Theoretical Insight into Nonadiabatic Proton- Coupled Electron

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Theoretical Insight into Nonadiabatic Proton-Coupled Electron Transfer Mechanism of Reduced Flavin Oxygenation Yanling Luo, and Ya-Jun Liu J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.9b02084 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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The Journal of Physical Chemistry

Theoretical

Insight

into

Nonadiabatic

Proton-

Coupled Electron Transfer Mechanism of Reduced Flavin Oxygenation Yanling Luo, Ya-Jun Liu* Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, China.

ACS Paragon Plus Environment

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ABSTRACT. The oxygenation of reduced flavin has been remaining a fascinating research hotspot in flavin-dependent proteins, because it plays an indispensable role in cellular metabolism and has potential applications in biocatalysis. This spin-forbidden reaction of high efficiency is far from being fully understood. Although investigation on the flavin chemistry has been going on for more than sixty years, there is few mechanistic explanations for the reaction of the singlet reduced flavin with triplet oxygen. In this paper, the reaction between oxygen and the model of free reduced flavin (reduced lumiflavin anion) was studied by density functional and multi-reference calculations in details. The results reveal that the reaction proceeds by an electronically nonadiabatic protoncoupled electron transfer (PCET) mechanism. The intersystem crossing point has been captured.


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INTRODUCTION Flavins are a group of organic compounds with 7,8-dimethyl-10-alkylisoalloxazine basic structure, of which flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are two common enzymatically active ones (Scheme 1).1, 2 Thousands of flavin-dependent proteins use flavins as a cofactor or substrate.3-5 These proteins are indispensable redox mediators participating in a wide range of biological processes, including cellular redox metabolism, biocatalysis, biosynthesis, bioluminescence and homeostasis related to the generation of reactive oxygen species (ROS).3-9 Notably, many functions would involve the mysterious oxygen activation.5 Oxygen is an excellent and abundant oxidizing agent in the atmosphere. However, the ground-state oxygen is in its triplet (T1) state, which prevents the spontaneous reaction with organic molecules generally in the ground singlet (S0) state. Flavin-dependent proteins are one particular kind of the few biocatalysts for oxygen activation, which could promote the spin-forbidden reactions between oxygen and organic molecules effectively without the aid of metal ions.5, 10-13 Flavins are extremely versatile cofactors, since they could participate in both one-electron and two-electron transfer processes. There are three different redox states of flavins, which are the oxidized flavin (FLox), the one-electron reduced flavin (flavin semiquinone, FLsq) and the twoelectron reduced flavin (FLred) (Figure S1 in the Supporting Information).2 Flavin-dependent proteins activate oxygen by alternating different redox states of flavins. According to the reactions with oxygen, flavin-dependent proteins can be divided into three categories, of which monooxygenases and oxidases are two efficiently reactive ones (Scheme 1).5,

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Monooxygenases with FLred react with O2 to form a quasi-stable C4a-(hydro)peroxyflavin intermediate, which would subsequently incorporate a single oxygen atom into an organic substance. On the other hand, most oxidases with FLred rapidly react with O2 to produce FLox and


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hydrogen peroxide (H2O2) directly, but with some exceptions. For example, C4ahydroperoxyflavin intermediate has been detected before H2O2 elimination in the pyranose 2oxidase.14-16 The different reaction pathways are believed due to the diverse active-site microenvironment of proteins, which result a variety functions of flavin-dependent proteins. Nevertheless, the C4a-(hydro)peroxyflavin intermediate is crucial for understanding the enigmatic reaction between FLred and O2.

Scheme 1. Reactions of FLred with O2. For most oxidases, FLred reacts with O2 generating FLox and H2O2 directly. For monooxygenases and certain oxidases, the reaction forms a C4a(hydro)peroxyflavin intermediate, which can further perform monooxygenation or H2O2 elimination. The chemical structures of FMN and FAD with 7,8-dimethyl-10-alkylisoalloxazine (in violet) are in orange and cyan frames, respectively.


