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.

The amino acid sequence and isoelectric point of Cyt c suggests that many positively charged residues are located on the protein surface, as confirmed by solving the three-dimensional structures of Cyt c^1,2, allowing us to speculate that the electrostatic interactions contribute to the formation of the ET complex between Cyt c and CcO. The contributions of electrostatic interactions are supported by a previous docking simulation between Cyt c and CcO³, but the simulation study on the binding of a PDZ domain, a common structural domain found in signaling proteins, to the target peptide showed that the hydrophobic interactions are the major thermodynamic factors stabilizing the protein complex and that the thermodynamic contribution of the electrostatic interactions to the stability of the protein complex was negligible⁴. The thermodynamic contributions of hydrophobic and electrostatic interactions to the formation of the ET complex between Cyt c and CcO, therefore, remain controversial and detailed experimental characterization of the interprotein interactions in the ET complex has been quite limited.

Recently, however, we successfully identified the interaction site for CcO on Cyt c using NMR spectroscopy⁵. Our NMR analysis using chemical shift perturbations clearly indicated positively charged residues including several Lys residues (Lys5, Lys7, Lys8, Lys13, Lys79, Lys86, Lys87, and Lys88), located in the interaction site for CcO on Cyt c, as we expected. In addition to the positively charged residues, several negatively charged residues such as Glu4, Glu89, Glu90, and Asp93 and some hydrophobic residues such as Ile9, Ile11, Met12, and Ile81 are also present in the interaction site for CcO, indicating that both electrostatic and hydrophobic interactions would contribute to ET complex formation between CcO and Cyt.

While we have successfully determined the interaction site of Cyt c for CcO, 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. Furthermore, our previous NMR analysis revealed the CcO interaction site on Cyt c, but no information about the Cyt c interaction site on CcO has been obtained, and a detailed analysis of the interactions such as the energetic components stabilizing the ET complex, particularly transiently formed ET complexes where the ET reaction from Cyt c to CcO is induced under turnover conditions, has not yet been conducted.

Based on the structural information about the CcO interaction site on Cyt c⁵, here we constructed the ET complexes between Cyt c and CcO by performing docking simulation. We also mutated the amino acid residues located in the CcO interaction site on Cyt c, and determined the Michaelis constant, K_m, for the ET reaction from Cyt c to CcO by monitoring the oxidation of reduced Cyt c by CcO. The predicted protein complexes were computationally validated by the K_m values for wild-type and mutant Cyt c and a reasonable complex structure under turnover conditions was selected. Together with the mutational effects on the ET reaction and the predicted ET complex, the interactions essential for the formation of the Cyt c – CcO complex under turnover conditions for the ET reaction were energetically characterized.

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