FTIR studies of the CO and cyanide adducts of fully reduced bovine cytochrome c oxidase.

Photolysis spectra of the CO and cyanide adducts of reduced bovine cytochrome c oxidase have been studied by FTIR difference spectroscopy. Bound CO is predominantly in a single 1963 cm(-1) form whereas cyanide is bound in at least two forms (2058/2045 cm(-1)). These forms are pH-independent between pH 6.5 and 8.5, indicating that there is no titratable protonatable group that influences significantly their binding in this pH range. Photolysis spectra of the cyanide adduct have a positive band around 2090 cm(-1) in H(2)O due at least in part to free HCN and at 1880 cm(-1) in D(2)O due to free DCN. The frequency of the positive band around 2090 cm(-1), and its persistence in D(2)O media, raises the possibility that a transient cyanide-Cu(B) adduct also contributes to this signal, equivalent to the CO-Cu(B) species that is formed when CO is photolyzed. Photolysis produces changes throughout the 1000-1800 cm(-1) region. Reduced minus (reduced + CO) photolysis spectra in H(2)O exhibit a pH-independent and symmetrical peak/trough at 1749/1741 cm(-1). A related feature in homologous oxidases has been suggested to arise from a conserved glutamic acid. However, only around one-third of the feature is shifted to lower frequencies by incubation in D(2)O media, and an additional fraction is shifted if catalytic turnover occurs in D(2)O. Reduced minus (reduced + cyanide) photolysis spectra exhibit multiple features in H(2)O in this region with peaks at 1752, 1725, and 1708 cm(-1) and troughs at 1740, 1715, and 1698 cm(-1). Again, only a part of these features shift in D(2)O, even with catalytic turnover. A variety of additional H/D-sensitive features in the 1700-1000 cm(-1) region of the spectra can be discerned, one of which in cyanide photolysis spectra is tentatively assigned to a conserved tyrosine, Y244. Data are discussed in relation to the structure of the binuclear center and protonatable groups in its vicinity.