DF2t, a de novo designed protein that mimicks the active-site structure of many non-heme biferrous enzymes, has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD. The active site of DF2t is found to have one five-coordinate iron and one four-coordinate iron, which are weakly antiferromagnetically coupled through a mu-1,3 carboxylate bridge. These results bear a strong resemblance to the spectra of Escherichia coli ribonucleotide reductase (R2), and density functional theory calculations were conducted on the W48F/D84E R2 mutant in order to determine the energetics of formation of a monodentate end-on-bound O-2 to one iron in the binuclear site. The mu-1,3 carboxylate bridges found in O-2-activating enzymes lack efficient superexchange pathways for the second electron transfer (i.e., the OH/oxo bridge in hemerythrin), and simulations of the binding of O-2 in a monodentate end-on manner revealed that the bridging carboxylate ligands do not appear capable of transferring an electron to O-2 from the remote Fe. Comparison of the results from previous studies of the mu-1,2 biferric-peroxo structure, which bridges both irons, finds that the end-on superoxide mixed-valent species is considerably higher in energy than the bridging peroxo-diferric species. Thus, one of the differences between O-2-activating and O-2-binding proteins appears to be the ability of O-2 to bridge both Fe centers to generate a peroxo intermediate capable of further reactivity.