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

摘要

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 Fe^III2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe^II2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn^III2-Y• cofactor in their NrdF subunit. Mn^II2-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 Mn^III2-Y• cofactor. Here we investigate the mechanism of assembly of the Mn^III2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn^II2-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 Mn^II₂-NrdF to form a Mn^IIIMn^IV intermediate (2.2 ± 0.4 s⁻¹), and the Mn^IIIMn^IV species oxidizes tyrosine to Y• (0.08-0.15 s⁻¹). Controlled production of O₂^•− by NrdI_hq during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn^II₂ cluster with O₂ and satisfies the requirement for an “extra” reducing equivalent in Y• generation.