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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

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journal contribution
posted on 13.03.2013, 00:00 by Joseph A. Cotruvo, Troy A. Stich, R. David Britt, JoAnne Stubbe
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 FeIII2-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. MnII2-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 MnIII2-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 MnII2 cluster with O2 and satisfies the requirement for an “extra” reducing equivalent in Y generation.