Isolation, subunit composition, and site of synthesis of human cytochrome c oxidase

Abstract

Cytochrome c oxidase, the terminal oxidase of the respiratory chain in eukaryotic cells, was purified from human placenta mitochondria. Seven polypeptides were identified reproducibly by high-resolution electrophoresis of the enzyme complex through sodium dodecyl sulfate (NaDodSO4)-urea polyacrylamide gels; these correspond closely in size to the subunits of beef heart cytochrome c oxidase. When HeLa cells, grown in suspension culture, were pulse-labeled with [35S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis and chased with an excess of unlabeled methionine in the absence of the drug, the mitochondrially synthesized polypeptides were resolved into at least 17 components by NaDodSO4-urea polyacrylamide gel electrophoresis. After labeled HeLa mitochondria were mixed with human placenta mitochondria and the cytochrome c oxidase was isolated, 3 of the labeled components copurified with the 3 largest subunits of the complex. Human cytochrome c oxidase apparently contains 7 subunits, the 3 largest of which are synthesized on mitochondrial ribosomes, while the other 4 are synthesized in the cytoplasm.