Ribonucleotide reductase (RR) catalyzes the first committed and rate-determining step in DNA biosynthesis, the reduction of ribonucleotides to deoxyribonucleotides. Fe-II binding to the binuclear non-heme iron active site has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopies. These studies show that the two sites have significantly different metal binding affinities. This has also allowed a (MnFeII)-Fe-II derivative to be prepared and studied by the above spectroscopies. The spectral features of the individual irons provide geometric and electronic structural insight into each metal site. Density functional calculations on reduced RR an correlated to the spectral features to obtain insight into its electronic structure. Parallel calculations are also performed on reduced stearoyl-acyl carrier protein Delta(9) desaturase (Delta(9)D) to correlate to prior spectral data and to the active site of RR. Differences in their dioxygen reactivities are investigated through reaction of these reduced sites with dioxygen, and possible electron-transfer pathways are evaluated. These results show that the active site of reduced RR consists of one 5- and one 4-coordinate iron with the 5C center having a higher binding affinity. Compared to reduced Delta(9)D, the presence of the 4C site energetically destabilizes reduced RR. Reaction of reduced RR with dioxygen to form a superoxide intermediate is energetically up hill as it results in an excited quartet state on the oxygenated iron, while the formation of a bridged peroxo intermediate is energetically favorable. Formation of peroxo-RR is more favorable than peroxo-Delta(9)D due to ligand field differences that can control the overlap of the redox active orbitals of the reduced sites with the pi* orbitals of dioxygen. This parallels experimental differences in the dioxygen reactivity of the reduced RR and Delta(9)D active sites.