Key Interactions for Electron Transfer from Cytochrome C to Cytochrome c Oxidase in Respiratory Chain: Thermodynamic Characterization of Complex Formation for Electron Transfer

The electron transfer (ET) from cytochrome bc₁ to cytochrome c oxidase (CcO) is the final and crucial ET process in the respiratory chain, which is mediated by a small hemoprotein, cytochrome c (Cyt c) (Fig.1). Despite its biological significance, the lack of the structural information prevents us from understanding the molecular mechanism for the electron transfer. Recently, we utilized ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled Cyt c in the absence and presence of unlabeled CcO, and successfully identified the interaction site for CcO on Cyt c¹. As we previously reported¹, the interaction site is constituted by the charged and hydrophobic residues in Cyt c, suggesting that both of electrostatic and hydrophobic interactions contribute to the formation of the Cyt c – CcO complex.  Although the mutations of the charged residue resulted in the significant defects of the electron transfer activity in Cyt c, the substitution of the hydrophobic residues did not affect the dissociation constant for the complex formation and electron transfer rate from Cyt c to CcO. 

To examine the contribution of the hydrophobic interactions to the electron transfer reaction from Cyt c to CcO, we determined the thermodynamic parameters for the complex formation between Cyt c and CcO.  The isothermal titration calorimetry (ITC) measurements have revealed that the complex formation of Cyt c with CcO induces the positive enthalpy (ΔH > 0) and positive entropy changes (ΔS > 0), showing that the complex formation is an endothermic and entropy-driven reaction.  It is quite interesting that the protein-protein complex formation reaction is entropy-drivenand the analysis of the entropy change suggests that one of the factors to increase the entropy in the complex formation is dehydration of hydrophobic amino acid residues located in the interaction site. 

While the experimental confirmation of the dehydration of hydrophobic amino acid residues is rather difficult, we examined osmotic pressure effects on the dissociation constant for the complex formation between Cyt c and CcO.  Based on thermodynamics, the dependence of the dissociation constant on the osmotic pressure is correlated with the volume change in the reaction of the complex formation.  Such a volume change in the complex formation can be supposed to originate from the dissociation of hydrated water molecules from the protein-protein interaction site, associated with the complex formation between Cyt c and CcO.  The dissociation constant was decreased with the increase of the osmotic pressure, corresponding to the negative volume change in the complex formation.  Because the negative volume change is one of the characteristics of the dehydration of hydrophobic amino acid residues, the osmotic pressure dependence of the dissociation constant for the complex formation between Cyt c and CcO clearly demonstrated that water molecules are dehydrated from hydrophobic amino acid residues in the interaction site between Cyt c and CcO, confirming that the hydrophobic interactions are key interactions for the formation of the electron transfer complex. To determine the amino acid residues responsible for the dehydration and examine the contribution of the dehydration to the electron transfer reaction rate from Cyt c to CcO, some mutational studies on Cyt c are now in progress.

References

[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.