Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans.: Evidence from FTIR difference spectroscopy and site-directed mutagenesis

By specific C-13 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., Mantele, W., and Michel, H. (1998) Biochemistry 37, 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 a(3) might undergo conformational changes or, less likely, act as a proton donor.