The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) employs a MnIV/FeIII cofactor in each monomer of its β2 subunit to initiate nucleotide reduction. The cofactor forms by reaction of MnII/FeII-β2 with O 2. Previously, in vitro cofactor assembly from apo β2 and divalent metal ions produced a mixture of two forms, with Mn at site 1 (MnIV/FeIII) or site 2 (FeIII/MnIV), of which the more active MnIV/FeIII product predominates. Here we have addressed the basis for metal site selectivity by determining X-ray crystal structures of apo, MnII, and MnII/Fe II complexes of Ct β2. A structure obtained anaerobically with equimolar MnII, FeII, and apoprotein reveals exclusive incorporation of MnII at site 1 and FeII at site 2, in contrast to the more modest site selectivity achieved previously. Site specificity is controlled thermodynamically by the apoprotein structure, as only minor adjustments of ligands occur upon metal binding. Additional structures imply that, by itself, MnII binds in either site. Together, the structures are consistent with a model for in vitro cofactor assembly in which FeII specificity for site 2 drives assembly of the appropriately configured heterobimetallic center, provided that FeII is substoichiometric. This model suggests that use of a MnIV/Fe III cofactor in vivo could be an adaptation to FeII limitation. A 1.8 Å resolution model of the MnII/Fe II-β2 complex reveals additional structural determinants for activation of the cofactor, including a proposed site for side-on (η2) addition of O2 to FeII and a short (3.2 Å) MnII-FeII interionic distance, promoting formation of the MnIV/FeIV activation intermediate.
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