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Flavins along with the physical and chemical basis underlying the biocatalysis has been a significant subject of intensive research for more than 60 years. Most of the current understanding of flavin chemistry is based on the works by Bruice and Massey.11, 17-19 But there have always been extraordinary results. For example, a newly discovered flavin-dependent protein could react with oxygen forming the unusual N5-peroxide intermediate, which goes beyond the conventional classification of flavin-oxygen reactions.20, 21 Still the exploration of flavins is ongoing for their complexity and importance, and not all aspects have been well explained. One major issue is about the enigmatic reaction mechanism of its oxygen activation.5 It is proved that FLsq could react with the superoxide anion (O2-) to form flavin 4a-peroxide.22 And a species resembling FLsq has been captured by fast kinetics studies in the glycolate oxidase.23 Beyond that, numerous studies have found that a conserved alkaline amino acid is usually located near the flavin ring in flavin-dependent proteins, and mutations of this residue will lead to dramatically decrease in oxygen activation reaction rate.24-27 Although there has been some experimental evidences, there is little direct explanation to the intrinsic reaction of FLred with oxygen, and the details are still poorly understood. In order to explore the efficient and fascinating spin-forbidden reaction, we studied the formation of the vital C4a-peroxyflavin intermediate in the gas and solutions phases by calculations of density functional theory (DFT) and multi-reference approaches. COMPUTATIONAL DETAILS In the DFT calculations, the CAM-B3LYP functional28 with 6-311G** basis set29,

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was

employed. The larger basis sets (6-311+G** and 6-311++G**) and the wB97XD functional, which contains the dispersion correction, have been tested and showed no improvement in the geometry optimization (Figures S2-4 and Table S1). In addition, the polarizable continuum model (PCM)31,


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and conductor-like polarized continuum model (C-PCM)33, 34 were used to describe nonpolar

and polar solvents effect respectively. The unrestricted open-shell CAMB3LYP (UCAM-B3LYP) with broken-symmetry technology were used to optimize the stationary points (reactants, products and the transition state), analyze the frequencies of the stationary points, and calculate the intrinsic reaction coordinate (IRC). The intersystem crossing (ISC) point was optimized at the complete active space self-consistent field (CASSCF)/6-31G* computational level.35-37 The ANO-RCCVDZP basis set38 was used for single-point multi-state complete active space second-order perturbation (MS-CASPT2) calculations. An active space of 12-in-9 was chosen (Figures S5 and S6). The ionization potential-electron affinity (IPEA) shift was set to zero,39 and the imaginary shift technique (0.1 a.u.) was employed to avoid intruder-state issue.40 The DFT calculations were performed using Gaussian 0941 program package, while the multi-reference calculations were performed with MOLCAS 8.042. RESULTS AND DISCUSSION Since the long ribose phosphate side-chains of FMN and FAD are not involved in catalysis,1 and there is no hydrogen-bond network anchoring its conformation in the gas phase or in solutions, we replaced it by methyl group. Apparently, the simplification to lumiflavin (LF) is reasonable and sufficiently sound to reflect the nature of the oxygenation reaction. Studies have proposed that the oxygen activation reaction is more beneficial from the reduced flavin anion than the electroneutral one.43 Also in general, the anionic reduced flavin is the active form bound to the flavin-dependent proteins.12, 16, 44 So the reduced lumiflavin anion (LF-red) was adopted in the study. Chaiyen et al.3 summarized that the ‘face-on’ attack may be a general feature of monooxygenases forming the C4a-(hydro)peroxyflavin intermediate, because a well-defined cavity always locates above the C4a atom. Meanwhile, –OOH moiety of the C4a-hydroperoxyflavin intermediate is also revealed as the


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‘face-on’ configuration in the special pyranose 2-oxidase.16 Therefore, we optimized the reactant (reduced flavin anion with oxygen) and product (C4a-peroxyflavin) from the initial geometry of perpendicular face-on conformation. Optimized Reactants and Products in S0 and T1 states. As the reaction involves the singlet reduced flavin and the triplet oxygen, we optimized the reactants and products in both the S0 and T1 states. The optimized geometries and relative energies of the reactants and products in the different states (S0 and T1) and different environments (gas phase, benzene, dimethylsulfoxide (DMSO) and water solvents) are shown in Figures S7 and S8. Two kinds of products were located in solutions, which are the directly adduct (LFHOO-) and the proton-transferred one (LFOOH-). In the gas phase, only the latter was located. Compared the relative energies of the two different products, the LFOOH- is more stable than the LFHOO- no matter in polar solvents (DMSO and water) and the nonpolar solvent (benzene). It seems reasonable, because the electronegativity of atom oxygen is stronger than atom nitrogen, which leads to the energy drop of product LFOOH- by transferring the H5 from the N5 to the Ob (Scheme 2). Similarly, other researches had also located the LFOOH- as the product for the reaction between O2 and free reduced flavin.45 So we think the proton-transferred adduct LFOOH- is the preferred product for the oxygenation reaction of free reduced lumiflavin anion. In addition, the reactants in the T1 state are more stable than the ones in the S0 state, while the products LFOOH- are on the contrary, which is consistent with flavin chemistry. So overall, the reaction of the reduced lumiflavin anion with oxygen is a spin inversion process from the triplet reactant 3(LF-red…O2) to the singlet product 1LFOOH- (Scheme 2).


