Immunological Studies on Cytochrome c Oxidase: Arrangements of Protein Subunits in the Solubilized and Membrane‐Bound Enzyme

Abstract

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, I1 and 111) and guanidine hydrochloride (subunits V, VT and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly crosslinked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = 1, 36700; 11, 24300; 111, 20400; IV, 17300; V, 12300; VI, 8700; and VII, 5100. Monospecific rabbit antisera were obtained against subunits 1, IV, V, VI and V11 and a mixture of subunits I1 and 111. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits I1 + 111, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25 % inhibition while anti-I immunoglobulin did not inhibit the activity.The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(I1 + 111) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using '251-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits I1 and 111 are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and V1 are buried within the membrane, not exposed on either side.Cytochrome c oxidase, the terminal enzyme of the electron transport chain, is an integral component of the mitochondrial inner membrane. Earlier studies on the structure of the enzyme indicated that it isa complex unit containing two hemes a, two copper atoms and multiple dissimilar protein subunits [I -31. Experiments using antibodies against the native oxidase [4,5] and chemical labelling with bovine heart mitochondria [4,6] have shown that cytochrome oxidase spans the mitochondrial membrane asymmetrically, which is the basis of the chemiosmotic hypothesis .of oxidative phosphorylation. Based on differential reactivities of normal and inverted mitochondrial membranes towards surface-labelling reagent, Eytan et al. ourselves that the interpretation based on chemical labelling experiments does not suffer from relatively low specific activity of labelled products as well as possible damaging effects on membranes by the chemical labels, we decided to use individual subunitspecific antibodies to establish the arrangements of cytochrome c oxidase subunits in mitochondrial membrane. It is generally acknowledged that antibody molecules ...

Structure of the cytochrome c oxidase complex of rat liver

“…Model on the structural and functional organization of polypeptides in the mammalian cytochrome c oxidase complex cytochrome c 1161. The same polypeptides have been found in this study to be oriented to the cytosolic phase, the binding site for cytochrome c. Two of these polypeptides (VIa and VIc) formed cross-links with polypeptide 11,two (VIII,VIIb) with polypeptide I, and one (VIIa) did not form any crosslink 1151. Polypeptides I and I1 must be located close together in the enzyme complex, since the distance between heme u and u3 amounts to 120-160 nm 1261 and their location was suggested in polypeptides I1 and I, respectively [I -4, 271.…”

“…Therefore we suggest a cleavage of polypeptide VIc by pronase, whereas at the same position a cleavage product of polypeptide VIa appears. Most cleavable polypeptides have disappeared after incubation with 125 pg protease/mg mitoplast protein (lanes 5,11). Polypeptide I1 in contrast, seems to be cleaved only at higher protease concentrations.…”

“…Mitoplasts were incubated with increasing concentrations of subtilisin (lanes 2-7) and pronase (8 -14). Proteasejmitoplast protein ratios (pg/mg) were: 0 (lanes 2, 8); 5 (lanes 3,9); 25 (lanes 4, 10); 125 (lanes 5,11); 500 (lanes 6,12); 1000 (lanes 7, 13). Lane 1 and 14: isolated rat liver cytochrorne c oxidase.…”

“…Cleavage of cytochrome c oxidase polypeptides in mitoplasts by suhtilisin as studied by specific antisera. Mitoplasts were incubated with (lanes 2, 4, 6,8,10,12) and without (lanes 1,3,5,7,9,11) subtilisin (1 mg/mg mitoplast protein) separated by SDS gel electrophoresis and transferred to nitrocellulose as described under Materials and Methods. The same sheet was successively incubated with antisera against polypeptide VIII (lanes 1, 2); VIIabc (lanes 3,4); VIa + VIbc (lanes 5, 6); Vab (lanes 7,8); IV (lanes 9, 10) and I1 (lanes 11, 12) each followed by incubation with fluorescein-isothiocyanate-conjugated protein A and photographic documentation.…”

The effect of specific antibodies on oxygen uptake and H+ pumping by cytochrome c oxidase vesicles

“…Anti-III and anti-oxidase were raised and gamma globulin isolated as in [16], except that the gamma globulin preparations were used as stock solutions of 40-50 mg/ml in 50 mM KzS04 (pH 7.5) and the data were recalculated to 20 mg gammagiobulin/ml. Incubation of these antibodies or non-immune gammaglobulin with the COV prior to oxygen uptake assay was for 15-20 min at 4°C in 50 mM K2S04/l mM MOPS/pH 7.2 to a total volume of 2OO/rl of which 20 ~1 were COV.…”