Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase

Abstract

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 sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y[subscript 122]•) cofactor which, in the presence of S and E, oxidizes C[subscript 439] in α to a thiyl radical, C[subscript 439]•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y[subscript 122]• --> W[subscript 48 --> Y[subscript 356] in β2 to Y[subscript 731] --> Y[subscript 730] --> C[subscript 439] in α2. 3-Aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH[subscript 2]Y•) 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[subscript 356]F-β2:Y[subscript 731]NH[subscript 2]Y-α2, Y[subscript 356]F-β2:Y[subscript 730]NH[subscript 2]Y-α2, and wt-β2:Y[subscript 731]F/Y[subscript 730]NH[subscript 2]Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH[subscript 2]Y• was detected. To study off-pathway oxidation, Y[subscript 413], located 5 Å from Y[subscript 730] and Y[subscript 731] but not implicated in long-range oxidation, was examined. Evidence for NH[subscript 2]Y[subscript 413]• was sought in three complexes: wt-β2:Y[subscript 413]NH[subscript 2]Y-α2 (a), wt-β2:Y[subscript 731]F/Y[subscript 413]NH[subscript 2]Y-α2 (b), and Y[subscript 356]F-β2:Y[subscript 413]NH[subscript 2]Y-α2 (c). With (a), NH[subscript 2]Y• 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 for NH[subscript 2]Y• formation was 0.2−0.3% of that observed at 731 and 730, and with (c), no NH[subscript 2]Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C[subscript 439].