Moving a phenol hydroxyl group from the surface to the interior of a protein: effects on the phenol potential and pK(A).

De novo protein design and electrochemistry were used to measure changes in the potential and pK(A) of a phenol when its OH group is moved from a solvent-exposed to a sequestered protein position. A "phenol rotation strategy" was adopted in which phenols, containing a SH in position 4, 3, or 2 relative to the OH group, were bound to a buried protein site. The alpha(3)C protein used here is a tryptophan to cysteine variant of the structurally defined alpha(3)W protein (Dai et al. (2002) J. Am. Chem. Soc. 124, 10952-10953). The protein characteristics of alpha(3)C and the three mercaptophenol-alpha(3)C (MP-alpha(3)C) proteins are shown to be close to those of alpha(3)W. Moreover, the phenol OH group is fully solvent exposed in 4MP-alpha(3)C and more sequestered in 3MP-alpha(3)C and 2MP-alpha(3)C. Here we compare the redox properties of the three mercaptophenols when bound to alpha(3)C and to cysteine free in water. The pK(A) and E(peak) values are essential identical when 4MP is ligated to alpha(3)C relative to when it is free in solution. In contrast, these values are increased in 3MP-alpha(3)C and 2MP-alpha(3)C relative to the solvated compounds. The E(peak) vs pH plots all display a approximately 59 mV/pH unit dependence. We conclude that interactions with the OH group dominate the phenol redox characteristics. In 3MP-alpha(3)C and 2MP-alpha(3)C, hydrogen bonds between the protein and the bound phenols appear to either stabilize the reduced phenol or destabilize the radical, relative to the aqueous buffer, raising the potential by 0.11 and 0.12 V, respectively.