Evidence for the presence of two conformations of the heme a ₃-Cu_B pocket of cytochrome caa ₃ from Thermus thermophilus

Resonance Raman (RR) and "light" minus "dark" Fourier transform infrared (FTIR) difference spectra are reported for the CO-bound caa₃ oxidase from Thermus thermophilus. Two Fe-CO stretching modes at 518 and 507 cm^-1, the Fe-C-O bending mode at 570 cm^-1, and three C-O modes of heme a₃ at 1958, 1967, and 1973 cm ^-1 have been identified in the RR and FTIR spectra, respectively. The FTIR "light" minus "dark" spectrum indicates the formation of Cu_BCO as revealed by its ν(CO) at 2060/2065 cm^-1. We assign the bands at 518 (ν_Fe-CO) and 1967/1973 cm^-1 (ν_C-O) as the α-conformation. We also assign the bands at 507 and 1958 cm^-1 (ν_C-O) as originating from the β-conformation of the enzyme. A frequency upshift of the heme a₃ Fe-His mode is observed subsequent to CO photolysis from 209 cm^-1 in the equilibrium deoxy enzyme to 214 cm^-1 in the photoproduct. The caa₃ data, distinctly different from those of ba₃ oxidase, are discussed in terms of the coupling of the α- and β-conformations that occur in heme-copper oxidases with catalytic function. The dynamics between the heme a₃ and heme a propionates as revealed by the perturbation of the bending vibrations δ_prop of hemes a and a₃ at 385 and 392 cm^-1, respectively, induced upon CO binding to heme a₃ is discussed in terms of the protonic connectivity between the heme a ring-D propionate/Arg site with that of the heme a ₃ ring-D propionate-H₂O site that leads to the highly conserved in the heme-copper oxidases water pool.