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

Abstract

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-E243DI and 6H-I67NI) 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 D2O. The bands shifted to 1736/1762cm−1 in 6H-E243DI, establishing that the carboxyl group affected by CO binding in mitochondrial CcOs is E243. In 6H-I67NI, 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).