Cytochrome-c oxidase from Saccharomyces cerevisiae

This chapter discusses the methods for the isolation and characterization of the nine-subunit yeast holo-cytochrome-c oxidase and its subunits as well as procedures for producing and analyzing mutant cytochrome-c oxidases. Two methods for the isolation of a nine-subunit yeast cytochrome-c oxidase are described. These methods use submitochondrial particles (SMPs) as starting material. Both methods involve detergent solubilization of SMPs, ammonium sulfate fractionation of the detergent extracts, and chromatography. Two types of assay can be used to measure the activity of yeast cytochrome-c oxidase. One––a polarographic assay––follows the rate of oxygen consumption by cytochrome-c oxidase in the presence of excess reducing equivalents provided by the redox dye N, N, N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD). The other is a spectrophotometric assay that follows the rate of cytochrome-c oxidation. A reduction in cytochrome-c oxidase activity in mutant strains can result from a decreased number of assembled holoenzyme molecules, a decrease in the catalytic activity of each holoenzyme molecule, or a decrease in both the number of assembled holoenzyme molecules and their catalytic activity. © 1995 Elsevier Inc.