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Ribonucleotide reductase (RR) catalyzes the first committed and rate-determining step in DNA biosynthesis, the reduction of ribonucleotides to deoxyribonucleotides. FeII 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 MnIIFeII 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 are correlated to the spectral features to obtain insight into its electronic structure. Parallel calculations are also performed on reduced stearoyl-acyl carrier protein Δ9 desaturase (Δ9D) 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 Δ9D, 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-Δ9D due to ligand field differences that can control the overlap of the redox active orbitals of the reduced sites with the π* orbitals of dioxygen. This parallels experimental differences in the dioxygen reactivity of the reduced RR and Δ9D active sites.

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Circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD have been used to probe the biferrous active site of two variants of ribonucleotide reductase. The aspartate to glutamate substitution (R2-D84E) at the binuclear iron site modifies the endogenous ligand set of ribonucleotide reductase to match that of the binuclear center in the hydroxylase component of methane monooxygenase (MMOH). The crystal structure of chemically reduced R2-D84E suggests that the active-site structure parallels that of MMOH. However, CD, MCD, and VTVH MCD data combined with spin-Hamiltonian analysis of reduced R2-D84E indicate a different coordination environment relative to reduced MMOH, with no mu-(1,1)(eta(1),eta(2)) carboxylate bridge. To further understand the variations in geometry of the active site, which lead to differences in reactivity, density functional theory (DFT) calculations have been carried out to identify active-site structures for R2-wt and R2-D84E consistent with these spectroscopic data. The effects of varying the ligand set, positions of bound and free waters, and additional protein constraints on the geometry and energy of the binuclear site of both R2-wt and variant R2s are also explored to identify the contributions to their structural differences and their relation to reduced MMOH.

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