Isolated beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) contains four or five molecules of tightly bound diphosphatidylglycerol per monomer (2-heme complex). This lipid could be removed in part, or wholly, by mixing the enzyme with high concentrations of Triton X-100 and then centrifuging the mixture through a glycerol gradient equilibrated in the same detergent. Cytochrome c oxidase retaining three or more diphosphatidylglycerol molecules per monomer was fully active when assayed in 1-oleoyl lysophosphatidylcholine. Upon removal of one or more of these diphosphatidylglycerols, enzymic activity was lost. Full activation could be obtained by adding diphosphatidylglycerol to the assay mixture along with lysophosphatidylcholine but not by adding phosphatidylcholine or phosphatidylethanolamine. Direct binding experiments, kinetic studies, and previous work using arylazidocytochrome c derivatives [Bisson, R., Jacobs, B. & Capaldi, R. A. (1980) Biochemistry 10, 4173-4178], indicate that diphosphatidylglycerol is involved in binding of substrate cytochrome c to cytochrome c oxidase.

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.

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)

Purified oat phytochrome was labeled with 125 I without altering the photoreversibility or absorbance properties of the pigment. The radiolabeled phytochrome was used in experiments in vitro to quantitate the binding of the pigment to both crude and purified membrane preparations from oat tissue. After the membranes were allowed to react with 125 I-labeled phytochrome, washed free of unbound material, and pelleted, they were found to have significant levels of radioactivity bound to them. Qualitative identification of phytochrome as the bound radioactive species was confirmed by autoradiography of sodium dodecyl sulfate gels after electrophoresis of the proteins contained in the washed membranes. Data supporting the specificity of the binding are that the binding shows saturation kinetics and that unlabeled phytochrome, but not bovine serum albumin, will competitively inhibit the binding of labeled phytochrome. This technique permits the detection of less than a nanogram of phytochrome and provides a new method for quantifying bound phytochrome that is independent of the spectral detectability of the pigment. It should be useful in elucidating the nature of phytochrome attachment to cellular membranes.

in restoring the initial molecular activity of cytochrome c oxidase. Other PL's, even those having a net negative charge, i.e., phosphatidylglycerol (PG), phosphatidylserine (DS), and phosphatidic acid (PA), were unable to stimulate the activity more than 5-10%. One possible explanation for the apparent specificity for DPG is its unique structure: two phosphatidic acids joined by a central glycerol molecule. This structure might permit DPG to form a bridge between two separate hydrophobic sites which none of the other PL's would be able to do. Another structural difference between DPG and the other PL's is its high percentage of linoleic acid (bovine DPG contains 90% C18:2 fatty acyl chains). To assess the