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Dual Photochemical Reaction Pathway in Flavin-Based Photoreceptor LOV Domain: A Combined Quantum-Mechanics/Molecular-Mechanics Investigation Setsuko Nakagawa, Oliver Weingart, and Christel M. Marian J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b09207 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 21, 2017

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The Journal of Physical Chemistry

Dual Photochemical Reaction Pathway in Flavin-based Photoreceptor LOV Domain: A Combined Quantum-Mechanics/Molecular-Mechanics Investigation

Setsuko Nakagawa,*,† Oliver Weingart, ‡ Christel M. Marian‡

Department of Human Life and Environment, Kinjo Gakuin University, Omori, Moriyama-ku, Nagoya 463-8521, Japan, and Institute of Theoretical and Computational Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany

* † ‡

To whom correspondence should be addressed. E-mail: [email protected]. Kinjo Gakuin University. Heinrich-Heine Universität Düsseldorf.

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ABSTRACT The primary photochemical reaction of the light, oxygen, and voltage (LOV) domain of the blue-light photo-sensor YtvA of Bacillus subtilis were investigated using high-level QM(DFT/MRCI)/MM methods. After blue-light excitation, the Sγ atom of the reactive cysteine forms a covalent bond with the C4a of the flavin mononucleotide (FMN) ring. Two conformations for the side chain of reactive cysteine with occupancies of 70% (conf A) and 30% (conf B) are observed in the X-ray crystallographic structures of the YtvA-LOV (Möglich, A. and Moffat, K. J. Mol. Biol. 2007, 373, 112-126). In conf A, the thiol group is directed toward the dimethylbenzene moiety of the FMN ring whereas it is placed directly above the N5 atom of the FMN ring in conf B. Starting from both conformations, the singlet and triplet excited pathways were evaluated. The singlet states excited from conf A decay nonradiatively to the triplet states by intersystem crossing (ISC). After the formation of a neutral bi-radical, the triplet states cross over to the electronic ground state by a second ISC and the adducts are efficiently formed. The singlet states excited from conf B are located near the S1/S0 conical intersection (CIn). A major fraction returns to the initial states through the CIn. The rest may directly reach the adduct state. Thus, the photo-excitation has a dual reaction pathway. In YtvA-LOV, it is inferred that the efficient triplet excitation from conf A was chosen by bypassing the less efficient singlet excitation from conf B.

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INTRODUCTION Flavin-based photoreceptor proteins of LOV (light, oxygen and voltage) families have attracted scientific attention due to the key role in blue light-sensing signal transduction in plants, algae, fungi and bacteria.1-5 LOV proteins were identified initially in plants as phototropin. LOV domains are found in regulatory proteins for phototropism, chloroplast relocation, bacterial stress responses, and many other diverse biological responses. Each LOV domain contains one oxidized flavin mononucleotide (FMN) as a light-absorbing chromophore having a characteristic band within the visible spectral range.1-7 In the dark form of the LOV domains, FMN is non-covalently incorporated into the protein as a cofactor. The form is denoted as LOV-447 according to the characteristic absorption band at 447 nm. Upon illumination with blue light, LOV domains undergo a photocycle. The dark form is excited to the singlet states. The lowest-lying singlet excited state decays either by intersystem crossing (ISC) to the triplet excited state (LOV-660 or LOV-715) or by fluorescence back to the ground state. This photoreaction progresses within 0.1-10 ns. The FMN triplet decays with a time constant in the range of 100 ns to 5 ms. The only photoreceptors whose long-lived triplet state has been identified is the LOV domains. A covalent bond is formed between the sulfur atom of a conserved cysteine residue of LOV and the C4a atom of the FMN ring. This conformational change generates structural changes that propagate to the domain surface. The adduct shows a single broad absorption band at 390 nm (LOV-390). At room temperature in the dark, the photoproduct slowly reverts to LOV-447. The metastable cysteine adduct decays thermally on the timescale from seconds to hours. The LOV domain core has a α/β PAS (PER-ARNT-SIM) fold which consists of a five-stranded antiparallel β-sheet and helical connector elements (Fig.1). X-ray crystallography has shown that there are three conformational features concerning the dark state and the bright state.3,4,8 The first is the presence of rotamers of the reactive cysteine side chain in the dark state. One conformation is the thiol group orienting toward the dimethylbenzene moiety of the FMN ring (conf A). In another conformation, the thiol group is situated directly above the N5 atom of the FMN ring (conf B). A conformation close to conf B is necessary for adduct formation. The second feature regards the structure of the FMN ring. The FMN is planar in the dark state, whereas the planarity is broken in the bright state by the formation of a covalent bond between the Sγ of the reactive Cys and the C4a atom of the FMN ring. The third corresponds to the side chain conformation of conserved Gln near the O4 atom of the FMN ring. In the 3



