Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: Enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate

Joseph A. Cotruvo, Troy A. Stich, R. David Britt, Joanne Stubbe

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Abstract

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y) generated by oxidation of a reduced dinuclear metal cluster. The FeIII 2-Y cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y cofactor in their NrdF subunit. MnII 2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII 2-Y cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•- (40-48 s-1, 0.6 mM O2), the O2 •- channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s -1), and the MnIIIMnIV species oxidizes tyrosine to Y (0.08-0.15 s-1). Controlled production of O2•- by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII 2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y generation.

Original languageEnglish (US)
Pages (from-to)4027-4039
Number of pages13
JournalJournal of the American Chemical Society
Volume135
Issue number10
DOIs
StatePublished - Mar 13 2013

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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