Unraveling proton-coupled electron transfer in cofactor-free oxidase- and oxygenase-catalyzed oxygen activation: a theoretical view

Abstract

Oxygen plays a crucial role in the metabolic processes of non-anaerobic organisms. However, a detailed understanding of how triplet oxygen participates in the enzymatic oxidation of organic compounds involved in life processes is still lacking. It is noteworthy that recent studies have found that cofactor-free oxidase- and oxygenase-catalyzed oxygen activation occurs through proton-coupled electron transfer (PCET), which is significantly different from the previously proposed single electron transfer (SET) mechanism. Herein, we summarize the recent advances in the general mechanism of catalytic activation reactions of triplet oxygen by these enzymes. We believe that this review not only helps in providing a deep understanding of the processes involved in oxygen metabolism in organisms but also provides valuable theoretical reference data for designing more efficient enzyme mutants for treating diseases and handling environmental pollution in the future.

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Qian-Qian Wang

Qian-Qian Wang was born in 2000 in Henan, China. She obtained a bachelor's degree from Zhengzhou Normal University and is currently pursuing her master's degree at Zhengzhou University under the guidance of Prof. Donghui Wei. Her research interests are centered on the mechanistic studies of enzyme and organic catalyst-catalyzed reactions.

Yan Qiao

Yan Qiao, born in 1986 in Henan, China, received her BSc in Chemistry from Zhengzhou University in 2008 and her PhD in Physical Chemistry from the Dalian Institute of Chemical Physics in 2014. She conducted joint research at the University of Kentucky (2011–2013). Now an Associate Professor at Zhengzhou University, her research focuses on cancer chemoprevention, integrating computational biology and experimental methods to discover therapeutic targets for esophageal cancer.

Donghui Wei

Donghui Wei was born in 1983 in Henan, China. He graduated from Zhengzhou University (2006) and received his PhD (2012) from Zhengzhou University. While obtaining his PhD degree, he studied as a visiting scholar (2010–2012) with Prof. Chang-Guo Zhan as his advisor at the University of Kentucky, KY, USA. He joined the faculty of Zhengzhou University in 2013 and is currently a professor in the College of Chemistry. He performed a series of theoretical studies on the mechanisms of enzyme-, organocatalyst-, and transition metal-catalyzed reactions.

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1 Introduction

As we all know, the metabolic processes of non-anaerobic organisms cannot occur efficiently without the participation of oxygen (O₂). Thus, the mechanistic studies of triplet O₂-involving enzymatic reactions are highly valuable in the fields of chemistry, life sciences, medicine, and pharmacy.^1,2 Enzymes capable of activating triplet O₂ are divided into two types: oxidases that use oxygen as an oxidant to produce hydrogen peroxide or water and oxygenases that incorporate one or two oxygen atoms (monooxygenases or dioxygenases) into the substrate.³ Commonly, O₂-dependent oxidase- and oxygenase-catalyzed reactions in living organisms require the participation of metal ions or organic cofactors.^3–17 In the presence of these cofactors or metal ions, the activation process of triplet O₂ is efficiently promoted. However, a significant number of cofactor- and metal-free enzymes can also activate triplet O₂. These include cofactor-free oxidases such as uricase,¹⁸ CPO,^19,20 and PqqC^21,22 as well as cofactor-free monooxygenases and dioxygenases, including ActVA-Orf6,²³ DpgC,²⁴ HOD,^25,26 and RluC²⁷ (Fig. 1). At the same time, the mechanism by which triplet O₂ is activated without cofactors and metal ions has attracted increasing experimental and theoretical attention.^{8,19,21,22,28–36}

Fig. 1 Crystal structures of selected cofactor-free oxidases and oxygenases, including uricase (PDB ID: 4N9M³⁷), PqqC (PDB ID: 1OTW²¹), CPO (PDB ID: 2AEX²⁰), ActVA-Orf6 (PDB ID: 1N5S²³), RluC (PDB ID: 2PSJ²⁷), DpgC (PDB ID: 2NP9³⁸), HOD (PDB ID: 2WJ4³⁹), TnmJ (PDB ID: 8G5S⁴⁰), and TnmK2 (PDB ID: 8G5T⁴⁰).

