Osmotic Pressure Effects Reveal a Specific Dehydration-Induced Hydrophobic Electron Transfer Structure Comprising Cytochrome C and Cytochrome C Oxidase
Osmotic pressure was increased by addition of sugars or polyols having no specific interactions with proteins and Kd was estimated by the Michaelis-Menten analysis of the steady state kinetics for the electron transfer from Cyt c to CcO.
As Figure 1 shows, the Kd value is decreased with increasing the osmotic pressure, corresponding to the negative ΔVW of – 300 ± 78 mL mol-1. Assuming that the partial molar volume change of one hydrated water molecule around hydrophobic residues would be－17.5 mL mol-1, the volume change we obtained here corresponds to the dehydration of 17 ± 4 water molecules. Considering that the entropy change from the dehydration (dehydΔS) of one water molecule was estimated to be 5.5 JK-1mol-1, dehydΔS of 17 water molecules corresponds to ~90 JK-1mol-1, which is about 75% of total entropy change for the Cyt c-CcO complex formation. The dehydration is, therefore, one of the major factors to increase the entropy for the complex formation.
To identify the dehydration sites from hydrophobic residues in the interaction site for CcO on Cyt c, we mutated Ile11 or Ile81, hydrophobic amino acid residues in the interaction site for CcO , to Ala, an amino acid residue having a smaller side chain and less number of the hydrated water molecules. As clearly illustrated in Figure 1, the slope of the osmotic pressure dependence of Kd for the Ile mutant is less steep than that for the wild type protein. The estimated numbers of the dehydrated water molecules were reduced to 13 ± 3 and 8 ± 2 for the Ile11 and Ile81 mutants, respectively. The reduced numbers of the dehydrated water molecules in these Ile mutants indicate that the hydrated water molecules around Ile11 and Ile81 were expelled to the solvent in the complex formation between Cyt c and CcO.
In conclusion, we successfully identified that about 17 water molecules are dehydrated in the Cyt c-CcO complex formation and the primary dehydration sites are Ile11 and Ile81. Such dehydration from the hydrophobic residues can be a major factor to increase the entropy, facilitating the complex formation and the electron transfer from Cyt c to CcO.
 Sakamoto, K. et al., PNAS., 108, (2011), 12271