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initial dark state, Nε2 of the Gln side chain is hydrogen bonded to O4 and N5 of the FMN ring. In the adduct state, Oε1 of the Gln side chain is presumed to point to the N5 atom of the FMN ring by the side chain flip.8 The light-induced conformational changes which are concentrated in the β-scaffold of the LOV core are thought to be significant for the early signal propagation. The blue-light signal induces the unfolding of flanking helices and the rotation and dimerization of LOV modules.2,4 The photocycle of the LOV domains (Fig. 2) has been established relatively well experimentally, but some key issues remain unresolved. The first one concerns the reaction pathway of singlet excited FMN. In the LOV2 domains of phototropin from Chlamydomonas reinhardtii (Crphot-LOV2), the quantum yield of adduct formation was found to be higher than that of triplet formation. 9, 10 Therefore, a photoreaction proceeding via a singlet excited state electron transfer in parallel with the triplet formation has been proposed. The singlet excited-state electron transfer (1FMN* + Cys-SH → FMN・- + Cys-SH+) might be followed by proton transfer (→ FMNH・+ Cys-S・) (or combined electron and proton transfer). The triplet formation which is observed in most of the LOV domains requires singlet-triplet ISC. However, the adduct is formed directly without ISC. This direct singlet excited pathway is thought to be a special case of Crphot-LOV2.1 The second key issue regards the process of the adduct formation between the triplet excited FMN ring and the adjacent Cys residue. This photochemical reaction involves the bond breaking of Sγ-H, the bond formation of N5-H and Sγ-C4a, and the change from double-bond to single-bond character between N5 and C4a. Triplet-singlet ISC is again required for this process. A protonated triplet (3FMNH+),11 radical anion (2FMN・-)12 and a neutral semiquinone radical (2FMNH・) 13-16 have been proposed as the intermediates in different experimental methods.17 Although an ionic mechanism on the bond formation was excluded, the details of the mechanism still remain unclear, because the intermediates can hardly be detected.12 Third, there is a wide range of timescales of recovery to the dark state.4 Recent studies support the involvement of an organized water cluster that acts as the proton acceptor of N5-H.18 A number of studies on the signaling mechanism and its involvement of the Jα helix, which remains as an unresolved problem, are now in progress.4,19,20 Several theoretical studies on the photocycle of LOV domains have been reported in the literature. The quantum chemical calculations using the FMN and cysteine model compound appeared in 2003.21, 22 A neutral radical mechanism and a concerted mechanism were proposed. So far, seven electronic structures have been determined theoretically in the photocycle. In addition to the structures of the five states shown in Fig. 2 (S0 dark, S1, T1, T1 birad, and S0 add), the structures of two transition states (S0 ts 4