For the catalytic mechanism of cofactor-free oxidases and oxygenases, the activation of triplet O₂ may occur via two possible pathways, including single electron transfer (SET) and proton-coupled electron transfer (PCET), as depicted in Scheme 1A.^28–35 The SET pathway involves direct transfer of a single electron from the substrate ion (Sub⁻) to triplet O₂, resulting in the generation of ˙Sub and ˙O₂⁻ radicals. The SET mechanism has been widely investigated for the oxidase- and oxygenase-catalyzed activation of O₂ in a significant number of mechanistic studies, providing a deep understanding of the metabolic processes of non-anaerobic organisms.^{2,3,28–31,34,41,42} On the other hand, the PCET involves the deprotonation of the substrate (Sub-H) coupled with electron transfer to O₂.

Scheme 1 (A) Possible SET and PCET pathways of cofactor-free oxidase- and oxygenase-catalyzed activation of O₂. (B)–(D) SET and PCET pathways of cofactor-free uricase-, ActVA-Orf6-, and HOD-catalyzed activation of O₂. (E) Possible SET and PCET pathways of flavin-dependent oxygenase-catalyzed activation of O₂. (F) SET and PCET pathways of flavin-dependent RutA-catalyzed activation of O₂. Dots represent single electrons on O₂, Sub-H represents substrates, and Fla-H represents flavin cofactor.

It is worth mentioning that the PCET concept and mechanism have been widely studied in other reaction systems,^43–57 but they have been less explored in the cofactor-free oxidase and oxygenase systems. In the previous studies, triplet O₂ has always been considered and simulated to directly react with the substrate ions (Sub⁻) and not in the deprotonation process of substrates, causing the PCET mechanism to be rarely mentioned for a long duration. Until recently, the possible PCET pathways for the cofactor-free oxidases and oxygenases shown in Scheme 1B–D have gradually attracted increasing attention from researchers.^32,33,35

Besides, regio- and stereospecific oxygen insertions by flavin-dependent oxidases and oxygenases are useful for the production of pharmaceutical ingredients and fine chemicals in industrial applications.^58–67 Therefore, it is very important to study the mechanism of flavin-dependent oxidase and oxygenase-catalyzed reactions involving triplet O₂. The flavin-dependent oxidase and oxygenase-promoted reactions may also include two different pathways including SET and PCET pathways to activate O₂.^9,68–81 As shown in Scheme 1E, the SET mechanism involves the direct transfer of a single electron from flavin ion (Fla⁻) to triplet O₂, resulting in the generation of ˙Fla radical and ˙O₂⁻ radical. Alternatively, the PCET mechanism has been reported via the deprotonation of flavin (Fla-H) coupled with an electron transfer to O₂ in the flavin-dependent oxygenase-mediated reaction, as shown in Scheme 1F.

Understanding the cofactor-free oxygenases, cofactor-free oxidases, and flavin-dependent oxygenases, catalytic mechanisms can provide valuable theoretical reference data for designing more efficient enzyme mutants to treat hyperuricemia-related diseases and address environmental pollution. Therefore, we are motivated to contribute this review on recent advances in the mechanistic studies of oxidase- and oxygenase-catalyzed reactions from a theoretical perspective. In this review, in order to better understand the metabolic processes in oxygen-dependent metabolism in organisms, we mainly summarize the latest progress on the activation of O₂ by cofactor-free oxygenases, cofactor-free oxidases, and flavin-dependent oxygenases via the PCET pathway.

