Enthalpic and Entropic Contributions to the Mutational Changes in the Reduction Potential of Azurin

Abstract

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted 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 degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.

Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions

“…These observations are consistent with the predictions based on Coulomb's law as discussed above. Moreover, the sign of DH°(i.e., negative DH°) appears to be consistent with electrostatic considerations in other proteins [10,11,12].…”

“…Most notably for these two charged mutations, the entropic contribution to the reduction potential is larger and opposite in sign relative to the enthalpic contribution. This phenomenon has been observed for other electron transfer proteins ( [12] and references therein).…”

“…However, this law is difficult to apply to complex molecules like proteins, particularly the dielectric constant. The role of the medium that separates charges has been investigated through various methods, including solvent [8] and temperature [5,7,9,10,11,12]. Temperature alters solvent-solute interactions, with solvent more efficiently packed at lower temperature.…”

Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations

“…Multiple studies have suggested that the remaining influences on the potential of the Cu site are from hydrophobic and hydrogen bonding interactions in the secondary coordination sphere [9,32,[35][36][37][38][39][40], however these hypotheses require empirical verification [1]. Compiled works have suggested that the secondary coordination sphere can tune the reduction potential through factors such as varied water solvation, protein rigidity, electrostatics and dipole moments, and exogenous free ions which leads to a complex interplay of the entropic and enthalpic terms of each redox state [4,9,28,35,36,[41][42][43][44][45][46][47][48][49].…”

Effects of axial ligand mutation of the type I copper site in bilirubin oxidase on direct electron transfer-type bioelectrocatalytic reduction of dioxygen

“…Site-directed point mutation of the axial ligand has been carried out for blue copper proteins. It has been reported that the replacement of Met at the axial ligand by Gln causes the negative shift in the formal potential of the type I Cu site in blue copper proteins [7][8][9][10][11]. In contrast, the replacement of Gln by Met causes the positive shift in the formal potential [12,13].…”