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and T1 ts) were determined. Domratcheva et al. studied the energy profile of six states (S0 dark, T1, T1 ts, T1 birad, S0 add, and S0 ts) by high-level MCQDPT2/CASSCF methods using the methanethiol-lumiflavin complex model.23 Zenichowski et al. investigated five states (S0, T1, T1 ts, T1 birad and S0 add) on the DFT/B3LYP and MCQDPT2 level.24 They observed strong spin-orbit coupling in the neutral biradicals generated after the transition state (T1 ts), and ISC from the lowest triplet state (T1 birad) to the singlet state (S0 add). Dittrich et al. performed combined quantum mechanical/molecular mechanical (QM/MM) calculations with the single-configurational methods of HF and DFT on five states (S0 dark, T1, T1ts, T1 birad, S0 add).25 These quantum mechanical studies all supported the neutral radical mechanism in the photochemical reaction of the adduct formation. Salzmann et al. studied the ground state and the low-lying excited states (S0 and S1) using the QM/MM method with high-level DFT/MRCI calculations.26 They expected the transition from the lowest singlet state (S1) to the second lowest triplet state (T2) to play an important role for the first ISC in the photoreaction. Recently, the photoreactions of dark- and light-adapted states were investigated at the QM(MS-CASPT2)/MM level of theory.27 Today, a realistic photochemical reaction model which includes a chromophore, protein and surrounding water can be calculated by the QM/MM approach. From the results of the detailed energy profile calculations, we can obtain a wealth of information such as the assignment of the spectrum and vibrational modes, and the rate constant of ISC. Molecular dynamics (MD) simulations of the LOV domains give also clues for the signaling mechanism.28 In this work, the energy pathways of the photocycle in the LOV domain of the blue-light photo-sensor YtvA of Bacillus subtilis were examined in detail using the QM/MM method. Our previous work, which treated the events that take place immediately after the blue-light absorption, has shown that the QM/MM approach using DFT/MRCI is reliable for the estimation of excitation energies.26 Therefore, we have performed the energy pathway search starting from conf A and conf B by using similar systems. We discuss a dual pathway in the primary photoreaction of the LOV domain from the theoretical point of view.

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METHODS AND COMPUTATIONAL DETAILS System Setup. The initial geometry was constructed based on an X-ray crystallographic structure of YtvA-LOV (PDB 2PR5).8 The core LOV domain (residues 25-126) of monomer A was used for our investigation. An N-terminal segment and a linker region are not included. The two orientations for the side chain of Cys62 were observed in the X-ray crystallographic structure (conf A and conf B). The occupancies of the conf A and conf B are 70% and 30%, respectively. The sulfur atom of Cys62, denoted here as Sγ, is closer to the C4a atom of the FMN ring in conf B as compared to conf A. Both conformations were adopted here as initial structures of the photoreaction. The starting structures of the photocycle were taken from the previous work.26 The present model system comprises the LOV domain, consisting of 102 amino acids, the FMN cofactor, 240 crystallographic water molecules, and 10 sodium ions, solvated in a 35 Å water sphere. The force-field parameters for the FMN cofactor were also taken from the previous work.26 All non-hydrogen atoms of protein and FMN were frozen during system setup in order to preserve the X-ray structure. During these initial molecular mechanics (MM) relaxation steps, only water molecules and hydrogen atoms were able to move. Since the X-ray structure shows reasonable average coordinates for non-hydrogen atoms in proteins, MD simulation was not performed. The optically active part of the LOV domains is the isoalloxazine core of the FMN. A lumiflavin and the Cys62 model molecules are included in the QM region, because a metastable covalent bond is formed between the Sγ atom of Cys62 and the C4a atom of the FMN ring in the bright state. The -SγH group forms a hydrogen bond with one of the crystal water molecules in conf A. In contrast, it forms a hydrogen bond with the carbonyl group of Cys62 in conf B. Therefore, the crystal water molecule and the peptide group including the carbonyl group are added to the QM region. Since the polar side chains of Gln123 are expected to affect the photophysics of the cofactor, the functional groups of the residue are also incorporated into the QM region (see S0 structures of conf A and conf B in Fig. 3). In this case, there are four covalent bonds between the QM and the MM parts, which were saturated by hydrogen link atoms. QM/MM Calculations. The QM/MM calculations were carried out using the ChemShell package.29 We applied the CHARMM/TIP3P force field30,31 to the MM part using the DL_POLY code32 in ChemShell. By including all MM point charges in the one-electron part of the QM Hamiltonian, the electrostatic interaction between QM and MM atoms was incorporated. A link atom approach with the charge-shift scheme29, 33 was used for boundary region. All QM/MM geometry optimizations of the ground state and the excited state were performed with time dependent density functional theory 6