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Scheme 2. The oxygenation reaction of free reduced lumiflavin anion.

S0 Reaction Pathway. We first calculated the reaction on the S0 potential energy surface (PES) by the DFT method. From 1(LF-red…O2) to 1LFOOH-, one transition state (TS) labelled TSS (the subscript s indicates singlet) was located. Vibrational analysis indicates that the imaginary vibrational mode of TSS (602i cm-1) corresponds to the simultaneous stretchings of C4a-Oa and ObH5. The IRC calculation was performed to ensure that TSS connects the correct reactant and product. The potential energy curve (PEC), key structures, changes of NPA charge and geometric parameters along the reaction path are depicted in Figure 1.


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Figure 1. The description of singlet reaction pathway between oxygen and reduced lumiflavin anion in the gas phase. The PEC (a), the key structures (b), the NPA charge of O2 moiety (c) and the geometric parameters (d) were computed at the UCAM-B3LYP/6-311G** level. The key atoms (N1, C10a, C4a, N5, H5, Oa and Ob) are highlighted in black, and the dihedral angle (N5−C4a−C10a−N1) in fuchsia. Here and hereunder, gray, blue, white, and red balls represent carbon, nitrogen, hydrogen, and oxygen atoms, respectively. The units are angstrom (Å) for bond distance, degrees for the dihedral angle, and kcal/mol for Gibbs energy change (ΔG).

As shown in Figure 1, the 1.233 Å Oa-Ob bond and the 176.5° N5-C4a-C10a-N1 dihedral angle in 1(LF-

red…O2)

imply a weak interaction between the reduced lumiflavin anion and oxygen. For


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convinence, we describe the raction process from 1(LF-red…O2) to 1LFOOH- in three stages (Figure 1(a)). Stage 1 from -8.475 to -5.050 amu1/2·bohr, the reaction starts with fine-tuning of the reactant structure. Stage 2 from -5.050 to 1.904 amu1/2·bohr, the C4a-Oa distance is noticebly tapering off along with the access of O2 to the tricyclic isoalloxazine ring. Meanwhile, the distance of Ob-H5 and the NPA charge of the O2 moiety decrease dramatically and synchronously. The H5 atom is attracted by the electronegative Ob forming the hydrogen bond when the distance between C4a and Oa reaches 2.385 Å. Subsequently, the Ob-H5 bond forms after the TSS, and the NPA charge of O2 moiety changes to -0.89|e|. This indicates an electron transfer from the reduced lumiflavin anion to the electroneutral oxygen in the original reactant. The electron transfer also lengthens the OaOb bond from 1.233 to 1.413 Å. In short, the hydroperoxyl radical (HOO·) does generate throuth both the proton transfer and the electron transfer in this stage. Then in stage 3 from 1.904 to 8.375 amu1/2·bohr, the HOO· radical attacks at the C4a reaction site leading to the formation of C4a-Oa bond and relaxing to product 1LFOOH-. Besides the bond formation, this process is accompanied by a small partial charge transfer from the O2 moiety back to the lumiflavin moiety. The change in N5-C4a-C10a-N1 dihedral angle is typical for the C4a hybridization altering from sp2 to sp3, and C4a-C10a is to a certain extent stretched from a double bond to a single one. The expectation values of S2 operator () of the three stationary points are also shown in Figure 1. The 0.96 value demonstrates that 1(LF-red…O2) has obvious biradical characteristic. This is donated by the two singly occupied π* orbitals of O2 via the natural orbital analysis. Along with the reaction path, the biradical character of the system disappears gradually (0.24 for the TS, and absolute 0 for the product). Collectively, the reaction from 1(LF-red…O2) to 1LFOOH- proceeds by the proton-coupled electron transfer (PCET)46-48 mechanism. In the reaction process, the electron transfer and the proton


