Pulsed Multifrequency Electron Paramagnetic Resonance Spectroscopy Reveals Key Branch Points for One- vs Two-Electron Reactivity in Mn/Fe Proteins

Traditionally, the ferritin-like superfamily of proteins was thought to exclusively use a diiron active site in catalyzing a diverse array of oxygen-dependent reactions. In recent years, novel redox-active cofactors featuring heterobimetallic Mn/Fe active sites have been discovered in both the radical-generating R2 subunit of class Ic (R2c) ribonucleotide reductases (RNRs) and the related R2 -like ligand-binding oxidases (R2lox). However, the protein-specific factors that differentiate the radical reactivity of R2c from the C-H activation reactions of R2lox remain unknown. In this work, multifrequency pulsed electron paramagnetic resonance (EPR) spectroscopy and ligand hyperfine techniques in conjunction with broken-symmetry density functional theory calculations are used to characterize the molecular and electronic structures of two EPR-active intermediates trapped during aerobic assembly of the R2lox Mn/Fe cofactor. A Mn-III(mu-O)(mu-OH)Fe-III species is identified as the first EPR-active species and represents a common state between the two classes of redox-active Mn/Fe proteins. The species downstream from the MnIII(mu-O)(mu-OH)FeIII state exhibits unique EPR properties, including unprecedented spectral breadth and isotope-dependent g-tensors, which are attributed to a weakly coupled, hydrogen-bonded Mn-III(mu-OH)Fe-III species. This final intermediate precedes formation of the Mn-III/Fe-III resting state and is suggested to be relevant to understanding the endogenous reactivity of R2lox.