Electrochemical and Structural Properties of a Protein System Designed To Generate Tyrosine Pourbaix Diagrams

This report describes a model protein specifically tailored to electrochemically study the reduction potential of protein tyrosine radicals as a function of pH. The model system is based on the 67-residue alpha Y-3 three-helix bundle, alpha Y-3 contains a single buried tyrosine at position 32 and displays structural properties inherent to a protein. The present report presents differential pulse voltammograms obtained from alpha Y-3 at both:acidic (pH 5.6) and alkaline (pH 8.3) Conditions. The. observed Faradaic. response is uniquely associated. with Y32, as shown by site-directed mutagenesis. This is the first time voltammetry is successfully applied to detect a redox-active tyrosine residing in a structured protein environment. Tyrosine is a proton coupled electron transfer cofactor making voltammetry-based pH titrations a central experimental approach. A second set of experiments was performed to demonstrate that pH-dependent studies can be conducted on the redox-active tyrosine without introducing large-scale structural changes in the protein scaffold alpha Y-3 was re-engineered-with the specific aim to place the imidazole group of a histidine close to the Y32 phenol ring alpha Y-3-K29H and alpha Y-3-K36H each contain a histidine residue whose protonation perturbs the fluorescence of Y32. We show that these variants are stable and well-folded proteins whose helical: content, tertiary structure, solution aggregation state, and solvent-sequestered position of Y32 remain pH insensitive across a range of at least 3-4 pH units. These results confirm that the local environment of Y32 can be altered and the resulting radical site studied by voltammetry over a broad pH range without interference from long-range structural effects.