The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn(2)(III)-tyrosyl radical (Y center dot) or a Fe(2)(III)-Y center dot cofactor in the NrdF subunit. Whereas Fe(2)(III)-Y center dot can self-assemble from Fe(2)(II)-NrdF and O(2), activation of Mn(2)(II)-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O(2). The crystal structures reported here of E. coli Mn(2)(II)-NrdF and Fe(2)(II)-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn(2)(II)-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF Mn(2)(II) active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn(2)(III)-Y center dot cofactor assembly.