title: Structural and functional analysis of deficient mutants in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae.
abstract: Four point mutations in subunit I of cytochrome c oxidase from Saccharomyces cerevisiae that had been selected for respiratory incompetence but still contained spectrally detectable haem aa3 were analysed. The isolated mutant enzymes exhibited minor band shifts in their optical spectra and contained all eleven subunits. However, steady state activities were only a few percent compared to wild type enzyme. Using a comprehensive experimental approach, we first checked the integrity of the enzyme preparations and then identified the specific functional defect. The results are discussed using information from the recently solved structures of cytochrome c oxidase at 2.8 A. Mutation 167N is positioned between haem a and a conserved glutamate residue (E243). It caused a distortion of the EPR signal of haem a and shifted its midpoint potential by 54 mV to the negative. The high-resolution structure suggests that the primary reason for the low activity of the mutant enzyme could be that asparagine in position 67 might form a stable hydrogen bond to E243, which is part of a proposed proton channel. Cytochrome c oxidase isolated from mutant T316K did not meet our criteria for homogeneity and was therefore omitted from further analysis. Mutants G352V and V380M exhibited an impairment of electron transfer from haem a to a3 and ligand binding to the binuclear centre was affected. In mutant V380M also the midpoint potential of CuB was shifted by 65 mV to the positive. The results indicated for these two mutants changes primarily associated with the binuclear centre, possibly associated with an interference in the routes and/or sites of protonation which are required for stable formation of the catalytic intermediates. This interpretation is discussed in the light of the high resolution structure.
authors.affiliation: Universitätsklinikum Frankfurt, Zentrum der Biologischen Chemie, Theodor-Stern-Kai 7, Frankfurt am Main, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Ortwein
authors.forename: T A
authors.initials: TA
authors.lastname: Link
authors.forename: B
authors.initials: B
authors.lastname: Meunier
authors.forename: A M
authors.initials: AM
authors.lastname: Colson-Corbisier
authors.forename: P R
authors.initials: PR
authors.lastname: Rich
authors.forename: U
authors.initials: U
authors.lastname: Brandt
mesh_terms: D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D003576:Electron Transport Complex IV