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transfer from the reduced lumiflavin anion to the oxygen occur simultaneously. This facilitates the formation of the unstable one-electron reduced lumiflavin (LFsq) and HOO· radical pair. Finally, the radical pair recombines to form the closed-shell product 1LFOOH with a small amount of back charge transfer. The energy barrier is 10.13 kcal/mol, which is liable to be overcome at room temperature. In addition, the currently predicted energy barrier is consistent with the experimental kinetic data52. T1 Reaction Pathway and Solvent Effects. We also calculated the reaction from 3(LF-red…O2) to 3LFOO H

on the T1 PES and compared the S0 and T1 PECs in Figure S9. At beginning, the triplet

reactant has a lower energy than the singlet one. As the reaction goes on, the triplet energies rise sharply after -2.002 amu1/2·bohr. Around TSS, the energies of S0 and T1 are degenerate. For considering solvent effects, we investigated the reaction on both the S0 and T1 PESs in benzene, DMSO and water. As the results listed in Table S2 and Figures S10-15, the reaction also carries out in accordance with the PCET mechanism in solutions as in the gas phase. Along with the environmental polarity increases, the reaction barrier declines (Table S2). So the oxygenation reaction of the reduced lumiflavin anion would be influenced by the environments, which is the polar surrounding profits. This is consistent with the conclusion of the oxygenation reaction in firefly squid.49 Intersystem Crossing. In order to obtain more accurate energies, we performed single-point calculations on the S0 and T1 PECs by the method of MS-CASPT2. The results are depicted in Figures 2 and S16. The MS-CASPT2 calculated PECs are qualitatively consistent with the DFT predicted ones, but the former have lower energies. As the itinerary of green arrows shown in Figure 2, for thermodynamic preference, LF-red oxygenation reaction starts at the T1 PES, but switches to the S0 PES after an ISC around 0 amu1/2·bohr. The ISC point was located by CASSCF


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optimization. The information of electronic configurations and the main geometric parameters of the ISC point are displayed in Table S3 and Figure 2, respectively. The distance between C4a and Oa is 2.182 Å, and the bond length of Oa-Ob is 1.251 Å, which is longer than it in 3O2 (1.205 Å). Additionally, Ob and H5 forms a hydrogen bond with 1.972 Å length. A considerable spin-orbit coupling of 45.24 cm-1 at the MS-CASPT2 level assists the ISC process.

Figure 2. The S0- and T1- PECs for the oxygenation reaction of reduced lumiflavin anion in the gas phase at the MS-CASPT2//CAM-B3LYP computational level, and the structure of ISC point optimized via the CASSCF method.


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Combined the reaction process on the S0 PES discussed above, we qualitatively outlined the reaction route in Figure 3. In short, the spin-forbidden reaction between the singlet reduced lumiflavin anion and the triplet oxygen proceeds by the electronically nonadiabatic PCET mechanism. The gradually proton and electron transfers from the reduced lumiflavin anion to the oxygen firstly form the unstable singlet radical pair of LFsq and HOO·. And the radical pair annihilates to the product 1LFOOH- instantly. The spin reverison from the T1 state to the S0 state takes place during the PCET reaction process via an ISC with a considerable spin-orbit coupling. The electronically nonadiabatic PCET mechanism has been identified in phenoxyl/phenol selfexchange reaction.50, 51

Figure 3. Theoretically proposed reaction route between the triplet oxygen and singlet reduced lumiflavin anion.


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The currently proposed mechanism is partially different from the previously proposed mechanism of single electron transfer (SET).3,

