Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L4MnIV(μO)2FeIIIL4 (metal–metal distance of ~ 2.75 Å, L = ligand) prevailing in metal-grown R2, or L4MnIV(μO)(μOH)FeIIIL4 (~ 2.90 Å) dominating in metal- reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~ 2.55 Å) with a L4FeIV(μO)2FeIIIL3 core structure. Several Mn,Fe(III)Fe(III) (~ 2.9–3.1 Å) and Mn,Fe(III)Fe(II) species (~ 3.3–3.4 Å) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal–metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the MnIV(μO)(μOH)FeIII core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.