FTIR study of conformational substates in the CO adduct of cytochrome c oxidase from Rhodobacter sphaeroides.

Fourier transform infrared (FTIR) spectroscopy of cytochrome c oxidase from Rhodobacter sphaeroides reveals multiple CO stretch bands that are associated with different conformational substates of the enzyme. Here we report the temperature dependence of the infrared bands for the CO bound to the Fea3 heme iron and to CuB. We have also studied the kinetics of ligand return from Fea3 to CuB using temperature derivative spectroscopy (TDS). Two classes of substates (alpha/beta) can be distinguished from their different properties with regard to the width of the IR band, the temperature dependence of the peak position, and the peak of the enthalpy distribution. The pronounced temperature dependence of the stretch frequencies in the beta conformation and the lack thereof in the alpha conformation implies very different dynamic behavior in the active site and reflects structural differences between the two conformations, most likely a shift of the position of CuB in response to a change in its stereochemical environment. Similar conformational changes will be necessary during the catalytic cycle of the enzyme when dioxygen is bound in the active site.