11, 52

According to the conventional SET mechanism, the

reaction between the reduced flavin anion and oxygen proceeds in the order: (1) the SET from reduced flavin anion to O2 to form caged radical pair of FLsq and O2-; (2) the recombination between the FLsq and O2- producing the anionic C4a-peroxyflavin intermediate; (3) the protonation of anionic intermediate to give the product C4a-hydroperoxyflavin. But later, a positively charged residue around the flavin has been found to be recurrent but crucial for maintaining the reactivity of flavin-dependent protein.5 Specifically, the PCET is put forward in some specific flavindependent oxidases and monooxygenases,16, 53, 54 which provides a new view for oxygen activation in flavin chemistry. In the PCET mechanism, the electron transfer (step 1 in the SET mechanism) is coupled with the proton transfer (step 3 in the SET mechanism). So the oxygen activation reaction is divided into only two steps: (1) the PCET from the electron donor (reduced flavin anion) and proton donor (the residue in proteins) to the O2 forming caged radical pair of FLsq and HOO·; (2) the recombination between the FLsq and HOO· giving the oxygenated product C4ahydroperoxyflavin. Both the SET and PCET are two popular conjectures for the enigmatic reaction between the singlet reduced flavin anion and the triplet oxygen. They are both theoretically efficient for the inherent spin forbidden reaction, because the specific electron transfer in the step (1) could be a nonadiabatic pathway overcoming the spin-inversed barrier.48, 55 According to our current model study, we prefer that the PCET mechanism triggers the oxygenation reaction of reduced flavin anion. Firstly, The PCET has advantages in terms of reaction energy barrier by avoiding high-energy intermediates compared with the SET.46, 47, 51 Additionally, the positively charged residues conserving in flavin-dependent proteins are important for maintaing the reaction rate,24-27 which proves the PECT mechanism either, because the proton transfer is directly related


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to the rate-limiting step of the radical pair formation in the PCET. We have investigated the oxygenation reaction in bacterial luciferase, which is a speculiar flavin-dependent monooxygenase involving the bioluminescence phenomenon.56, 57 The systematic results indicate that the reaction between reduced flavin anion and oxygen proceeds via the PCET mechanism in the luciferase, but not the commonly supposed SET mechanism. And the proton is provided by the conserved residue HIP44+.57 Comparison of Oxygenation Reactions for Free Reduced Flavin and Flavin-Dependent Proteins. The oxygenation of free reduced flavin and reduced flavin in proteins could both comply with the PCET mechanism, but they are different. In most investigated flavin-dependent proteins, the oxygenation reaction follows the multiple site-electron proton transfer (MS-EPT) mechanism, which is a special PCET pathway that the electron-proton acceptor (oxygen for this case) simultaneously accepts the eletron and the proton from different donors (reduced flavin and some residue). The positively charged residue is the proton source.16, 53, 54, 57 So the mutations of the vital residue would result in a dramatical decrease in reactivity.24-27 As discussed above, for the oxygenation reaction of free reduced flavin, the proton is transferred from the reduced flavin itself to the oxygen since there is no other proton sources. Therefore, the reaction kinetics of oxygen activation in flavin-dependent proteins and in bulk solutions vary considerably.5, 53 Additionaly, it has been reported that hydrogen bond connecting to N5 of flavin position can stabilize the oxygenated intermediate in flavin-dependent proteins.3, 58 So it is rational to speculate that the oxygenated product from self-proton transfer of free reduced flavin is less stable than it in protein. Last but not least, the radical pair of FLsq and HOO· could be captured as an intermediate in the flavin-dependent proteins.16, 57 But according to this investigation of the free reduced flavin (in the


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gas phase or in solutions), the radical pair is unstable and would form the product 1LFOOHimmediately without the protein fixation. CONCLUSIONS The magical and efficient reaction between the free reduced lumiflavin at singlet state and oxygen at triplet state was theoretically investigated in this study. The reaction from the triplet reactant 3(LF-

red…O2)

proceeds to the singlet product 1LFOOH- via an unstable FLsq and HOO· complex

formed by the PCET. The triplet-singlet crossing is facilitated during the reaction, which is assisted by a considerable spin-orbit coupling. In a word, the electronically nonadiabatic PCET initiates the oxygenation reaction of free reduced lumiflavin. The study provides an in-depth prospective on the molecular basis and electronic-state level for the oxygenation of free reduced flavin. The oxygenation mechanisms of free reduced flavin and reduced flavin in proteins have similarities and differences, which has been compared and discussed. ASSOCIATED CONTENT Supporting Information. Additional structures, IRC paths, active space orbitals, absolute energies and Cartesian coordinates. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Notes The authors declare no competing financial interests.


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ACKNOWLEDGMENTS This work has been supported by grants from the National Natural Science Foundation of China (21673020, 21911530094 and 21421003). REFERENCES (1)

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Theoretical Insight into Nonadiabatic Proton- Coupled Electron

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