Use of 3-aminotyrosine to examine the pathway dependence of radical propagation in Escherichia coli ribonucleotide reductase

Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 50-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y₁₂₂ ^•) cofactor which, in the presence of S and E, oxidizes C ₄₃₉ in α to a thiyl radical, C₄₃₉^•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y₁₂₂^• → W₄₈ → Y₃₅₆ in β2 to Y₇₃₁ → Y₇₃₀ → C₄₃₉ in α2. 3-Aminotyrosine (NH2Y) has been sitespecifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH₂Y^•) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y ₃₅₆F-β2:Y₇₃₁NH₂Y-α2, Y ₃₅₆F-β2:Y₇₃₀NH₂Y-α2, and wt-β2:Y₇₃₁F/Y₇₃₀NH₂Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH₂Y^• was detected. To study off-pathway oxidation, Y₄₁₃, located 5Å from Y ₇₃₀ and Y₇₃₁ but not implicated in long-range oxidation, was examined. Evidence for NH₂Y₄₁₃^• was sought in three complexes: wt-β2:Y₄₁₃NH₂Y-α2 (a), wt-β2:Y₇₃₁F/Y₄₁₃NH₂Y-α2 (b), and Y₃₅₆F-β2: Y₄₁₃NH₂Y-α2 (c). With (a), NH2Y^• was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant forNH₂Y^• formation was 0.2-0.3% of that observed at 731 and 730, and with (c), noNH ₂Y^• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C₄₃₉.