Reversible Phenol Oxidation and Reduction in the Structurally Well-Defined 2-Mercaptophenol-α 3 C Protein

2-mercaptophenol-α3C serves as a biomimetic model for enzymes that use tyrosine residues in redox catalysis and multistep electron transfer. This model protein was tailored for electrochemical studies of phenol oxidation-reduction with specific emphasis on the redox-driven protonic reactions occurring at the phenol oxygen. This protein contains a covalently modified 2-mercaptophenol-cysteine residue. The radical site and the phenol compound were specifically chosen to bury the phenol OH group inside the protein. A solution nuclear magnetic resonance structural analysis: (i) demonstrates that the synthetic 2-mercaptophenol-α3C model protein behaves structurally as a natural protein, (ii) confirms the design of the radical site, (iii) reveals that the ligated phenol forms an inter-helical hydrogen bond to glutamate-13 (phenol oxygen/carboxyl oxygen distance 3.2 ± 0.5 Å), and (iv) suggests a proton-transfer pathway from the buried phenol OH (average solvent accessible surface area of 3 ± 5%) via glutamate-13 (average solvent accessible surface area of the carboxyl oxygens 37 ± 18%) to the bulk solvent. A square-wave voltammetry analysis of 2-mercaptophenol-α3C further demonstrates: (v) that the phenol oxidation-reduction cycle is reversible, (vi) that formal reduction potentials can be obtained, and (vii) that the phenol-O• state is long lived with an estimated lifetime of ≥ 180 milliseconds. These properties make 2-mercaptophenol-α3C a unique system to characterize phenol-based proton-coupled electron transfer in a low dielectric and structured protein environment.