Electron Transfer from Cytochrome C to Cytochrome C Oxidase: Contribution of Hydrophobic “Breakwater” to Electron Transfer
Authors: Ishimori Koichiro 
Published: May 2020

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. To elucidate regulation mechanisms for the ET reaction from Cyt c to CcO, we determined interaction sites of Cyt c to CcO by NMR measurements and revealed that some hydrophobic amino acid residues including Ile11, Ile81 and Val83 near the exposed heme periphery, in addition to the positively charged amino acid residues as previously reported, are located in the CcO interaction site [1]. Combined with the structural information of the CcO interaction site on Cyt c and the Michaelis-Menten analysis of the steady state kinetics of the ET reaction from Cyt c to CcO, we estimated the structure of the ET complex under turnover conditions using the protein docking simulation. Based on the energetic analysis of the protein-protein interaction in the simulated structure of the ET complex [2], the interactions energetically essential for the complex formation were found to be hydrophobic interactions mediated by several hydrophobic amino acid residues surrounding the exposed heme periphery, which forms a “molecular breakwater”. The formation of such a breakwater would expel hydrated water molecules around the redox centers to form the hydrophobic and effective ET pathway between Cyt c and CcO. The pathway analysis of the ET pathway from Cyt c to CcO in the simulated ET complex between Cyt c and CcO also showed that the ET pathway consists of many hydrophobic amino acid residues in the interaction site and only a few hydrophilic amino acid residues contribute to the ET pathay. To confirm the contribution of the hydrophobic environments to the efficiency of the ET reaction, we mutated one of the hydrophobic amino acid residues constituting the breakwater, Ile81, to a hydrophilic amino acid residue, Ser, and found that the ET rate for the mutant (I81S) was significantly retarded. The simulated structure of the ET complex between I81S Cyt c and CcO also indicated that the reduced stabilization energy from the hydrophobic interactions and disruption of the “breakwater“, due to the invasion of the bulk water to the hydrophobic interaction site between Cyt c and CcO. Less hydrophobic environments of the interaction sites were also supported by the pathway analysis showing more hydrophilic amino acid residues are included in the ET pathway of the I81S mutant complex, compared to that of the wild type complex. However, the products of the decay parameters for the ET pathway, ε
tot, reflecting the electronic coupling constant, T
DA, for the ET reaction, was increased for the ET complex between I81S Cyt c and CcO, implying that the ET rate should be accelerate by introducing the hydrophilic amino acid residue into the interaction site. On the other hand, recent DFT calculation revealed that direct electron tunneling from the heme moiety of Cyt c to Trp104 near the copper A site of CcO and the lower electron transmission probability, estimated from the Green’s function calculations at the level of DFT, was calculated for the ET reaction in the complex between I81S Cyt c and CcO. Although we have not yet found out the reasons for the discrepancy between the experimental ET rates and the the electronic coupling constant, it is more plausible that electrons would be directly transferred from Cyt c to CcO and formation of such a breakwater would expel hydrated water molecules around the redox centers to form the hydrophobic interaction site to facilitate the effective ET reaction from Cyt c to CcO.
References
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[2] W. Sato, S. Hitaoka, K. Inoue, M. Imai, T. Saio, T. Uchida, K. Shinzawa-Itoh, S. Yoshikawa, K. Yoshizawa and K. Ishimori, J. Biol. Chem. Vol. 291 (2016), p. 15320