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(TDDFT/B3LYP).34-36 Some optimizations of the lowest triplet state (T1) were carried out at the level of unrestricted density functional theory (UDFT/ B3LYP). The HDLC optimizer in ChemShell was used for the minimum and transition state searches.37 The starting structures of the optimization were constructed by side chain χ1 and χ2 rotation of Cys62 along the expected reaction path. The optimization has been performed with the default HDLC optimization parameters, but the maximum gradient component convergence is set to 0.00135~0.00405 a.u. which is less strict than the default value (0.00045 a.u.). The optimized structures can be considered as local minima of the limited space or as passing points of the reaction. We optimized the QM region and all MM residues with a distance of less than 9 Å from cofactor.26 It is well known that DFT with standard functionals does not correctly describe the energy position and the long range behavior of charge transfer (CT) states in the excited states.38 Therefore, additional single-point calculations embedded in the MM charges using the combined density functional theory/multireference configuration interaction (DFT/MRCI) method39 were performed. From subsequent single-point calculations, vertical electronic excitation energies and oscillator strengths were obtained. The numbers of roots determined in the MRCI calculations on the singlet and triplet manifold were chosen to be 6 and 8, respectively.26 The TZVP basis set was used for all atoms. In the total energy estimation of QM(DFT/MRCI)/MM, the MM energy is added to the DFT/MRCI energy. The TURBOMOLE 6.3 program package40 was used for the QM/MM calculations. The spin-orbit coupling kit (SPOCK) 41,42,43 was used for the spin-orbit matrix elements (SOMEs) between the correlated DFT/MRCI wave functions.

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RESULTS AND DISCUSSION Starting from the conf A and conf B of Cys62, minimum nuclear arrangements of the S0, S1, S2, T1 and T2 states were determined. In the S0 minimized structure of conf A, the thiol group of Cys62 forms a hydrogen bond with the oxygen of a crystal water (Fig. 3) whereas in conf B an intramolecular hydrogen bond with the carbonyl oxygen of Cys62 is formed (Fig. 3). In the course of the photoreaction, the hydrogen atom of the thiol group, denoted here as Hγ, should be placed directly above the N5-C4a bond of the FMN ring (see Fig. 2 for atom labels) by rotating the χ1 and χ2 dihedral angles of Cys62 side chain. After the Hγ atom has been transferred toward the N5, Sγ forms a covalent bond with the C4a atom. The reaction coordinate (RC) in the i-th structure is defined as the sum of distance differences of Sγ-Hγ, N5-Hγ, C4a-N5, and Sγ-C4a from the starting S0 structure of conf A (‫ݎ‬௜ோ஼ = ∆‫ݎ‬௜ ሺܵ‫ ܪ‬ሻ − ∆‫ݎ‬௜ ሺܰ‫ܪ‬ሻ + ∆‫ݎ‬௜ ሺ‫ܰܥ‬ሻ − ∆‫ݎ‬௜ ሺܵ‫ ܥ‬ሻ). With the progress of the photoreaction, the Sγ-Hγ and C4a-N5 bonds are elongated and the N5-Hγ and Sγ-C4 bonds are shortened. The QM(DFT/MRCI)/MM energy profiles starting from conf A and conf B are shown in Fig. 4a and 4b, respectively. The QM(TDDFT)/MM energy profiles are shown in Fig. S1 of the supporting information (SI). In conf A, the RC changes from 0.0 Å at the S0 starting structure to 8.06 Å at the adduct structure. For conf B, the RC of the starting structure is 1.37 Å. This means that the starting structure of conf B is closer to the reaction site than conf A. The energy difference between the starting structures of conf A and conf B is less than 1 kcal/mol. The selected optimized structures according to the reaction pathway and the χ1 and χ2 dihedral angles of Cys62 side chain are shown in Fig. 3. A full list of the optimized structures and the frontier orbitals are shown in Fig. S2 of the SI. A full list of the QM/MM energies, the maximum gradients and the distances among the Sγ, Hγ, N5 and C4a atoms are shown in Table S1 of the SI. Reaction Profile of the Dark State In the starting structure of conf A, the HOMO is a lone-pair orbital at the sulfur center of Cys62 (pSH) and the HOMO-1 is a π orbital (πH-1) that is delocalized over the whole ring system (Fig. 5). In the HOMO of conf B, the π orbital of the FMN ring mixes with the pS (π/pS) (Fig. 6). The HOMO-1 is almost the pS. The energy differences between the HOMO and HOMO-1 orbital are only 0.1 eV in both conformers (Fig. S2a). The HOMO-2 of conf A and conf B are the π orbitals that are delocalized over the dimethylbenzene moiety of the FMN ring (πH-2). The LUMOs are the π orbitals (π*L) that are delocalized over the whole FMN ring in conf A and conf B. A metastable structure in which the Hγ atom of Cys62 is located above the N5 atom of the FMN ring was found at RC 1.97 Å (Fig. S2a). The distance of Hγ-N5 was 2.81 Å. 8


