Two distinct mechanisms of inactivation of the class Ic ribonucleotide reductase from chlamydia trachomatis by hydroxyurea: Implications for the protein gating of intersubunit electron transfer

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 Mn^IV/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 Mn^IV/Fe^III cofactor to give two distinct products: the previously described homogeneous Mn^III/ Fe^III-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 Mn^IV/Fe^III-β₂ only. Formation of Mn^III/Fe^III-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 Mn^IV/Fe^IV 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/Fe^III → Mn^III/Fe^III reduction by intercepting a Y^• that forms when the ready holoenzyme complex is assembled, the ET gate is opened, and the Mn^IV oxidizes either Y222 or Y338.