A pH-dependent polarity change at the heme-copper binuclear center of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides has been identified by low-temperature FTIR difference spectroscopy. 'Light'-minus- 'dark' FTIR difference spectra of the fully reduced CO enzyme adduct were recorded at a range of pH, and the dominance of different populations of bound CO, α and β, was found to vary with pH. An apparent pK(a) of about 7.3 for the transition was obtained. The α and β 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 α3 Fe or to Cu(B). Several site-directed mutants in the vicinity of the heme- copper center are shown to favor either the α or the β forms of the enzyme, suggesting that what is being monitored is an equilibrium between two conformations of the reduced form of the oxidase. Recent resonance Raman evidence has been presented demonstrating that the α and β forms of the R. sphaeroides oxidase exist at room temperature; therefore, the pH-dependent change in the polarity in the vicinity of the heme-copper center may be functionally significant.
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