Kinetic properties and ligand binding of the eleven-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-c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 mmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.-W. & Capaldi, R.A. (1992) J. Biol. Chem. 267, 22,481-22,485]. 15 of the N-terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (M(r) 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome-c oxidase contains eleven rather than twelve subunits. We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane-spanning helix. The purified enzyme had a turnover number of 1500 s-1 and the ionic-strength dependence of the Km value for cytochrome-c was similar to that described 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 resistant to formate incubation, indicating that chloride was bound to the binuclear 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 optical and EPR spectra for cytochrome-c oxidase purified with our method.
Universitätsklinikum Frankfurt, Zentrum der Biologischen Chemie, Germany.
B M
BM
Geier
H
H
Schägger
C
C
Ortwein
T A
TA
Link
W R
WR
Hagen
U
U
Brandt
G
G
Von Jagow
D000595:Amino Acid Sequence; D003486:Cyanides / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013057:Spectrum Analysis; D013379:Substrate Specificity
D003486:Cyanides; D008024:Ligands; D003576:Electron Transport Complex IV