Identification of <scp>CO</scp> ‐binding modes in the <scp> Cu _B </scp> site of <i>bovine</i> cytochrome <i>c</i> oxidase by spatial mapping of binding energy

Cytochrome c oxidase (CcO) is the terminal enzyme of the electron-transfer system and reduces an oxygen molecule to two water molecules. The trigger of this reaction is the binding of an oxygen molecule to the binuclear center (BNC) comprising the Cu_B site and heme a₃. Due to the difficulty in obtaining the crystal structure of the complex with an oxygen molecule, other ligand molecules have been utilized to investigate the ligand-binding mechanism. In the previous studies, crystal structures of complexes with CO, NO, and CN^- ligands were determined, suggesting dynamic changes in helix X induced by ligand binding according to time-resolved infrared spectroscopic analysis. In this study, we employed ab initio quantum mechanical calculations to elucidate the ligand-recognition mechanisms of the Cu_B site and systematically analyzed the potential fields comprising the BNC and ligands. Additionally, we evaluated the effect of Tyr244 and Val243 located close to the BNC site on the potential fields, identifying Val243 as a critical factor in determining the configuration of the CO ligand bound to the Cu_B site by inducing hybridization between the 2p orbital of the O atom (CO) and the 3d orbital of the Fe atom (heme a₃). Furthermore, the Val243 model indicated the existence of two CO ligand configurations, which were consistent with experimental Fourier-transform infrared spectroscopy data. To the best of our knowledge, this represents the first elucidation of the functional role of Val243.