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

• Published in: Biochimica et biophysica acta. Bioenergetics Vol. 1864; no. 4; p. 149000
• Main Authors: Baserga, Federico, Storm, Julian, Schlesinger, Ramona, Heberle, Joachim, Stripp, Sven T.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2023
• Subjects: Carbon monoxide; Cytochrome c oxidase; Infrared spectroscopy; Ligation; Nanodiscs; Cytochrome c oxidase; Infrared spectroscopy; Ligation; Carbon monoxide; Nanodiscs
• ISSN: 0005-2728; 1879-2650
• DOI: 10.1016/j.bbabio.2023.149000

Cytochrome c oxidase (CcO) is a transmembrane heme‑copper metalloenzyme that catalyzes the reduction of O2 to H2O 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 R2CO state as well as the fully-reduced R4 and R4CO states and induced the “active ready” oxidized state OH. These experiments are performed in the dark yielding FTIR difference spectra exclusively triggered by exposure to O2, the natural substrate of CcO. Our data demonstrate that the presence of CO at heme a3 does not impair the catalytic oxidation of CcO when the cycle starts from the fully-reduced states. Interestingly, when starting from the R2CO 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 a3 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. •In situ gas titrations benchmark reactions of cytochrome c oxidase.•Reactions can be performed using molecular O2 and without the need for light.•CO ligation does not influence the oxidation of fully-reduced cytochrome c oxidase.•Ligand photolysis or purging in the mixed-valence state are indistinguishable.