Time-resolved step-scan fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo₃ from Escherichia coli

We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of heme-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a₃-Cu_B binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu_B. Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo₃, the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu_B and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa₃ under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo₃. Furthermore, using time-resolved (5 μs resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa₃ and bo₃. In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo₃), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu_B-CO signal in bo₃ is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O₂ in flow-flash experiments.