A PH-DEPENDENT POLARITY CHANGE AT THE BINUCLEAR CENTER OF REDUCED CYTOCHROME-C-OXIDASE DETECTED BY FTIR DIFFERENCE SPECTROSCOPY OF THE CO ADDUCT

A pH-dependent polarity change at the heme-copper binuclear center of the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides has b een identified by low-temperature FTIR difference spectroscopy. ''Ligh t''-minus-''dark'' FTIR difference spectra of the fully reduced CO-enz yme adduct were recorded at a range of pH, and the dominance of differ ent populations of bound CO, alpha and beta, was found to vary with pH . An apparent pK(a) of about 7.3 for the transition was obtained. The alpha and beta forms are differentiated by different polarities at the heme-copper binuclear center of the enzyme, sensed by the stretching frequencies of CO bound either to the heme a(3) Fe or to Cu-B. Several site-directed mutants in the vicinity of the heme-copper center are s hown to favor either the alpha or the beta forms of the enyzme, sugges ting that what is being monitored is an equilibrium between two confor mations of the reduced form of the oxidase. Recent resonance Raman evi dence has been presented demonstrating that the alpha and beta forms o f the R. sphaeroides oxidase exist at room temperature; therefore, the pH-dependent change in the polarity in the vicinity of the heme-coppe r center may be functionally significant.