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The QM(DFT/MRCI)/MM energy was higher than the starting structure of conf A by 7 kcal/mol (Fig. 4a). Such metastable structures are thought to exist thermally. The HOMO and HOMO-1 are admixtures between pS and π orbitals (pS/π and π/pS) (Fig. S2a). In the preliminary study using a smaller QM region (including lumiflavin and methanethiol), the transition state (imaginary frequency 391i cm-1) was found at the RC 5.66 Å (N5-Hγ 1.05 Å, Sγ-Hγ 2.19 Å, Sγ-N5 2.87 Å, χ1=-153°, χ2=-139°). The barrier height estimated by QM(DFT/MRCI)/MM was 35.9 kcal/mol. However, in the current larger system, we could not find a transition state with only one imaginary frequency. Rather, a second-order saddle point at RC 5.58 Å (N5-Hγ 1.03 Å, Sγ-Hγ 2.24 Å, Sγ-N5 2.84 Å, χ1=-151°, χ2=-132°) close to the transition state was obtained (Fig. S2a). The true barrier height of the dark-state reaction is considered to be somewhat higher than the value of 27 kcal/mol shown in Fig. 4. Similar barriers (28.1-36.3 kcal/mol) had been obtained by QM(MS-CASPT2)/MM,27 supporting our assignment. The barrier is too high for the reaction to proceed thermally in the dark. The adduct (add) in which the thioester bond was formed between the Sγ and the C4a of the FMN ring was found at RC 8.06 Å (Fig. 3). In this structure, the distance between the O4 of the FMN ring and the Nε2 of Gln123 (O4-Nε2) is shortened from 3.12 Å of the conf A starting structure to 3.05 Å of add. The distance between the N5 of the FMN ring and the Nε2 of Gln123 (N5-Nε2) extended from 3.61 Å of the conf A starting structure to 3.85 Å of add. Since the side chain flip of Gln123 was presumed by the X-ray crystallographic study,8 the minimization was performed using the side chain 180 degrees flipped model. The RC of the minimized adduct flip (add flip) structure was also 8.06 Å (Fig. S2a). The distances between O4 and Oε1 of Gln123 (O4- Oε1) and between N5 and Oε1 of Gln123 (N5-Oε1) are 3.37 Å and 3.30 Å, respectively. The N5-Oε1 corresponds with 3.2 Å of the X-ray crystallographic study.8 The frontier orbitals of the adduct (and the add flip) are shown in Fig. 6 (and Fig. S2a). HOMO and HOMO-1 are the π orbitals and HOMO-2 is the pS orbital. LUMO is an admixture of π and pS orbitals. The QM(DFT/MRCI)/MM energy of the adduct was 6.7 kcal/mol (0.29 eV) more stable than the energy of the adduct flip (Table 1). It is unnecessary to flip the side chain by 180 degrees. According to the MD simulation, the mobility of the Gln side chain induced by the adduct formation is thought to be significant for primary signaling mechanisms to regulate a C-terminal effector domain.28 The weakening of the interaction between the N5 of the FMN ring and the Nε2 of Gln123 by the adduct formation brings the flexibility of the Gln123 side chain. Recent MD simulations 9



