Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms.
Biochem J
; 444(2): 199-204, 2012 Jun 01.
Article
em En
| MEDLINE
| ID: mdl-22394221
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Mutação Puntual
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Complexo IV da Cadeia de Transporte de Elétrons
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Proteínas de Saccharomyces cerevisiae
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Histidina
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Mitocôndrias
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article