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 alpha Y-3 model protein has allowed the first determination of formal reduction potentials (E') for a Y residing within a protein (Berry, B. W.; Martinez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 97399743). Using Schultzs technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into alpha Y-3. The global protein properties of the resulting alpha(3)(3,5)F2Y, alpha(3)(2,3,5)F3Y, alpha(3)(2,3)F2Y and alpha(3)(2,3,6)F3Y variants are essentially identical to those of alpha Y-3. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and Es of the very high-potential alpha 3FnY proteins. E'(pH 5.5; alpha 3FnY (O center dot/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 FnY 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 alpha 3FnY Delta E-o's to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.