title: Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms.
abstract: Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s(-1) (electrons per s)], UV-visible signatures of oxidized and reduced states and ability to form the P(M) ['peroxy' (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.
authors.affiliation: Centre de Génétique Moléculaire du CNRS, UPR, Gif-sur-Yvette, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Meunier
authors.forename: Amandine
authors.initials: A
authors.lastname: Maréchal
authors.forename: Peter R
authors.initials: PR
authors.lastname: Rich
mesh_terms: D003576:Electron Transport Complex IV / Q000235:genetics* / Q000302:isolation & purification; D006639:Histidine / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics*; D009687:Nuclear Proteins / Q000138:chemical synthesis / Q000302:isolation & purification; D017354:Point Mutation / Q000235:genetics*; D021122:Protein Subunits / Q000138:chemical synthesis / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D009687:Nuclear Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D003576:Electron Transport Complex IV