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

Abstract

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 R2like 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 (μ-O)(μ-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 Mn III (μ-O)(μ-OH)Fe III 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 (μ-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.

Enzymatic Hydroxylation of Aliphatic C-H Bonds by a Mn/Fe Cofactor

“…This latter signal (Fig 3F) contained well-resolved features that are noticeably perturbed when isotopically enriched 57 Fe was incorporated into the sample. These spectra are similar to the STOT = 1/2, antiferromagnetically coupled Mn(III)-Fe(III) forms of C. trachomatis ribonucleotide reductase and R2lox (39,40). Indeed, spectral simulations revealed g-(2.03 2.03 2.02) and 55 Mn hyperfine (AMn = [300 250 380] MHz) tensors consistent with the respective spin and oxidation state assignments.…”

Enzymatic Hydroxylation of Aliphatic C–H Bonds by a Mn/Fe Cofactor

“…These spectra are similar to the S TOT = 1/2, antiferromagnetically coupled Mn­(III)–Fe­(III) forms of C . trachomatis ribonucleotide reductase and R2lox. ,, Indeed, spectral simulations revealed g - (2.03 2.03 2.02) and 55 Mn hyperfine ( A Mn = [300 250 380] MHz) tensors consistent with the respective spin and oxidation state assignments. The isotropic 57 Fe hyperfine tensor ( A Fe = [−70 −70 −70] MHz) further supported the assignment of a high-spin Fe­(III) oxidation state.…”

Bioinformatic Discovery of a Cambialistic Monooxygenase

“…These differences are analogous to those found in the conversion of Mn/Fe R2lox between the resting and I 3 states and were interpreted as arising from a longer metal−metal distance and a weaker antiferromagnetic coupling between the Mn and Fe centers. 28 We hence speculate that AEP may coordinate to one or more redox states of the cofactors, whereas the SO 3 −containing molecules occupy sites within the secondary coordination sphere of the cofactor.…”