Generation of a stable, aminotyrosyl radical-induced α2β2 complex of Escherichia coli class Ia ribonucleotide reductase

Abstract

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) in the RNR small (β2) subunit over a 35-Å pathway of redox-active amino acids: Y[subscript 122]• ↔ [W[subscript 48]?] ↔ Y[subscript 356] in β2 to Y[subscript 731] ↔ Y[subscript 730] ↔ C[subscript 439] in α2. When 3-aminotyrosine (NH[subscript 2]Y) is incorporated in place of Y[subscript 730], a long-lived NH[subscript 2]Y[subscript 730]• is generated in α2 in the presence of wild-type (wt)-β2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH[subscript 2]Y[subscript 730]• induces formation of a kinetically stable α2β2 complex. Under conditions that generate NH[subscript 2]Y[subscript 730]•, binding between Y[subscript 730]NH[subscript 2]Y-α2 and wt-β2 is 25-fold tighter (K[subscript d] = 7 nM) than for wt-α2|wt-β2 and is cooperative. Stopped-flow fluorescence experiments establish that the dissociation rate constant for the Y[subscript 730]NH[subscript 2]Y-α2|wt-β2 interaction is ~10[superscript 4]-fold slower than for the wt subunits (~60 s[superscript −1]). EM and small-angle X-ray scattering studies indicate that the stabilized species is a compact globular α2β2, consistent with the structure predicted by Uhlin and Eklund’s docking model [Uhlin U, Eklund H (1994) Nature 370(6490):533–539]. These results present a structural and biochemical characterization of the active RNR complex “trapped” during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.