2 Cofactor-free oxidases and oxygenases

2.1 Cofactor-free oxidase uricase

Uric acid (UA) is the product of xanthine oxidase-catalyzed oxidation of xanthine in the human body, and it is typically maintained at a concentration of 3.6–8.3 mg dL⁻¹ in blood plasma.⁸² Elevated serum UA levels predominantly lead to hyperuricemia in the human body. Persistent hyperuricemia can certainly result in gouty arthritis and renal stones, which is characterized by the deposition of monosodium UA monohydrate crystals.^83–86 In the case of tumor lysis syndrome, UA levels become extremely high, resulting in the rapid loss of kidney function.^87–89 Uricase regulates the concentration of uric acids in the blood of birds and reptiles, and uricase mutants could also serve as potential exogenous protein drugs for effective treatment of patients suffering from hyperuricemia disorder and related diseases.^90–92 Therefore, it is necessary to understand the mechanism of cofactor-free oxidase uricase-catalyzed reactions.

According to the available knowledge in the literature, the uricase-catalyzed reaction starts from 8-HX.^93–97 In 2017, Zhan and co-workers reported on the PCET pathway for uricase-catalyzed activation of O₂, and the QM/MM-FE calculations were performed at the [B3LYP/6-31+G(d):AMBER]//[B3LYP/6-31G(d):AMBER] level.³² In these QM/MM-FE calculations, the QM/MM reaction coordinate calculations were followed by free energy perturbation (FEP) calculations on the protein environment to account for the dynamic effects of the protein environment on the free energy barriers for the enzymatic reaction.^98–102 The DFT method has been confirmed to be a good choice to study the reaction mechanism in QM part.^103–108Fig. 2 depicts the uricase-catalyzed oxidation reaction pathway for 8-HX in detail. In this process, the first step involves the transfer of protons from the substrate 8-HX to the N^z atom of the residue K9 with the assistance of residue T69, while electron transfer occurs from 8-HX to the oxygen molecule to activate O₂via transition state 2ts^t (10.0 kcal mol⁻¹). The second reaction step involves the transfer of protons from the protonated amino group of residue K9 to the O₂ atom through transition state 4ts^t (0.8 kcal mol⁻¹). Subsequently, diradical recombination occurs to form a C5–O1 bond. The third reaction step is the dissociation of H₂O₂ molecule through transition state 7ts^s (13.3 kcal mol⁻¹). Further, as reported,¹⁰⁹ the experimentally measured rate constant (k_cat) for uricase-catalyzed oxidation of UA ranges from 6.0 s⁻¹ to 70 s⁻¹ at T = 298.15 K for uricase. This value of calculated free energy barrier (16.2 kcal mol⁻¹) is close to experimental measurement free energy barrier (14.9–16.4 kcal mol⁻¹) for the rate-determining step of the uricase-catalyzed reaction. In addition, the residues K9 and T69 participate in the proton transfer process of oxygen activation catalyzed by uricase (Fig. 2).

Fig. 2 Reaction pathway for the catalytic oxidation of 8-HX by uricase and free energy profile for uricase-catalyzed oxidation of 8-HX (energy in kcal mol⁻¹). The superscript “t” represents a triplet state, the superscript “s” represents a singlet state, the superscript “os” represents an open-shell singlet state. The meanings represented by “t”, “s”, and “os” are the same in Fig. 3 to 8 as in this figure.

In almost all oxidation reactions involving molecular oxygen, changes in spin state are inevitable, involving quite complex dynamic processes that are cross correlated with various potential energy surfaces. Although the issue has not been deeply and widely explored in the cofactor-free oxidase and oxygenase-catalyzed reactions, the solutions have been mentioned in previous literature on the transformation of oxygen. For example, Bearpark et al. presented a method, which avoids the use of Lagrange multipliers, for the optimization of the lowest energy point of the intersection of two potential energy surfaces,¹¹⁰ and implemented in quantum chemistry packages. This method is also applied to the oxidation of flavin by O₂.¹¹¹

