The electron transfer (ET) reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. A series of ET reactions is terminated at cytochrome c oxidase (CcO), where molecular oxygen is reduced to water. Associated with the reduction of molecular oxygen, CcO functions as a proton pump across the membrane, and the proton gradient is the primary driving force for the generation of ATP.　In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at CcO are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c forms an ET complex with CcO to promote the ET reaction. While the reduction of molecular oxygen to water molecules requires four electrons, Cyt c can carry only one electron, implying that Cyt c repetitively associates with and dissociates from CcO and suggesting that the specific interprotein interactions between Cyt c and CcO to form the ET pathway from Cyt c to CcO.

To identify the specific interactions for the complex formation between Cyt c and CcO and estimate the ET pathway, we have determined the interaction site of Cyt c for CcO using NMR spectroscopy¹. However, the NMR measurements can be applied only for ET complexes such as oxidized Cyt c – fully oxidized CcO or reduced Cyt c – fully reduced CcO complexes, where the ET reaction does not occur. In addition, our previous NMR analysis¹ identified the CcO interaction site on Cyt c, while no information about the Cyt c interaction site on CcO has been obtained. A detailed analysis of the interactions essential for the formation of the ET pathway in the ET complexes under turnover conditions, where the ET reaction from Cyt c to CcO is induced, has not yet been conducted.

Based on the mutational effects on the steady state kinetics of the ET reaction and our NMR analysis of the interaction site, we examined the structure of the ET complex between Cyt c and CcO under turnover conditions using protein docking simulation², and energetically characterized the interactions essential for complex formation and estimated the ET pathway from Cyt c to CcO. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the ET reaction to CcO. The interaction analysis using the selected Cyt c – CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions which were almost cancelled out by large favorable (Columbic) electrostatic interactions, but resulting in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, while the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO.

To determine the ET pathway from Cyt c to CcO, the pathway analysis³ was applied to the predicted ET complex between Cyt c and CcO under turnover conditions. The estimated ET pathway was found to be constructed by many hydrophobic amino acid residues, including Trp104 of the subunit II of CcO, which is supposed to be the electron entry site for CcO from Cyt c. The contribution of many hydrophobic amino acid residues to formation of the ET pathway also supports the crucial role of the dehydration from hydrophobic amino acid residues associated with the complex formation between Cyt c and CcO, and suggests formation of a structure blocking water access from the bulk water phase (a “molecular breakwater”) surrounding the ET pathway across the Cyt c - CcO interface to avoid formation of hydrogen-bond mediated non-specific ET pathways.

1. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., Proc. Natl. Acad. Sci. USA 2011, 108, 12271-12276.
2. Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., J. Biol. Chem. 2016, 291, 15320-15331.
3. Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Ann. Rev. Biophys. Biomol. Struct. 1992, 21, 349-377.