Structural Factors for Regulation of Electron Transfer from Cytochrome C to Cytochrome C Oxidase

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The electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is the terminal ET reaction in the respiratory chain of mitochondria. By accepting four electrons from the repetitive binding of Cyt c, CcO reduces molecular oxygen to water molecules to promote the proton pumping from the matrix to inner membrane space in mitochondria, resulting in the formation of the proton gradient, which is the primary driving force for the ATP synthesis in the complex V. The ET reaction from Cyt c to CcO is, therefore, the crucial step for the energy generation system in the cellular level. Because the incomplete reduction of molecular oxygen generates reactive oxygen species, which is highly cytotoxic to damage cellular components, the ET reaction from Cyt c to CcO is thought to be strictly regulated and the formation of the specific ET pathway from Cyt c to CcO in the ET complex has been suggested. However, the ET pathway and its regulation mechanism are still controversial due to the lack of the structural information about the ET complex between Cyt c and CcO. To determine the structure of the Cyt c – CcO ET complex, we used the protein-protein docking simulation1 between Cyt c and CcO based on the NMR structural data for the interaction site for CcO on Cyt c 2. The predicted complex structure between Cyt c and CcO clearly showed that the interaction site between the two proteins is highly hydrophobic and the mutational study also revealed that the introduction of hydrophilic amino acid residues significantly reduced the ET rate from Cyt c to CcO3. The formation of such a hydrophobic interaction site was confirmed by the osmotic pressure experiments for the steady-state kinetic measurements of the ET reaction from Cyt c to CcO. Based on the osmotic pressure dependence of Michaelis constant, K m, for the ET reaction from Cyt c to CcO, we found out the significant dehydration from hydrophobic amino acid residues near the exposed hydrophobic heme periphery, facing to the hydrophobic amino acid residues of CcO, to form the hydrophobic interface between Cyt c and CcO in the predicted ET complex. The protein-protein docking simulation for the complex formation with a mutant having the hydrophilic amino acid residues in the interaction site for CcO indicated that a new hydrogen bond was formed in the interaction site between Cyt c and CcO, implying that the incomplete dehydration in the interaction site and/or invasion of the bulk water to the interaction site of the mutant. To determine the ET pathway from Cyt c to CcO, the conventional ET pathway analysis4 was applied to the predicted ET complex between Cyt c and CcO. Although the estimated ET pathway was constructed by many hydrophobic amino acid residues, the introduction of the hydrophobic amino acid residue into the interaction site resulted in the accelerated ET rate in the pathway analysis, which is inconsistent with the experimental data. We re-evaluate the ET pathway using the Green’s function and found that electron is directly transferred from the heme iron of Cyt c to the cupper ion of the subunit II of CcO through the tunneling mechanism, and the decreased hydrophobicity in the interaction site reduced the ET rate in this analysis. We, therefore, conclude that the formation of the hydrophobic interaction site by the dehydration around exposed heme periphery is essential for the regulation of the ET reaction from Cyt c to CcO in the respiratory chain. Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation. J. Biol. Chem. 2016, 291 (29), 15320-15331. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 2011, 108 (30), 12271-12276. Sato, W.; Uchida, T.; Saio, T.; Ishimori, K., Polyethylene glycol promotes autoxidation of cytochrome c. Biochim. Biophys. Acta 2018, 1862 (6), 1339-1349. Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Pathway Analysis of Protein Electron-Transfer Reactions. Ann. Rev. Biophys. Biomol. Struct. 1992, 21 (1), 349-377.