Topology of beef heart cytochrome c oxidase from studies on reconstituted membranes

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Abstract
The orientation of purified beef heart cytochrome c oxidase, incorporated into vesicles by the cholate dialysis procedure was 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-phenylendiamine. 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 was 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. Labeling experiments with [35S]DABS and proteinase digestions of intact membranes with trypsin and chymotrypsin were used to determine the topography of the subunits of cytochrome c oxidase. These studies confirm that subunits II and III are in the C domain and subunits IV and VII in the M domain and show for the 1st time that polypeptides b and c are on the C side of the mitochondrial inner membrane.