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

摘要

Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 5′-diphosphates. α2 houses the active site for nucleotide reduction and the binding site for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y122•) cofactor which, in the presence of S and E, oxidizes C439 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 site specifically 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 Y356F–β2:Y731NH2Y-α2, Y356F–β2:Y730NH₂Y–α2, and wt-β2:Y₇₃₁F/Y₇₃₀NH₂Y–α2, in which the non-oxidizable 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 5 Å from Y₇₃₀ 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 of 25-30% and an amplitude of 25% that observed for its formation at residues 731 and 730. With (b), the rate constant for NH₂Y• formation was 0.2-0.3% that observed at 731 and 730, and with (c), no NH₂Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C₄₃₉.