Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer.

Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized αY model protein has allowed the first determination of formal reduction potentials (E°') for a Y residing within a protein (Berry, B. W.; Martı́nez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9739-9743). Using Schultz's technology, a series of fluorotyrosines (FY, n = 2 or 3) was site-specifically incorporated into αY. The global protein properties of the resulting α(3,5)FY, α(2,3,5)FY, α(2,3)FY and α(2,3,6)FY variants are essentially identical to those of αY. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°'s of the very high-potential αFY proteins. E°'(pH 5.5; αFY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The FY analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined αFY ΔE°'s to model the thermodynamics of radical-transfer reactions in FY-RNRs and to experimentally test and support the key prediction made.