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Structure/function studies of yeast cytochrome oxidase by a combination of molecular genetic, biophysical and biochemical techniques

Objective


A study has been made of the structure and function of yeast cytochrome c oxidase using genetic, biochemical and biophysical techniques. Work has been carried out in the following areas:
establishment of a new optical screening method for the assay of oxidase levels in yeast strains by direct analysis of spots growing on agar plates (a very sensitive technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates);
studies on site directed mutants of the homologous cytochrome bo from Escherichia coli and development of a working model of the structure of subunit I (studies were extended to include analyses of mutant forms of a bacterial analogue of cytochrome oxidase, termed cytochrome bo);
generation and characterization of new yeast mutants of COXI, II and III from an intronless yeast strain (a large number of cytochrome oxidase mutants were isolated from the newly constructed intronless wild type strain of yeast)
generation of new yeast mutants of COXI from an intron containing yeast strain (172 cytochrome oxidase mutants were isolated from an intron containing strain, with mutations located in COXI and each of these was characterized genetically);
site directed mutagenesis of yeast oxidase (site directed mutagenesis was achieved by 'biolistics' of H234 in COXI and the resulting mutant did not contain detectable cytochrome oxidase);
generation and characterization of second site revertants from primary yeast mutants of COXI, II and III from an intronless yeast strain (success in generation of second site revertants has now been achieved);
purification and protein chemistry of yeast cytochrome oxidase (wild type and several mutant yeast strains have been purified, mutants were selected and good resolution of the polypeptide subunit pattern has been achieved);
biochemical/biophysical characterization of enzymes (wild type enzyme has been characterized in terms of subunit composition, ligand binding, turnover number and other kinetic parameters, spectral properties, behaviour after laser photolysis of carbon monoxide and cyanide, and steady state kinetic behaviour).
The aim of the work is to produce a mechanistic/structural model of cytochrome oxidase by studying functional aspects of a variety of yeast mutants with a modified cytochrome oxidase. Such mutants can be selected as revertants of deficient cytochrome oxidase mutants of known nucleotide changes which are localisated in the mitochondrial genes coding for subunits of cytochrome oxidase. Techniques for the rapid screening of a large number of such revertants will be developed so that those of particular interest can be identified. Selected revertants will be characterised in terms of change (s) in amino acid residues and a range of
biochemical and biophysical studies will be used to localise the functional alterations which are caused by such changes.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Glynn Research Foundation Ltd
Address
Glynn House
PL30 4AU Bodmin
United Kingdom

Participants (3)

CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE
France
Address
Avenue De La Terrasse, Bftiment 26
91198 Gif Sur Yvette
Klinikum der Johann-Wolfgang-Goethe Universität Frankfurt
Germany
Address
Theodor-stern-kai 7
60596 Frankfurt Am Main
UNIVERSITE CATHOLIQUE DE LOUVAIN
Belgium
Address
5/3,Place Croix Du Sud 5/3
1348 Louvain-la-neuve