Simultaneous resonance Raman detection of the heme a₃-Fe-CO and Cu_B-CO species in CO-bound ba₃-cytochrome c oxidase from Thermus thermophilus: Evidence for a charge transfer Cu_B-CO transition

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba₃-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the Cu_B-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba₃-oxidase. We assigned the bands at 507 and 568 cm ^-1 to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm ^-1 showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the α-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa₃-oxidase, are discussed in terms of the proposed coupling of the α-and β-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The Cu _B-CO complex was identified by its ν(CO) at 2053 cm^-1 and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent Cu_B(I) that is located at the O₂ delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.