Multielectron oxidation reactions catalyzed by earth-abundant metals such as iron are commonly observed in nature. As one example, soluble methane monooxygenase (sMMO) selectively converts methane to methanol, carrying out the two-electron oxidation process using a diiron (Fe/Fe) cofactor and a benign oxidant, O2. Reproducing the selectivity and specificity of metalloenzyme-catalyzed multielectron reactions has proven difficult, as such constraints are often mediated by residues in the outer coordination sphere, necessitating holistic mechanistic studies on such systems to elucidate these many levels of control. Multielectron oxidation reactions catalyzed by a heterometallic enzyme cofactor, e.g., a manganese-iron (Mn/Fe) site, represent an emergent field, though the specific determinants of two-electron reactivity in these systems remain unknown. In this work, we leverage the robust R2-like ligand-binding oxidase (R2lox) as a model Mn/Fe protein scaffold to gain a fundamental understanding of the oxygen activation and C-H bond oxidation mechanism along with effects of the outer coordination sphere on reactivity. We combine multifrequency pulsed EPR and XAS techniques in conjunction with broken-symmetry density functional theory calculations to investigate the electronic and geometric structures of the Mn/Fe cofactors of wild-type R2lox and variants with perturbed primary or secondary coordination spheres across different redox states. We find that (i) modulating the hydrogen bonding network in the secondary coordination sphere impacts the electronic interaction between Mn and Fe, which in turn influences the potency towards C–H bond activation; (ii) disrupting the primary coordination sphere almost entirely abolishes two-electron oxidation reactions; and (iii) altering proton and electron transfer routes in the two-electron oxidation process does not affect the electronic structure of the Mn/Fe center. These results highlight the significance of both the primary and secondary coordination spheres, which not only control metal binding affinities but also tune the electronic and magnetic properties of the metallocofactor to promote oxidative processes.