Walter Sebald, W. Machleidt, J. Otto

• Cytochrome oxidase isolated from N eurospora crassa was resolved into seven protein eomponents by eleetrophoresis in polyaerylamide gels eontaining sodium dodeeylsulfate. The apparent molecular weights were determined tobe 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the eomponents 1, 2, 3, 4, 5, 6, and 7, respectively. The components 1, 2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino aeids in the presenee of cyeloheximide. Amino-acidanalysis of the isolated components 1, 2 and 3Cytochrome oxidase isolated from N eurospora crassa was resolved into seven protein eomponents by eleetrophoresis in polyaerylamide gels eontaining sodium dodeeylsulfate. The apparent molecular weights were determined tobe 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the eomponents 1, 2, 3, 4, 5, 6, and 7, respectively. The components 1, 2 and 3 are synthesized on mitochondrial ribosomes as shown by the incorporation of radioactive amino aeids in the presenee of cyeloheximide. Amino-acidanalysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low eontent of basic amino aeids compared to an average amino-aeid eomposition of components 4-7. Components 1, 2 and 3 eontribute 27.9°/0, 18°/0 and 14.2°/0 to the whole eytoehrome oxidase protein. This was calculated from the contributions of the single eomponents to the totalleueine eontent of the enzyme and the leueine eontents (nmol leueine per mg protein) of the single eomponents as determined by amino-aeid analysis. Equimolar relations of the components 1, 2 and 3 are found by dividing the amounts of protein by their apparent molecular weights. A stoichiometry of 1:1:1 results assuming a minimal molecular weight of 150000 for the whole cytochrome oxidase protein. On the basis of the heme a content a molecular weight of about 70000 per heme group was determined, using an absorption coeffieient L1e605 (redueed minus oxidized) of 12 mM-1 cm-1• It is concluded that the smallest structural unit of eytochrome oxidase contains two heme groups.…