Exploring the catalytic promiscuity of enzymes is a longstanding challenge and a current topic of interest. Our group previously modified a cytochrome P450BM3 monooxygenase to perform peroxygenase activity with assistance from a rationally designed dual-functional small molecule (DFSM). However, the DFSM-facilitated P450-H2O2 system showed limited peroxidase activity. On the basis of a mechanistic analysis of the possible competitive oxidation pathways, the present work applies a protein engineering strategy of mutating redox-sensitive residues that enables the peroxygenase system to achieve efficient peroxidase activity. The engineered system exhibits efficient one-electron oxidation activity toward various substrates, including guaiacol, 2,6-dimethoxyphenol, o-phenylenediamine, and p-phenylenediamine. This system attains the best peroxidase activity of any P450 reported to date and rivals most natural peroxidases, suggesting significant potential for practical applications. This work provides insights and strategies relevant for expanding the catalytic promiscuity of P450s through combining the effects of protein engineering and exogenous molecules.

It is a great challenge to optionally access diverse hydroxylation products from a given substrate bearing multiple reaction sites of sp 3 and sp 2 CÀ H bonds. Herein, we report the highly selective divergent hydroxylation of alkylbenzenes by an engineered P450 peroxygenase driven by a dual-functional small molecule (DFSM). Using combinations of various P450BM3 variants with DFSMs enabled access to more than half of all possible hydroxylated products from each substrate with excellent regioselectivity (up to > 99 %), enantioselectivity (up to > 99 % ee), and high total turnover numbers (up to 80963). Crystal structure analysis, molecular dynamic simulations, and theoretical calculations revealed that synergistic effects between exogenous DFSMs and the protein environment controlled regio-and enantioselectivity. This work has implications for exogenous-molecule-modulated enzymatic regiodivergent and enantioselective hydroxylation with potential applications in synthetic chemistry.

Metrics & More Article Recommendations CONSPECTUS: 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. H 2 O 2 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 H 2 O 2 activation, some heme enzymes prefer the alternative Fentontype mechanism, which produces •OH radical intermediates. The fate of the •OH radical is controlled by the protein environment, using tight H-bonding networks around H 2 O 2 . The so-generated •OH radical is constrained by the surrounding H-bonding interactions, the orientation of which is targeted to perform Habstraction 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 Fentontype H 2 O 2 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 H 2 O 2 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 2 -dependent activations of LPMOs and pMMOs, in which the O 2 cosubstrate may be reduced to H 2 O 2 by external reductants. Our studies show that, generally, the H 2 O 2 activation is highly dependent on the protein environment, as well as on the presence/absence of substrates. Since H 2 O 2 is a highly flexible and hydrophilic molecule, the absence of suitable substrates may lead to unproductive binding or even to the release of H 2 O 2 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 H 2 O 2 activation. In the absence of a substrate, the hydrophilic active site of P450BM3 disfavors the binding and activation of H 2 O 2 and protects thereby the enzyme from the damage by the Fenton reaction. Due to the distinct coordination and reaction environment, the Fenton-type H 2 O 2 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 H 2 O 2 is not strategically constrained as in the enzymatic environment. As such, our Account describes...

pH buffer plays versatile roles in both biology and chemistry. In this study, we unravel the critical role of pH buffer in accelerating degradation of the lignin substrate in lignin peroxidase (LiP) using QM/MM MD simulations and the nonadiabatic electron transfer (ET) and proton-coupled electron transfer (PCET) theories. As a key enzyme involved in lignin degradation, LiP accomplishes the oxidation of lignin via two consecutive ET reactions and the subsequent C−C cleavage of the lignin cation radical. The first one involves ET from Trp171 to the active species of Compound I, while the second one involves ET from the lignin substrate to the Trp171 radical. Differing from the common view that pH = 3 may enhance the oxidizing power of Cpd I via protonation of the protein environment, our study shows that the intrinsic electric fields have minor effects on the first ET step. Instead, our study shows that the pH buffer of tartaric acid plays key roles during the second ET step. Our study shows that the pH buffer of tartaric acid can form a strong H-bond with Glu250, which can prevent the proton transfer from the Trp171-H •+ cation radical to Glu250, thereby stabilizing the Trp171-H •+ cation radical for the lignin oxidation. In addition, the pH buffer of tartaric acid can enhance the oxidizing power of the Trp171-H •+ cation radical via both the protonation of the proximal Asp264 and the second-sphere H-bond with Glu250. Such synergistic effects of pH buffer facilitate the thermodynamics of the second ET step and reduce the overall barrier of lignin degradation by ∼4.3 kcal/mol, which corresponds to a rate acceleration of 10 3 -fold that agrees with experiments. These findings not only expand our understanding on pH-dependent redox reactions in both biology and chemistry but also provide valuable insights into tryptophanmediated biological ET reactions.

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