It is worth mentioning that during the activation of O₂ catalyzed by uricase, protons gradually transfer from 8-HX to T69, and then to K9. Meanwhile, electron transfer occurs from 8-HX to a triplet O₂ molecule, occupying the anti-π (π*) orbital of the O₂ molecule. Due to this electron transfer, the spin density (SD) of the O₂ molecule varies from 1.83 for 1^t to 1.61 for 2ts^t and 1.02 for 3^t; the SD value of 8-HX changes from 0.16 for 1^t to 0.39 for 2ts^t and 0.99 for 3^t (Fig. 3). The spin density value of the proton involved in the PCET process is zero, so the transfer process of hydrogen atom should not be involved in the reactions. Therefore, after the first step of the reaction, both the substrate 8-HX and O₂ become free radicals, which is consistent with the experimental findings of electron spin resonance spectroscopy (ESR) that can capture substrate free radicals.¹¹²

Fig. 3 Spin densities of the substrate 8-HX, O₂, the residue K9(N^ZH₂), and the residue T69(O^γH^γ) in the critical transition state 2ts^t at the oxidation stage of the uricase-catalyzed reaction.

As noted above, the intermediate 3^t′ will not exist at all in the active site under aerobic conditions, because it can automatically transfer an electron to the nearby O₂ molecule and activate the O₂ molecule. As a result of the O₂ activation, the O–O distance (R_O1–O2) in the oxygen molecule changes from 1.23 Å in 1^t to 1.26 Å in 2ts^t and to 1.34 Å in 3^t. The first reaction step produces ˙UA⁻ + ˙O₂⁻, rather than UA²⁻ + O₂, due to the automatic electron transfer from UA²⁻ to O₂ coupled with the O₂ activation. The O₂ activation is associated with the increase in the O–O bond length (R_O1–O2). To further test this point, Zhan and co-workers carried out the aforementioned QM/MM reaction-coordinate calculations, but with the O–O bond length (R_O1–O2) frozen at that (∼1.23 Å) in the optimized 1^t structure.³² The QM/MM reaction-coordinate calculations with reaction coordinate R_O1–O2 frozen indeed led to UA²⁻ and triplet O₂ in the active site, but with a significantly higher energy barrier (12.9 kcal mol⁻¹, Fig. 2). The calculation results indicate that O₂ would not accept an electron from UA²⁻ when the O–O bond length was frozen and the reaction pathway via ˙UA⁻ + ˙O₂⁻ is the more favourable pathway. This is of great significance for understanding the metabolic process of O₂ in living organisms.

It is well known that computational design and discovery of a high-activity mutant of an enzyme are extremely challenging and labour-intensive, because a truly reliable mutant design must be based on more extensive QM/MM calculations of the free energy profiles of various possible hypothetical mutants. Therefore, before carrying out extensive computational design and experiments for the oxidase and oxygenase redesign, Zhan and co-workers ensured that the key residues could affect the catalytic activity of the enzyme. For example, according to the calculation results of Zhan et al.,³² residues Asn271 and Gln299 were identified as key residues during the calculation process. In order to verify the importance of Asn271 and Gln299, they examined the effects of Asn271Leu and Gln299Ile mutations on the catalytic activity (V_max) of Bacillus fastidiosus uricase by carrying out experimental studies including site-directed mutagenesis, protein expression, and in vitro enzyme activity assays. According to the in vitro experimental data, with the Asn271Leu mutation, the uricase activity was too low to be detected (or with V_max < 1% of V_max for the wild-type enzyme) within the detection limit of our activity assay. The Gln299Ile mutation also significantly decreased the V_max value of the enzyme by ∼13-fold; the Gln299Ile mutant had ∼8% activity compared to the wild-type uricase. The experimental data are consistent with the computationally revealed roles of Asn271 and Gln299 in the enzymatic reaction process. It is worth mentioning that most of the catalytic functions of enzymes are strictly controlled by electrostatic interactions.¹¹³ The design of point mutations is a highly challenging task because the mutated residue not only changes its own (electrostatic) interactions with the reaction site, but also slightly alters the structure of the enzyme near the mutation point, thereby changing the entire pattern of electrostatic interactions with the active site. This novel PCET mechanism of uricase-catalyzed activation of oxygen provides a mechanistic base for the rational design of uricase mutants with improved catalytic activity against uric acid as an enhanced enzyme therapy.

