Incorporation of Fluorotyrosines into Ribonucleotide Reductase Using an Evolved, Polyspecific Aminoacyl-tRNA Synthetase

Abstract

Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F[subscript n]Ys, n = 1–4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pK[subscript a] values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F[subscript 3]Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F[subscript n]Ys while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F[subscript n]Ys site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F[subscript n]Y(s), and the resulting proteins are isolated in good yields. F[subscript n]Ys incorporated at position 122 of β2, the site of a stable Y· in wild-type RNR, generate long-lived F[subscript n]Y·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y[subscript 122](2,3,5)F[subscript 3]Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y· can accumulate. The distinct EPR properties of the 2,3,5-F[subscript 3]Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward.