INTERACTION OF CYTOCHROME-C WITH CYTOCHROME-C-OXIDASE STUDIED BY MONOCLONAL-ANTIBODIES AND A PROTEIN MODIFYING REAGENT

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition, Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [C-14] glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface. These results, along with activity and binding studies, strongly support the concept that subunit II provides the primary electron-transfer site for cytochrome c on cytochrome oxidase. Since more than one cytochrome c binding site is blocked by a single antibody binding to subunit II, it is suggested that two sites are in close proximity.