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

3-Aminotyrosine (NHY) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NHY of key importance for its application in mechanistic studies. By combining the tRNA/NHY-RS suppression technology with a model protein tailored for amino acid redox studies (αX, X = NHY), the formal reduction potential of NHY(O/OH) ( E°' = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the Δ E°' between NHY(O/OH) and Y(O/OH) when measured under reversible conditions is ∼300-400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NHY and Y. We have also generated D-NHY-α2 of ribonucleotide reductase (RNR), which when incubated with β2/CDP/ATP generates the D-NHY-α2/β2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NHY-RNR incorporated by infidelity of the tRNA/NHY-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NHY probe to study any protein of interest and is employed to address our previously reported activity associated with NHY-substituted RNRs.