Properties of Site-Specifically Incorporated 3‑Aminotyrosine
in Proteins To Study Redox-Active Tyrosines: <i>Escherichia coli</i> Ribonucleotide Reductase as a Paradigm
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Abstract
3-Aminotyrosine
(NH<sub>2</sub>Y) has been a useful probe to study
the role of redox active tyrosines in enzymes. This report describes
properties of NH<sub>2</sub>Y of key importance for its application
in mechanistic studies. By combining the tRNA/NH<sub>2</sub>Y-RS suppression
technology with a model protein tailored for amino acid redox studies
(α<sub>3</sub>X, X = NH<sub>2</sub>Y), the formal reduction
potential of NH<sub>2</sub>Y<sub>32</sub>(O<sup>•</sup>/OH)
(<i><i>E</i>°′</i> = 395 ± 7
mV at pH 7.08 ± 0.05) could be determined using protein film
voltammetry. We find that the Δ<i><i>E</i>°′</i> between NH<sub>2</sub>Y<sub>32</sub>(O<sup>•</sup>/OH) and
Y<sub>32</sub>(O<sup>•</sup>/OH) when measured under reversible
conditions is ∼300–400 mV larger than earlier estimates
based on irreversible voltammograms obtained on aqueous NH<sub>2</sub>Y and Y. We have also generated D<sub>6</sub>-NH<sub>2</sub>Y<sub>731</sub>-α2 of ribonucleotide reductase (RNR), which when
incubated with β2/CDP/ATP generates the D<sub>6</sub>-NH<sub>2</sub>Y<sub>731</sub><sup>•</sup>-α2/β2 complex.
By multifrequency electron paramagnetic resonance (35, 94, and 263
GHz) and 34 GHz <sup>1</sup>H ENDOR spectroscopies, we determined
the hyperfine coupling (hfc) constants of the amino protons that establish
RNH<sub>2</sub><sup>•</sup> planarity and thus minimal perturbation
of the reduction potential by the protein environment. The amount
of Y in the isolated NH<sub>2</sub>Y-RNR incorporated by infidelity
of the tRNA/NH<sub>2</sub>Y-RS pair was determined by a generally
useful LC-MS method. This information is essential to the utility
of this NH<sub>2</sub>Y probe to study any protein of interest and
is employed to address our previously reported activity associated
with NH<sub>2</sub>Y-substituted RNRs
