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-center dot) generated by oxidation of a reduced dinuclear metal cluster. The Fe-2(III)-Y-center dot cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe-2(II)-NrdB, O-2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn-2(III)-Y-center dot cofactor in their NrdF subunit. Mn-2(II)-NrdF does not react with O-2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdI(hq), which, in the presence of O-2, reacts to form the Mn-2(III)-Y-center dot cofactor. Here we investigate the mechanism of assembly of the Mn-2(III)-Y-center dot cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn-2(II)-NrdF, NrdI(hq), and O-2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI(hq) reduces O-2 to O-2(center dot-) (40-48 s(-1), 0.6 mM O-2), the O-2(center dot-) channels to and reacts with Mn-2(II)-NrdF to form a (MnMnIV)-Mn-III intermediate (2.2 +/- 0.4 s(-1)), and the (MnMnIV)-Mn-III species oxidizes tyrosine to Y-center dot (0.08-0.15 s(-1)). Controlled production of O-2(center dot-) by NrdI(hq) during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn-2(II) cluster with O-2 and satisfies the requirement for an "extra" reducing equivalent in Y-center dot generation.