The catalytic reaction of cytochrome c oxidase probed by in situ gas titrations and FTIR difference spectroscopy |
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| Affiliation: | 1. Freie Universität Berlin, Experimental Molecular Biophysics, Arnimallee 14, D-14195 Berlin, Germany;2. Freie Universität Berlin, Genetic Biophysics, Arnimallee 14, D-14195 Berlin, Germany;3. Technische Universität Berlin, Division of Physical Chemistry, Strasse des 17. Juni 115, D-10623 Berlin, Germany;1. Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China;2. The Laboratory of Endocrinology and Metabolism, Guangzhou Women and Children''s Medical Center, Guangzhou Medical University, Guangzhou 510623, China;3. Department of Clinical Laboratory, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510655, China;4. Guangzhou Huayinkang Medical Laboratory Center Co., Ltd., Guangzhou 510515, China;1. Department of Molecular Microbiology and Immunology, CSIR-Central Drug Research Institute (CSIR-CDRI), Sector 10, Jankipuram Extension, Lucknow 226031, India;2. Science Department, University of Roma Tre, Via della Vasca Navale 84, Rome, Italy;3. Super Computing Applications and Innovation, CINECA, 40033, Casalecchio di Reno, Italy;4. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India;1. Department of Sciences, Roma Tre University, 00146 Roma, Italy;2. Servizio Grandi Strumentazioni and Core Facilities, Istituto Superiore di Sanità, 00185 Roma, Italy;3. Department of Chemical Sciences and Technologies, University of Roma “Tor Vergata”, 00133 Roma, Italy;4. IRCCS Fondazione Bietti, Roma, Italy;5. Accademia Nazionale dei Lincei, 00165 Roma, Italy;1. Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom;2. ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United Kingdom;3. Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, United Kingdom |
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| Abstract: | 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. |
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