Catalysis by a class I ribonucleotide reductase (RNR) begins when a cysteine (C) residue in the α2 subunit is oxidized to a thiyl radical (C•) by a cofactor ∼35 Å away in the β2 subunit. In a class Ia or Ib RNR, a stable tyrosyl radical (Y•) is the C oxidant, whereas a MnIV/Fe III cluster serves this function in the class Ic enzyme from Chlamydia trachomatis (Ct). It is thought that, in either case, a chain of Y residues spanning the two subunits mediates C oxidation by forming transient ";pathway"; Y•s in a multistep electron transfer (ET) process that is ";gated"; by the protein so that it occurs only in the ready holoenzyme complex. The drug hydroxyurea (HU) inactivates both Ia/b and Ic β2 subunits by reducing their C oxidants. Reduction of the stable cofactor Y• (Y122•) in Escherichia coli class Ia β2 is faster in the presence of α2 and a substrate (CDP), leading to speculation that HU might intercept a transient ET pathway Y• under these turnover conditions. Here we show that this mechanism is one of two that are operant in HU inactivation of the Ct enzyme. HU reacts with the MnIV/FeIII cofactor to give two distinct products: the previously described homogeneous MnIII/ FeIII-β2 complex, which forms only under turnover conditions (in the presence of α2 and the substrate), and a distinct, diamagnetic Mn/Fe cluster, which forms ∼900-fold less rapidly as a second phase in the reaction under turnover conditions and as the sole outcome in the reaction of MnIV/FeIII-β2 only. Formation of MnIII/FeIII-β2 also requires (i) either Y338, the subunit-interfacial ET pathway residue of β2, or Y222, the surface residue that relays the ";extra electron"; to the MnIV/FeIV intermediate during activation of β2 but is not part of the catalytic ET pathway, and (ii) W51, the cofactor-proximal residue required for efficient ET between either Y222 or Y338 and the cofactor. The combined requirements for the catalytic subunit, the substrate, and, most importantly, a functional surface-to-cofactor electron relay system imply that HU effects the Mn IV/FeIII → MnIII/FeIII reduction by intercepting a Y• that forms when the ready holoenzyme complex is assembled, the ET gate is opened, and the MnIV oxidizes either Y222 or Y338.
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