The electron transfer (ET) reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at cytochrome c oxidase (CcO) are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c is thought to form an ET complex with CcO to promote the ET reaction from the heme iron in Cyt c to the Cu_A site in CcO. 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 regulate the binding affinity and the ET rate from Cyt c to CcO.

By using NMR spectroscopy, we have successfully determined the interaction site of Cyt c to CcO and found that the hydrophobic interactions are crucial for the complex formation between Cyt c and CcO¹. To confirm the energetic contribution of the hydrophobic interaction to the complex formation, we determined the numbers and sites of the dehydrated water molecules associated with formation of the ES (E: CcO, S: reduced Cyt c) complex by the osmotic pressure analysis, presenting the critical contribution of the dehydration to the entropy increase for the ES complex formation. Based on the osmotic pressure dependence of kinetics for the ET from Cyt c to CcO, about 20 water molecules were found to be dehydrated in the complex formation under turnover conditions, and the systematic mutations in the interaction site of Cyt c for CcO revealed that nearly half of the dehydrated water molecules were located around Ile81, one of the hydrophobic amino acid residues near the exposed heme periphery of Cyt c. Such a specific dehydration from the hydrophobic residue, dominantly compensating for the entropy decrease due to the association of Cyt c with CcO, forms a structure blocking water access from the bulk water phase (a “molecular breakwater”) surrounding the ET pathway across the Cyt c – CcO interface in the ES complex to protect the ET pathway from the leak of electrons to the bulk water phase.

To get further insights into the ET pathway from Cyt c to CcO, we determined the structure of the electron transfer complex between Cyt c and CcO under turnover conditions by considering the mutational effects on the steady state kinetics of the electron transfer reaction and our NMR analysis of the interaction site, and energetically characterized the interactions essential for complex formation². 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 electron transfer 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.

Based on the simulated complex structure between Cyt c and CcO, we examined the contribution of hydrophobic interactions to the ET rate. The replacement of Ile81 in Cyt c, a key hydrophobic amino acid residue constituting the hydrophobic region in the protein interface between Cyt c and CcO in the ET complex, with Ser reduced the ET rate and the Marcus theory suggested that the significant decrease of electronic coupling constant (H_DA), corresponding to the perturbations in the ET pathway from Cyt c to CcO. The dehydration of Ile81, therefore, facilitates the ET reaction from Cyt c to CcO by constituting the interprotein hydrophobic region as well as entropically promotes the ET complex formation.

1. Sakamoto, K. et al., Proc. Natl. Acad. Sci. USA 108, 12271-12276, (2011).

2. Sato, W. et al., J. Biol. Chem. 291, 15320-15331, (2016).