title: Biochemical characterization of the OXI mutants of the yeast Saccharomyces cerevisiae.
abstract: OXI mutants in Saccharomyces cerevisiae lack a functional cytochrome c oxidase. Wild type and OXI mutants were grown in the presence of radioactive delta-amino[14C]levulinic acid, a precursor of porphyrin and heme, and [3H]mevalonic acid, a precursor of the alkyl side-chain of heme a. SDS polyacrylamide gel electrophoresis of the delipidated mitochondria showed that delta-amino[14C]levulinic acid was distributed into three bands migrating in the regions of Mr 28 000, 13 500, and 10 000, while [3H]mevalonic acid was found in a single band with apparent Mr of 10 000. The immunoprecipitates obtained by incubating the solubilized mitochondria of any OXI mutant with antibodies against cytochrome c oxidase, showed, after delipidation, a high specific radioactivity due to delta-amino[14C]levulinic acid and [3H]mevalonic acid. This suggested that a prophyrin a was present in all these OXI mutants. HCl fractionation confirmed the presence of porphyrin a in the apooxidase of these mutants. Atomic absorption spectra of the immunoprecipitate of cytochrome c oxidase showed that copper was not detectable in the mutant OXI IIIa which lacked subunit 1, but was present in the mutant OXI IIIb, which exhibited a minor alteration in the electrophoretic mobility of subunit 1. In OXI I and II mutants there was a 50% reduction in the amount of copper in the immunoprecipitated cytochrome c oxidase. These observations may be interpretable as follows: (1) alterations in polypeptide biosynthesis due to the OXI mutations lead to an improper configuration of cytochrome c oxidase, so that ferrochelatase cannot transfer iron into porphyrin a; (2) subunit I is the binding site for copper, but the mutations in subunits II and III alter the binding site of one of the two copper atoms in subunit I.
mesh_terms: D000622:Aminolevulinic Acid / Q000378:metabolism; D003300:Copper / Q000494:pharmacology; D004272:DNA, Mitochondrial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics*; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D013054:Spectrophotometry, Atomic
chemical_list: D004272:DNA, Mitochondrial; D046911:Macromolecular Substances; D003300:Copper; D000622:Aminolevulinic Acid; D003576:Electron Transport Complex IV