The catalytic reaction of cytochrome c oxidase probed by in situ gas titrations and FTIR difference spectroscopy.

Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O to HO at the reducing end of the respiratory electron transport chain. To understand this reaction, we followed the conversion of CcO from Rhodobacter sphaeroides between several active-ready and carbon monoxide-inhibited states via attenuated total reflection Fourier-transform infrared (ATR FTIR) difference spectroscopy. Utilizing a novel gas titration setup, we prepared the mixed-valence, CO-inhibited RCO state as well as the fully-reduced R and RCO states and induced the "active ready" oxidized state O. These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O, the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the RCO state, the release of the CO ligand upon purging with inert gas yield a product that is indistinguishable from photolysis-induced states. The observed changes at heme a in the catalytic binuclear center (BNC) result from the loss of CO and are unrelated to electronic excitation upon illumination. Based on our experiments, we re-evaluate the assignment of marker bands that appear in time-resolved photolysis and perfusion-induced experiments on CcO.