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

Wei Jiang, Jiajia Xie, Paul T. Varano, Carsten Krebs, J. Martin Bollinger

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22 Scopus citations

Abstract

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"; Ys 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/ FeIII2 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/FeIII2 only. Formation of MnIII/FeIII2 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.

Original languageEnglish (US)
Pages (from-to)5340-5349
Number of pages10
JournalBiochemistry
Volume49
Issue number25
DOIs
StatePublished - Jun 29 2010

All Science Journal Classification (ASJC) codes

  • Biochemistry

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