Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins to Study Redox-Active Tyrosines: E. coli Ribonucleotide Reductase as a Paradigm.

3-Aminotyrosine (NH2Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH2Y of key importance for its application in mechanistic studies. By combining the tRNA/NH2Y-RS suppression technology with a model protein tailored for amino acid redox studies (alpha X-3, X = NH2Y), the formal reduction potential of NH2Y32(O-center dot/OH) (E degrees' = 395 +/- 7 mV at pH 7.08 +/- 0.05) could be determined using protein film voltammetry. We find that the Delta E degrees' between NH2Y32(O-center dot/OH) and Y-32(O-center dot/OH) when measured under reversible conditions is 300-400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH2Y and Y. We have also generated D-6-NH2Y731-alpha 2 of ribonucleotide reductase (RNR), which when incubated with beta 2/CDP/ATP generates the D-6-NH2Y731 center dot-alpha 2/beta 2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz H-1 ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH2 center dot planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH2Y-RNR incorporated by infidelity of the tRNA/NH2Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH2Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH2Y-substituted RNRs.