ROLE OF NUCLEAR-ENCODED SUBUNITS OF MITOCHONDRIAL CYTOCHROME-C-OXIDASE IN PROTON-PUMPING REVEALED BY LIMITED ENZYMATIC PROTEOLYSIS

The role of supernumerary subunits of bovine heart cytochrome c oxidas e has been investigated by examining the effect on the enzymatic activ ity of limited proteolysis by chymotrypsin, thermolysin, and trypsin. All three proteases, when added to the soluble oxidase, digested subun its III, VIa, and VIb and caused inhibition of electron flow in the ox idase. In addition, trypsin and thermolysin also digested subunit IV. Trypsin cleaved off an N-terminal segment of seven residues; thermolys in cleaved only the first four residues at the N-terminus of subunit I V, Digestion of the soluble oxidase by trypsin but not by thermolysin caused decoupling of redox-linked proton pumping in the oxidase. It is concluded that the sequence V5-V6-K7 of the hydrophilic N-terminus of subunit IV, which protrudes out of the matrix side of the mitochondri al membrane, mediates the access of protons into the transmembrane pro ton translocating pathway in the oxidase.