2.2 Cofactor-free monooxygenase ActVA-Orf6

The ActVA-Orf6 protein is a homodimer quinone-forming monooxygenase with A and B subunits from Streptomyces coelicolor. It requires only molecular O₂ without any assistance from the prosthetic group (i.e., metal ions and cofactors). In addition, it can catalyze the oxygenation of 6-deoxydihydrokalafungin (DDHK) to dihydrokalafungin (DHK) by releasing H₂O in the actinorhodin biosynthetic pathway.²³ Many efforts have been made to study the oxygenation mechanism of ActVA-Orf6 monooxygenase through spectroscopic characterization, kinetic studies, and other determination experimentally.^114–118

Noteworthy, Wei and co-workers have studied a PCET mechanism for O₂ activation in cofactor-free monooxygenase ActVA-Orf6-catalyzed oxygenation of DDHK to form DHK and the QM/MM calculations were performed at the [B3LYP-D3/6-311++G(2df,2pd):AMBER]//[B3LYP/6-31G(d,p):AMBER] level.³³ As shown in Fig. 4, the first step is a PCET process via transition state 10ts^t (15.5 kcal mol⁻¹) to activate O₂, which is assisted by one water molecule forming intermediate 11^t. Subsequently, 11^t is easy to transform into an open-shell singlet intermediate 12^os, since the structures and energies are similar and almost degenerate, respectively. Notably, the spin-flipping process might be involved during the transformation process between singlet and triplet, and computations on the energy barrier of spin-flipping process should be good choice for solving this issue.¹¹⁹ Then the diradical complexation occurs via the transition state 13^os (10.6 kcal mol⁻¹) into a closed-shell singlet intermediate 14^s with the formation of a C13–O2 bond. The final step is a dehydration process via the transition state 15ts^s (20.7 kcal mol⁻¹) accompanied by the generation of monooxygenation DHK⁻ products. It is worth mentioning that since the substrate is identified to exist in a negative deprotonated state in the active center of enzyme catalysis, an intermolecular single electron transfer (SET) process is likely to occur. The relative Gibbs free energy for the SET process is 53.3 kcal mol⁻¹ in the QM computational modeling, indicating that the SET process is impossible to occur under experimental condition. In addition, as reported,^120,121 the reaction rate constant k_cat is equal to 4.17 × 10⁻² s⁻¹ at 303 K in experiments of ActVA-Orf6 monooxygenase-catalyzed oxidation of phenols to quinones. The value of the calculated energy barrier of 20.7 kcal mol⁻¹ is very close to the energy barrier 19.7 kcal mol⁻¹ estimated by experiments. It is worth mentioning that in the catalytic oxidation process of monooxygenase ActVA-Orf6, the residues R86 and W66 form hydrogen bonds with substrate DDHK⁻ in the active cavity (Fig. 4).

Fig. 4 Reaction pathway of catalytic oxidation of DDHK⁻ by monooxygenase ActVA-Orf6 and free energy profile for ActVA-Orf6-catalyzed oxidation of DDHK⁻ (energy in kcal mol⁻¹).

In addition, the PCET process has been confirmed by analyzing the SD value changes in the first step. As shown in Fig. 5, the SD value of O₂ decreases from 1.97 in 9^t to 1.21 in transition state 10ts^t, and finally to 1.01 in 11^t, while the SD value of DDHK⁻ increases from 0.02 in 9^t to 0.77 in transition state 10ts^t, and eventually to 0.99 in 11^t. This proves that the activation of O₂ occurs through the PCET process. This PCET mechanism also provides new insights into the mechanism of O₂ activation by monooxygenases.

Fig. 5 Spin densities of the substrate DDHK⁻, O₂, and H₂O via the critical transition state 10ts^t of the monooxygenase ActVA-Orf6-catalyzed oxidation reaction.

