; D006418:Heme; D004024:Dicyclohexylcarbodiimide; D003576:Electron Transport Complex IV; D002918:Chymotrypsin; D014357:Trypsin; D000218:Adamantane; C000469:diazobenzenesulfonic acid