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

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: Y₁₂₂• ↔ [W₄₈?] ↔ Y₃₅₆ in β2 to Y₇₃₁ ↔ Y₇₃₀ ↔ C₄₃₉ in α2. When 3-aminotyrosine (NH₂Y) is incorporated in place of Y₇₃₀, a long-lived NH₂Y₇₃₀• is generated in α2 in the presence of wildtype (wt)-β2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH₂Y₇₃₀• induces formation of a kinetically stable α2β2 complex. Under conditions that generate NH2Y730•, binding between Y730NH2Y-α2 and wt-β2 is 25-fold tighter (K_d = 7 nM) than for wt-α2|wt-β2 and is cooperative. Stopped-flow fluorescence experiments establish that the dissociation rate constant for the Y₇₃₀NH₂Y-α2|wt- β2 interaction is ∼10⁴-fold slower than for the wt subunits (∼60 s^-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.