2.3 Cofactor-free dioxygenase HOD

A prototypical cofactor-free dioxygenase is 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Rü61a.³⁹ HOD can effectively promote the degradation of quinoline pollutants in the natural environment. Moreover, HOD is also useful to reduce both virulence and bacterial growth of P. aeruginosa in leaf tissues in plants.³⁹ It catalyzes the oxygenolytic ring-opening of the N-heteroaromatic substrate (1-H-3-hydroxy-4-oxoquinaldine, QND) with concomitant release of carbon monoxide.^34,122–124

In the past, extensive experimental studies have been conducted on the reaction mechanism of HOD through spectroscopic characterization, kinetic studies, and various enzymatic determinations.^25,125 In 2023, Wei and coworkers proposed the PCET mechanism for the O₂ activation in HOD-catalyzed oxidation reaction of QND, and the QM/MM calculations were performed at the [B3LYP-D3/6-311+G(d,p):Amber]//[B3LYP/6-31G(d,p):Amber] level.³⁵

As depicted in Fig. 6, the first step is a PCET process to activate O₂ assisted by the H251/D126 dyad to form intermediate 19^tvia transition state 18ts^t (6.7 kcal mol⁻¹). It is followed by the recombination of diradical in 20^os into a closed-shell singlet intermediate 22^s with C2–O2 bond formation via transition state 21ts^os (0.3 kcal mol⁻¹). It should be noted that the structures of 19^t and 20^os are similar, and their energies are degenerate. After that, a ring-closure process occurs to form a bicyclic structure via transition state 23ts^s (4.9 kcal mol⁻¹). The final step is the release of CO via transition state 25ts^s (10.5 kcal mol⁻¹) followed by the formation of N-acetyl-anthranilate product. Further, the experimentally measured rate constant (k_cat)¹²⁶ for the HOD-catalyzed N-heteroaromatic ring cleavage reaction is 38.4 s⁻¹ for HOD, and the k_cat value can be used to estimate the energy barrier for the reaction. Wei and co-workers calculated the energy barrier (14.9 kcal mol⁻¹) for the entire HOD-catalyzed reaction to be close to the experimental observation¹²⁷ that the energy barrier is 15.5 kcal mol⁻¹. In the catalytic oxidation process of dioxygenase HOD, the residues D126 and H251 form hydrogen bonds with the substrate in the active cavity and can participate in the following proton transfer process, and the residues H102 form hydrogen bonds with intermediate 22S in the active cavity (Fig. 6).

Fig. 6 Reaction pathway for catalytic oxidation of QND by cofactor-free dioxygenase HOD and potential energy profile for the HOD-catalyzed N-heteroaromatic ring cleavage reaction (energy in kcal mol⁻¹).

It is worth mentioning that Fig. 7 clearly illustrates the PCET process in the first step of the reaction. As shown in Fig. 7, the SD value of the O₂ molecule changes from 1.97 in 17^t to 1.66 in 18ts^t and to 1.04 in 19^t, while the SD value of QND changes from 0.03 in 17^t to 0.34 in 18ts^t and to 0.96 in 19^t. Therefore, it can be concluded that both the substrate QND and O₂ become radicals after the first step of the reaction, and this result is consistent with the experimental observation that the existence of substrate radicals and superoxide anion radicals was detected in the electron spin resonance spectroscopy (ESR) studies. This indicates that the PCET mechanism could be a general mechanism for the cofactor-free dioxygenase-catalyzed activation of O₂.

Fig. 7 Spin densities (SDs) of the substrate QND, O₂, residue H251, and residue D126 via the critical transition state 18ts^t in the dioxygenase HOD-catalyzed reaction.

