Multielectron oxidation reactions catalyzed by earth-abundant metals such as iron are commonly observed in nature.1 As an example, soluble methane monooxygenase (sMMO) converts methane to methanol, carrying out the two-electron oxidation process in the active site using a diiron (Fe/Fe) cofactor and a benign oxidant, O2. However, multielectron oxidation reactions catalyzed by a heterometallic cofactor, for example, manganese-iron (Mn/Fe), are less common or known yet. Compared to Fe/Fe active sites, Mn/Fe centers possess distinct geometric and electronic structures, and determinants of their one- or two-electron oxidation reactivity remain unclear.2 Additionally, it has been difficult to imitate the oxidation reactivity catalyzed by Mn/Fe metalloenzymes with synthetic model compounds due to challenges in the synthesis of heterobimetallic complexes. In this work, we leverage a Mn/Fe R2-like ligand-binding oxidase (R2lox) as a model protein scaffold to increase our understanding of the effects of outer coordination sphere on the electronic structure and two-electron oxidation reactivity of the cofactor. We combine multifrequency electron paramagnetic resonance and X-ray absorption spectroscopy techniques in conjunction with broken–symmetry density functional theory calculations to investigate the electronic and geometric structures of the MnIII/FeIII cofactors of wild-type R2lox and variants with perturbed primary or secondary coordination sphere. Two-electron oxidation reactivity is assessed by quantifying a cross–linked peptide formed upon O2 activation. Experimental results show that (i) breaking the hydrogen bonding network in the secondary coordination sphere moderately affects the electronic structure and reduces the potency of cross-link formation and (ii) disrupting the primary coordination sphere changes the electronic structure of resting state to that of a proposed MnIII(μ-oxo)(μ-hydoxo)FeIII intermediate species. These findings underscore the importance of both primary and secondary coordination spheres in tuning two–electron oxidation reactivity through controlling the electronic structures of Mn/Fe cofactor.