How Do Metalloproteins Tame the Fenton Reaction and Utilize •OH Radicals in Constructive Manners?

This Account describes the manner whereby nature controls the Fenton-type reaction of O-O homolysis of hydrogen peroxide and harnesses it to carry out various useful oxidative transformations in metalloenzymes. H₂O₂ acts as the cosubstrate for the heme-dependent peroxidases, P450BM3, P450_SPα, P450_BSβ, and the P450 decarboxylase OleT, as well as the nonheme enzymes HppE and the copper-dependent lytic polysaccharide monooxygenases (LPMOs). Whereas heme peroxidases use the Poulos-Kraut heterolytic mechanism for H₂O₂ activation, some heme enzymes prefer the alternative Fenton-type mechanism, which produces •OH radical intermediates. The fate of the •OH radical is controlled by the protein environment, using tight H-bonding networks around H₂O₂. The so-generated •OH radical is constrained by the surrounding H-bonding interactions, the orientation of which is targeted to perform H-abstraction from the Fe(III)-OH group and thereby leading to the formation of the active species, called Compound I (Cpd I), Por^+•Fe(IV)═O, which performs oxidation of the substrate. Alternatively, for the nonheme HppE enzyme, the O-O homolysis catalyzed by the resting state Fe(II) generates an Fe(III)-OH species that effectively constrains the •OH radical species by a tight H-bonding network. The so-formed H-bonded •OH radical acts directly as the oxidant, since it is oriented to perform H-abstraction from the C-H bond of the substrate (S)-2-HPP. The Fenton-type H₂O₂ activation is strongly suggested by computations to occur also in copper-dependent LPMOs and pMMO. In LPMOs, the Cu(I)-catalyzed O-O homolysis of the H₂O₂ cosubstrate generates an •OH radical that abstracts a hydrogen atom from Cu(II)-OH and forms thereby the active species of the enzyme, Cu(II)-O•. Such Fenton-type O-O activation can be shared by both the O₂-dependent activations of LPMOs and pMMOs, in which the O₂ cosubstrate may be reduced to H₂O₂ by external reductants. Our studies show that, generally, the H₂O₂ activation is highly dependent on the protein environment, as well as on the presence/absence of substrates. Since H₂O₂ is a highly flexible and hydrophilic molecule, the absence of suitable substrates may lead to unproductive binding or even to the release of H₂O₂ from the active site, as has been suggested in P450cam and LPMOs, whereas the presence of the substrate seems to play a role in steering a Fenton-type H₂O₂ activation. In the absence of a substrate, the hydrophilic active site of P450BM3 disfavors the binding and activation of H₂O₂ and protects thereby the enzyme from the damage by the Fenton reaction. Due to the distinct coordination and reaction environment, the Fenton-type H₂O₂ activation mechanism by enzymes differs from the reaction in synthetic systems. In nonenzymatic reactions, the H-bonding networks are quite dynamic and flexible and the reactivity of H₂O₂ is not strategically constrained as in the enzymatic environment. As such, our Account describes the controlled Fenton-type mechanism in metalloenzymes, and the role of the protein environment in constraining the •OH radical against oxidative damage, while directing it to perform useful oxidative transformations.