Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin

The changes in the reduction potential of Pseudomonas aeruginosa and Alcali genes denitrificans azurins following point mutations and residue ionizatio ns were factorized into the enthalpic and entropic contributions through va riable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the c opper ion, as in the Met121Gln and Met121His variants of A. denitrificans a zurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met4 4Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proto n uptake/release in wild-type and mutated species could invariably be accou nted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E-O' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligibl e and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Sever al lines of evidence indicate that the entropic effects are notably influen ced by reduction-induced solvent reorganization effects. In particular, pro tein reduction tends to be favored on entropic grounds with increasing expo sure of the copper site to the solvent. Moreover, enthalpy-entropy compensa tion phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site o r alter the H-bonding network in the hydration shell of the molecule. There fore, in these cases, caution must be used in making predictions of E-O' ch anges simply based on Coulombic or coordination chemistry arguments.