期刊
BIOCHEMICAL JOURNAL
卷 444, 期 -, 页码 199-204出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20120116
关键词
cytochrome c oxidase; energy coupling; mutagenesis; proton channel; subunit I; yeast
资金
- Agence Nationale de la Recherche [ANR-07-BLAN-0360-02]
- Biotechnology and Biological Sciences Research Council U.K. [BB/H000097/1]
- Agence Nationale de la Recherche (ANR) [ANR-07-BLAN-0360] Funding Source: Agence Nationale de la Recherche (ANR)
- Biotechnology and Biological Sciences Research Council [BB/K001094/1, BB/H000097/1] Funding Source: researchfish
- BBSRC [BB/K001094/1, BB/H000097/1] Funding Source: UKRI
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 (His(6) 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 Cox 13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [approximate to 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-E243D(1) (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed similar to 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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