Equilibration of Tyrosyl Radicals (Y[• over 356], Y[• over 731], Y[• over 730]) in the Radical Propagation Pathway of the Escherichia coli Class Ia Ribonucleotide Reductase

Abstract

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y[• over 122]) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y• over 122] [arrows facing left and right] [W[subscript 48]?] [arrows facing left and right] Y[subscript 356] within β to Y[subscript 731] [arrows facing left and right] Y[subscript 730] [arrows facing left and right] C[subscript 439] within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO[subscript 2]Y[superscript •]; 1.2 radicals/β2) in place of Y[• over 122] in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y[superscript •] proposed to be located on Y[subscript 356] in β2. We now report three independent methods that establish that Y[subscript 356] is the predominant location (85–90%) of the radical, with the remaining 10–15% delocalized onto Y[subscript 731] and Y[subscript 730] in α2. Pulsed electron–electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO[subscript 2]Y[• over 122]–Y[• over 356], NO[subscript 2]Y[• over 122]–NO[subscript 2]Y[• over 122], and NO[subscript 2]Y[• over 122–Y[• over 731(730)], respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y[subscript 731](3,5-F[subscript 2]Y) or Y[subscript 730](3,5-F[subscript 2]Y)-α2, which revealed F[subscript 2]Y[superscript •], studies using globally incorporated [β-[superscript 2]H[subscript 2]]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y[superscript •] delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y[superscript •] radicals in the PCET pathway and their conformations.