Observation of the equilibrium Cu_B-CO complex and functional implications of the transient heme a₃ propionates in cytochrome ba₃-CO from Thermus thermophilus. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR studies

We report the first evidence for the existence of the equilibrium Cu_B¹⁺-CO species of CO-bound reduced cytochrome ba₃ from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu_B¹⁺-CO is located at 2053 cm^-1 and remains unchanged in H₂O/D₂O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu_B histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu_B-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient CU_B¹⁺-CO complex is 34.5 s^-1 and rebinding to heme a₃ occurs with k₂ = 28.6 s^-1. The rate of decay of the transient Cu_B¹⁺-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a₃ propionates (COOH). The ν(C-O) of the transient Cu_B¹⁺-CO species is the same as that of the equilibrium Cu_B¹⁺-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu_B between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discussed.