Spectroscopic and electronic structure investigations of the oxygen-activating intermediates of ribonucleotide reductase

Circular dichroism (CD) and magnetic circular dichroism (MCD) data, combined with spin-Hamiltonian analysis and correlated with density functional theory (DFT) calculations allow identification of the active site geometries for biferrous wt- and W48F/D84E-R2 subunit of ribonucleotide reductase. This study probes the effect of varying the ligand set, the positions of coordinated water molecules and additional protein constraints on the geometry and energy of the binuclear site.; The geometric and electronic structure of the peroxo intermediate of the W48F/D84E-R2 variant was studied and related to the putative kinetically masked peroxo intermediate of wt-R2. The spectroscopic signatures of the W48F/D84E-R2 peroxo intermediate were found to be consistent only with the cis -μ-1,2 peroxo coordination geometry. DFT geometry optimizations using a model including the constraints imposed by the wt- and W48F/D84E-R2 active site ligand sets identifies cis-μ-1,2 peroxo as the most stable intermediate structure. This study produces new insight into the electronic structure of the experimentally defined peroxo intermediate, structurally similar model complexes and other biological biferric peroxo intermediates.; The high-valent intermediates of oxygen activation are poorly characterized and their structures are still a matter of debate. To investigate the nature of the high-valent bis-μ-oxo core, the spectroscopic properties and electronic structure of the structurally defined Fe₂(III,IV) complex, [Fe ₂O₂(5-Et₃-TPA)₂](ClO₄) ₃ where 5-Et₃-TPA=tris(5-ethyl-2-pyridylmethyl)amine were explored. Analysis of absorption, MCD, resonance Raman profiles and VTVH MCD saturation behavior allows transition assignments and calibration of density functional calculations. The C_2h geometric distortion of the Fe ₂O₂ core is due to asymmetry in the capping ligand and results in a small (∼20%) difference in bond strength between adjacent Fe–O bonds. Density functional calculations also identify the orbital origins of the unique magnetic features of this core and identify the three singly-occupied π* metal-based orbitals that form strong superexchange pathways which lead to valence delocalization and the S = $32$ ground state. Frontier molecular orbital (FMO) theory identifies these orbitals as key to the observed reactivity of this complex as they overlap with the substrate C–H bonding orbital in the best trajectory results for the high-valent enzyme intermediates are discussed.