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Redox reactions; Bioinorganic chemistry; Peptides and proteins; Fourier transform infrared spectroscopy; Noncovalent interactions
Abstract:
By specific 13C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry37, 7400−7406]. To attribute these signals to the individual propionates, we have constructed seven cytochrome c oxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F, H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochrome c oxidase. The ring D propionate of heme a3 might undergo conformational changes or, less likely, act as a proton donor.
