The R2-like ligand-binding oxidase (R2lox) proteins are one of only two identified classes of redox-active Mn/Fe proteins, defying the Irving Williams series to assemble a heterobimetallic core. In the presence of Fe alone, R2lox assembles a more canonical Fe/Fe cofactor. Upon O2activation, R2lox is capable of executing multi-electron chemistry, performing C-H bond oxidation to generate an unprecedented tyrosine-valine crosslink within its scaffold, achieving this reaction with impressive control and selectivity. As such, R2lox presents a unique opportunity for investigating Mn/Fe cofactor assembly and potential chemical reactivity, as well as the molecular elements contributing to their control, within a single protein scaffold. In this work, we have utilized diverse spectroscopic methods along with molecular biology and protein engineering methods to address many of the questions surrounding the Mn/Fe protein systems. Initial examination of the WT Mn/Fe R2lox assembly mechanism using time-resolved spectroscopies, combined with global kinetic modeling, resulted in identification of two reaction intermediates with distinct kinetic profiles and spectral signatures. Further characterization using advanced pulsed electron paramagnetic resonance (EPR) has revealed two spin-coupled, EPR-active species with unique spectral and relaxation profiles. To further examine metal binding and reactivity, point mutations in the primary and secondary metal coordination spheres have been introduced into the R2lox scaffold. Data indicate different relative binding affinities for MnIIand FeIIalong with an influence of the hydrogen bonding network on the R2lox reaction mechanism. Taken together, this work provides characterization of the Mn/Fe R2lox assembly mechanism and reaction intermediates and sets the stage for using the R2lox scaffold to probe molecular determinants of heterobimetallic protein reactivity.

Spectroscopic investigations of metal binding and oxygen activation in the heterobimetallic Mn/Fe R2lox proteins

2019 Fall National Meeting

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