The iron(Ⅳ)-oxointermediate has been amply established as the principal oxidant in non-heme enzymes and the key player in C-H bond activations and functionalizations.In contrast to this status,our present QM/MM calculations rule out the iron(IV)-oxo as the reactive species in fosfomycin biosynthesisin HppE enzyme.[1]Thus,our study reveals that the iron(Ⅳ)-oxo species is converted,by protonation of the ion-oxo group,into a more reactive species,the iron-complexed hydroxyl radical(FeⅢ-(HO·)).The so-formed FeⅢ-(HO·)species is much more potent compared to the alternative oxidants,the ferryl and oxyl species,FeⅣ=O and FeⅢ-O·.The FeⅢ-(HO·)species is also highly selective and is the only oxidant that generates the final cis-fosfomycin product.Thus,the present work broadens the landscape of non-heme iron enzymes and makes a connection to Fenton chemistry,with implications on new potential biocatalysts that may harness hydroxyl radicals for C-H bond functionalizations.