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. HO acts as the cosubstrate for the heme-dependent peroxidases, P450BM3, P450, P450, 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 HO 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 HO. 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), PorFe(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 HO 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 HO 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 O-dependent activations of LPMOs and pMMOs, in which the O cosubstrate may be reduced to HO by external reductants. Our studies show that, generally, the HO activation is highly dependent on the protein environment, as well as on the presence/absence of substrates. Since HO is a highly flexible and hydrophilic molecule, the absence of suitable substrates may lead to unproductive binding or even to the release of HO 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 HO activation. In the absence of a substrate, the hydrophilic active site of P450BM3 disfavors the binding and activation of HO and protects thereby the enzyme from the damage by the Fenton reaction. Due to the distinct coordination and reaction environment, the Fenton-type HO 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 HO 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.