Cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain in mitochondria and is located within the mitochondrial membrane. CcO couples electron donation to proton transfer. This transfer occurs from the N-side to the P-side, maintaining a functional proton gradient across the membrane space. Likewise, it reduces molecular oxygen to water. The generation of the proton transmembrane gradient is used and regulated by the ATP synthase, which produces energy molecules called ATP (Adenosine-5′-triphosphate). Therefore, CcO has been studied for decades to answer the remaining questions about (i) how the redox state of its binuclear center (BNC) regulates proton uptake in its transition from oxidized to reduced state (O → R) via the K-channel, and (ii) how arginine 473I (R473I) is involved in electron-proton transfer. To address these questions, a surface cysteine-less (cysless) CcO homolog of Paracoccus denitrificans was expressed, and diverse mutations were introduced to study the proton and conformational dynamics. Additionally, a single mutation was introduced in the BNC region to examine changes in the local electric field upon reduction. To evaluate the hypothesis of cooperative proton uptake through a proton-collecting antenna involving different residues, three amino acids around the K-channel entrance were mutated to alanine, a nonpolar amino acid (H526IA, H73IIA, and E78IIA) in a surface cysteine-free background. These residues were selected because their side chains are protonable and are located near the K-channel entrance. They are also involved in forming a hydrogen-bond network that regulates proton uptake. These residues were mutated individually or in multiple combinations. Furthermore, a fluorescence probe was introduced via the thiol group of the cysteine mutation in the P301I residue. The fluorescence sensor aimed to monitor the local polarity, protonation, and conformational dynamics. Steady-state and time-resolved fluorescence spectroscopy measurements revealed different turnover rates for the single, double, and triple mutants, which were altered by the detergent gradient. Moreover, each mutation showed distinct protonation dynamics and conformational changes. The double mutation H526IA/H73IIA showed no change in the ∆pKa, a low turnover rate, and constrained conformational changes during the O → R transition upon K-channel activation. These findings suggest that the residues form a finely tuned hydrogen-bond network that controls proton uptake at the K-channel surface and affect long-range electrostatic changes from the BNC to the N-side surface. Subsequently, the role of R473I within the BNC was studied using UV-Vis and electrochemical infrared spectroscopy. Three different CcO proteins were used for this study: wild type (WT), surface cysless-WT (CS-WT), and R473IC. Likewise, carbon monoxide (CO) was used as a probe to monitor redox changes. The R473IC mutant exhibited a lower turnover rate. It also displayed notable changes in the distribution of heme bands in the UV-Vis spectra. These changes also occurred in the presence of CO in the reduced state, as observed in the UV-Vis assays. In contrast, electrochemical IR spectroscopy experiments did not reveal conclusive changes in the CO-binding spectra, the amide I and II bands, or the redox potential. This implies that the effects are primarily local electrostatic changes. These results suggest that R473I is not essential for the overall function of the CcO protein, though it is necessary for maintaining the optimal redox cycle equilibrium and electron transport in the BNC. Overall, these results propose the existence of a cooperative surface network, consisting of specific amino acids (histidines and glutamic acids), which regulates the proton uptake in the vicinity of the K-channel. Meanwhile, R473I modulates the electrostatic environment of the BNC. Together, these findings provide new insight into the mechanistic link between surface proton-collection dynamics and the internal electrostatic field contributing to redox-coupled proton pumping in cytochrome c oxidase.
