KINETIC-PROPERTIES AND LIGAND-BINDING OF THE 11-SUBUNIT CYTOCHROME-C-OXIDASE FROM SACCHAROMYCES-CEREVISIAE ISOLATED WITH A NOVEL LARGE-SCALE PURIFICATION METHOD

A novel, large-scale method for the purification of cytochrome-e oxida se from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The puri fied enzyme exhibited a heme a/protein ratio of 9.1 nmol/mg and reveal ed twelve protein bands after Tricine/SDS/PAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to tho se recently described by Taanman and Capaldi [Taanman, J.-W & Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-22485]. 15 of the N-terminal r esidues of the 12th band were identical to subunit VIII indicating tha t this band represents a dimer of subunit VIII (M(r) 5364). We conclud e that subunit XII postulated by Taanman and Capaldi is the subunit VI II dimer and that cytochrome-c oxidase contains eleven rather than twe lve subunits. We obtained the complete sequence of subunit VIa by Edma n degradation. The protein contains more than 25% of charged amino aci ds and hydropathy analysis predicts one membrane-spanning helix. The p urified enzyme had a turnover number of 1500 s(-1) and the ionic-stren gth dependence of the K-m value for cytochrome-c was similar to that d escribed for other preparations of cytochrome-c oxidase. This was also true for the cyanide-binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide-binding kinetics and was resistan t to formate incubation, indicating that chloride was bound to the bin uclear center. When the enzyme was isolated in the absence of chloride , approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of o ptical and EPR spectra for cytochrome-c oxidase purified with our meth od.