Proteolysis of Paracoccus denitrificans cytochrome oxidase by trypsin and chymotrypsin

Paracoccus oxidase containing only two subunits was subjected to proteolysis by trypsin and chymotrypsin. Both subunits of the purified enzyme were cleaved at only a few sites and enzymatic activity was not inhibited. The cleavage sites were identified by protein sequencing. Subunit I was cleaved near the amino-terminus and subunit II in the loop connecting the two predicted trans-membrane helices. In native membrane fragments, but not in intact spheroplasts, this loop was accessible to both proteases. These results provide experimental evidence for the folding of subunit II in the membrane.