3 Flavin-dependent monooxygenase RutA

RutA belongs to a large family of flavoprotein monooxygenases (FMOs) that are widely spread across all kingdoms of life and catalyze the uracil-ring degradation to yield ureidoacrylate, allowing bacteria to utilize pyrimidine rings as the nitrogen source.^60,128,129 Recent research by Wang and co-workers proposed the PCET mechanism for O₂ activation in the RutA-catalyzed reaction and the QM/MM calculations were performed at the [B3LYP-D3/def2-TZVP]//[B3LYP-D3/def2-SVP] level.⁷⁰ As shown in Fig. 8, the first step is the PCET process to activate O₂ assisted by FMN⁻ to form 29^tvia transition state 28ts^t (4.9 kcal mol⁻¹). Then the 29^t intermediate flips from the triplet state to the open-shell singlet state 30^os. Then it is followed by diradical recombination in 30^os into a closed-shell singlet intermediate 32^s with N5–O1 bond formation via transition state 31ts^os (17.9 kcal mol⁻¹). The third step is the cleavage of O1–O2 bond coupled with the formation of C4–O2 bond via transition state 34ts^s (1.6 kcal mol⁻¹). The fourth step is the ring-cleavage via transition state 36ts^s (0.4 kcal mol⁻¹). The final step involves a proton transfer coupled with the formation of 3-ureidoacrylate product via transition state 38ts^s (0.4 kcal mol⁻¹). This indicates that the PCET mechanism could be a general mechanism for the flavin-dependent oxygenase-catalyzed activation of O₂.

Fig. 8 Reaction pathway for the catalytic C–N cleavage of uracil by flavin-dependent monooxygenase RutA (free energy in kcal mol⁻¹).

Furthermore, the PCET mechanism in enzymes involving O₂ activation has been supported by the recent experimental study. For example, Zhang and co-workers reported that the interaction between an electron donor and a proton donor could overcome the barrier of direct O₂ activation via a proton-coupled electron transfer mechanism.¹³⁰ It is worth mentioning that our group have proposed and confirmed a new mechanistic model named relayed proton-coupled electron transfer (relayed-PCET) for diradical generation in NHC organocatalysis and organic reaction systems,^131–133 and Duan et al. has further confirmed the existence of relayed-PCET mechanism in the chiral phosphoric acid (CPA)-catalyzed reaction.¹³⁴ As shown in Scheme 2, the relayed-PCET transition state is an effective transformation process from nonradical species to diradical species, in which the proton transfer is coupled with an electron transfer from electron donor (D) to acceptor (A). It could open a new door for understanding the diradical generation mechanism in both organic and enzymatic reactions. Therefore, exploring the new PCET mechanisms in the enzymes could be an interesting research area in theoretical and computational chemistry fields.

Scheme 2 (A) and (B) Conceptual difference between standard PCET and relayed-PCET. In relayed-PCET transition state, electron donor (D), electron acceptor (A), and base (B) are all initially nonradical species.

4 Conclusion

This review summarizes the general PCET mechanisms of multiple cofactor-free oxidase- and oxygenase-catalyzed activations of O₂ and the universal rules of enzyme-catalyzed metabolism of organic compounds in living organisms, thereby providing a theoretical basis for designing more efficient novel enzyme mutants. In addition, this kind of PCET mechanism also exists in flavin-dependent oxygenases and biomimetic organocatalytic reactions. It is worth mentioning that the detailed mechanism of a triplet intermediate to open-shell singlet intermediate remains a challenge during the cofactor-free enzyme-catalyzed activation of O₂. In the process of enzyme-catalyzed activation of O₂, the PCET mechanism progresses keep in open-shell doublet system, and the remarkably different mechanism involving relayed-PCET transition states in enzyme-catalyzed reactions should be another important issue to explore in future.

Author contributions

Qian-Qian Wang: conceptualization, writing – original draft, literature arrangement. Yan Qiao: discussion, literature research, writing – review & editing. Donghui Wei: conceptualization, supervision, funding acquisition, writing – review & editing.

Data availability

The data supporting this review can be found in the cited reference part.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We acknowledge financial support from the Key Project of the Joint Fund for Science and Technology Research and Development in Henan Province (no. 232301420008), the National Natural Science Fund of China (no. 22473100 and 21773214), the Basic Research and Cultivation Project for Young Teachers at Zhengzhou University (no. JC23261010), and the Training Plan for Young Key Teachers in Colleges and Universities in Henan Province (2020GGJS016).

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