The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 angstrom. The metal site contains an oxo/hydroxo-bridged manganese dinner, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia colt. Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled (MnMnIII)-Mn-III dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor ((MnMnY center dot)-Mn-III-Y-III) generation is proposed. H2O2 (HO2-) instead of O-2 is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H2O2 dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib.