RESUMO
Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E(m) = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the Cu(B) center. We assigned bands to the Cu(A) center that showed a pH-independent E(m) of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E(m) of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E(m) values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-)(1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.
