Assignment of the CO-sensitive carboxyl group in mitochondrial forms of cytochrome c oxidase using yeast mutants

Point mutations of E243D and I67N were introduced into subunit I of a 6histidine-tagged (6H-WT) form of yeast Saccharomyces cerevisiae mitochondrial cytochrome c oxidase. The two mutants (6H-E243D(I) and 6H-I67N(I)) were purified and showed ≈50 and 10% of the 6H-WT turnover number. Light-induced CO photolysis FTIR difference spectra of the 6H-WT showed a peak/trough at 1749/1740cm(-1), as seen in bovine CcO, which downshifted by 7cm(-1) in D(2)O. The bands shifted to 1736/1762cm(-1) in 6H-E243D(I), establishing that the carboxyl group affected by CO binding in mitochondrial CcOs is E243. In 6H-I67N(I), the trough at 1740cm(-1) was shifted to 1743cm(-1) and its accompanying peak intensity was greatly reduced. This confirms that the I67N mutation interferes with conformational alterations around E243. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).