Products of mitochondrial protein synthesis in Neurospora crassa. Determination of equimolar amounts of three products in cytochrome oxidase on the basis of amino-acid analysis.

Abstract

Cytochrome oxidase isolated from Neurospora crassa was resolved into seven protein components by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate. The apparent molecular weights were determined to be 41000, 28500, 21000, 16000, 14000, 11500 and 10000 for the components 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 acids in the presence of cycloheximide.Amino‐acid analysis of the isolated components 1, 2 and 3 revealed a high content of apolar amino acids and a low content of basic amino acids compared to an average amino‐acid composition of components 4–7.Components 1, 2 and 3 contribute 27.9%, 18% and 14.2% to the whole cytochrome oxidase protein. This was calculated from the contributions of the single components to the total leucine content of the enzyme and the leucine contents (nmol leucine per mg protein) of the single components as determined by amino‐acid 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 coefficient Δɛ605 (reduced minus oxidized) of 12 mM−1 cm−1. It is concluded that the smallest structural unit of cytochrome oxidase contains two heme groups.