Studien an Cytochrom-c-Oxidase, I. Reinigung und Charakterisierung des Enzyms aus Rinderherzen und Identifizierung der im Komplex enthaltenen Peptidketten.

Abstract

As part of the preliminary work for the structural elucidation of cytochrome c oxidase [EC 1.9.3.1], the enzyme complex was isolated from bovine heart muscle and characterized chemically. The enzyme contains 10-11 nmol heme a, and 12-13 nmol Cu/mg protein. The solubilized active enzyme also contains 5% phospholipid, comprising .apprx. 2 mol each of cardiolipin and phosphatidylethanolamine per mol heme a. 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 MW 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 7 intact polypeptide chain 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 2 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 may be 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 phenylalanine in nonstoichiometric amounts. Analysis gives a minimal protein MW of 130,000 (65,000/heme a) for the 2 heme and 2 Cu-containing monomers. The MW of the moiety preliminarily defined as enzymatic is .apprx. 48,000. The chemical characterization provides data for the strategy of the subsequent sequence analysis of the polypeptides.