Free-radical formation in the pulse-radiolysis oxidation of inactive escherichia coli met-R2 ribonucleotide reductase

Abstract

The regeneration of a tyrosyl radical from the inactive Escherichia coli met-R2 enzyme using pulse radiolytically generated azide radicals N3˙ has been studied. Tryptophan and tyrosine amino acid residues on the protein are reactive towards N3˙ with the formation of either a tryptophan radical (peak at 510 nm) or a tyrosyl radical (peak at 410 nm). The combined rate constant (19 °C) obtained for the formation of both these deprotonated species in N2O-saturated solution, [N3–]= 0.012 M, pH 7 (40 mM phosphate), and I= 0.100 M, is 1.75 × 109 M–1 s–1. Biphasic decay of the tryptophan radical is then observed, rate constants 2.9 × 103 and 7.3 × 102 s–1, which are assigned as intramolecular electron-transfer steps. In the faster of these the tryptophan radical receives an electron from a tyrosine to give a tyrosyl radical product. The tyrosyl radicals formed in both the primary and secondary processes decay within 1 s in contrast to the much longer-lived tyrosyl-122 radical present in active R2 enzyme. Similar results were obtained on pulse radiolysis of the mutant Tyr122Phe R2 protein. Thus the stable Tyr-122 radical form of R2 does not appear to be formed in any of these reactions, and the specificity of the regeneration of the radical in the native enzyme involving reaction of the diiron(II) site with O2 is highlighted.