Studies on cytochrome c oxidase, I. Purification and characterization of bovine myocardial enzyme and identification of peptide chains in the complex]

As part of the preliminary work for the structural elucidation of cytochrome c oxidase, the enzyme complex was isolated from bovine heart muscle and characterised chemically. The enzyme contains 10-11 nmol haem a, and 12-13 nmol copper per mg protein. The solubilised active enzyme also contains 5% phospholipid, comprising about 2 mol each of cardiolipin and phosphatidylethanolamine per mol haem a. In addition, the preparation contains a small number of detergent molecules (Tween-80). Eight polypeptide components were isolated by preparative dodecylsulphate gel electrophoresis, gel filtration on Biogel P-60, and counter current distribution. The apparent molecular weights of these components were I - 36 000, II - 28 000 (21 000), III - 19 000, IV - 14 000, V - 12 500, VI - 11 000, VII - 10 000 and VIII - 6000. At least seven intact polypeptide chains contribute to the structure of the enzyme complex of the terminal oxidase. On the basis of amino acid analysis and end group determination, they can be divided into two groups. The high molecular weight peptides I -III are hydrophobic and their amino acid compositions differ markedly from those of known enzyme proteins, especially with respect to their contents of leucine and methionine. Components I and II have formyl methionine at their N-termini. They are therefore possibly mitochondrial membrane components from complex 4 of the respiratory chain. Polypeptides IV - VII resemble functional enzyme subunits in their amino acid composition. Some of them possess free N-termini (alanine). The low molecular weight component VIII is heterogeneous and contains the N-terminal amino acids isoleucine, serine and phenylelanine in non-stoichiometric amounts. Analysis gives a minimal protein molecular weight of 130 000 (65 000 per haem a) for the two haem and two copper-containing "monomers". The molecular weight of the moiety preliminarily defined as enzymatic is about 48 000. The chemical characterisation provides data for the strategy of the subsequent sequence analysis of the polypeptides.