Solvent-based deuterium isotope effects on the redox thermodynamics of cytochrome c.

The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D(2)O with respect to H(2)O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E degrees ' occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions.