Generation of a stable, aminotyrosyl radical-induced α2β2 complex of <i>Escherichia coli</i> 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 122 • ↔ [W 48 ?] ↔ Y 356 in β2 to Y 731 ↔ Y 730 ↔ C 439 in α2. When 3-aminotyrosine (NH 2 Y) is incorporated in place of Y 730 , a long-lived NH 2 Y 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 2 Y 730 • induces formation of a kinetically stable α2β2 complex. Under conditions that generate NH 2 Y 730 •, binding between Y 730 NH 2 Y-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 Y 730 NH 2 Y-α2|wt-β2 interaction is ∼10 4 -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.