A hot oxidant, 3-NO[subscript 2]Y[subscript 122] radical, unmasks conformational gating in reductase

Abstract

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y[superscript •]) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y[subscript 122][superscript •]→W[subscript 48]→Y[subscript 356] within β to Y[subscript 731]→Y[subscript 730]→C[subscript 439] within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO[subscript 2]Y) has recently been incorporated site-specifically in place of Y[subscript 122] in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO[subscript 2]Y[superscript •]) and was inactive. In the present paper we show that incubation of apo-Y[subscript 122]NO[subscript 2]Y-β2 with Fe[superscript 2+] and O[subscript 2] generates a diferric-NO[subscript 2]Y[superscript •] that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO[subscript 2]Y[superscript •]/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO[subscript 2]Y[superscript •] is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s[superscript −1]), that dCDP is produced at 107 s[superscript −1], and that a new Y[superscript •] is produced at 97 s[superscript −1]. Studies with pathway mutants suggest that the new Y[superscript •] is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y[subscript 122]NO[subscript 2]Y-β2, a mechanism for PCET uncoupling in NO[subscript 2]Y[superscript •]-RNR is proposed.