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 [EC 1.9.3.1] from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulfate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analyzed by highly cross-linked sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of urea, the apparent MW were = I, 36,700; II, 24,300; III, 20,400; IV, 17,300; V, 12,300; VI, 8700; and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. 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 II + III, 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-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. Results of using 125I-labeled 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 II and III 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 VI are buried within the membrane, not exposed on either side.