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proposed a mechanism in which the side chain of the conserved Gln flips between two strands, maintaining two hydrogen bonds (N5 Hγ---Nε2 and O4---H Nε2).44 Although mutations of the conserved amino acids around the FMN core were done, the detailed signaling mechanism of YtvA is still little understood.4,45 Compared to the starting structures of conf A and conf B, the cysteine adduct was found to be energetically unstable by 14.8 and 15.7 kcal/mol (0.64 and 0.68 eV), respectively (Fig. 4). These QM(DFT/MRCI)/MM energies somewhat underestimate the experimental photocalorimetric result of 1.17 eV (Table 1).7 It can be said that our QM (DFT / MRCI) / MM model well reproduces the ground state energies of YtvA-LOV compared to the previous theoretical studies.21-25 The barrier height of the recovery reaction from the adduct is 12 kcal/mol in Fig. 4. The C4a-Sγ bond breaking and the Hγ transfer from N5 to Sγ will occur thermally. In the actual YtvA-LOV system, the adduct decays thermally with lifetime 43 min.46 Reaction Profile of the Bright State The dark-adapted form is transferred to the singlet excited states after the blue-light excitation. ISC to the triplet excited state occurs within 2 ns.46 The lowest-lying singlet state decays simultaneously by fluorescence back to the ground state (2.2 ns).7 The first ISC has not been investigated theoretically so far except for our previous study.26 The lowest-lying triplet state of YtvA decays with a lifetime of 2 µs. 7,45,46 Accompanied by a second ISC to the singlet ground state, the adduct is formed between the chromophore and a nearby Cys62. Here, the photoexcitation reaction pathways of conf A and B are studied separately in detail. Photoexcitation from the S0 state of conf A. First, we have investigated the reaction pathway of conf A. The DFT/MRCI vertical excitation energies of the starting S0 state are compiled in Table 2. The excitation of the lowest-lying singlet state (S1) corresponds to the optically bright π→π*transition. The vertical excitation energy was 2.80 eV. The oscillator strength (f) was relatively large (0.23). The energy is in excellent agreement with the experimental absorption maximum of 2.77 eV. 46 The electronic structure of the second excited singlet states (S2) corresponds to low-energy charge transfer (CT) excitations (pS→π*). The S2 energy was 3.03 eV with an oscillator strength of 0.12. An experimental absorption spectrum around 422 nm (2.94 eV),46 not appearing for FMN in solution, can be assigned to this excitation. A π→π* excitation was also found to dominate the fourth excited singlet state (S4) with a vertical excitation energy of 3.51 eV. The S4 energy was a little higher than the experimental absorption maximum of 3.40 eV.46 In our previous study in which different models with several amino acid side chains near the FMN ring are included, the CT state was situated 1 eV 10


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above the lowest πH→π*L state.26 Inclusion of the hydrogen-bonded crystal water in the QM region in the current study leads to a stabilization of the CT state (S2), placing it only 0.23 eV above the lowest πH-1→π*L state (S1). The vertical excitation energy levels are also shown in Fig 7a. The π→π* excitation energies of T1 and T2 were lower than the excitation energies of S1 and S2. The CT state may contribute to the photoreaction. The excitation energies at RC 1.97 Å are also shown in Table 2. The lowest vertical excitation energy was 2.72 eV. The value is somewhat red-shifted compared to the starting structure of conf A. The second excitation energies (3.19 eV) is blue-shifted compared to the starting structure. These are excitations from the admixture (pS/π) to π*. The S3 energy was 3.38 eV and was close to the experimental absorption maximum of 3.40 eV. Singlet energy surface of conf A. In the S1 minimized structure of conf A, the RC has a negative value (-0.29 Å). The bond length of Sγ-Hγ is expanded by 0.04 Å toward the oxygen of crystal water. The QM(DFT/MRCI)/MM energy at RC -0.29 Å is 63.2 kcal/mol higher than that of the S0 starting structure (Fig. 4a). The reaction proceeds by the side chain χ1 and χ2 rotation of Cys62. Two metastable S1 excited structures in which the Hγ are located above the dimethylbenzene moiety and the pyrazine moiety of the FMN ring were found at RC 1.28 Å and 3.23 Å, respectively (Fig. 3, Fig. S2b). The QM(DFT/RCI)/MM energies of RC 1.28 Å and 3.23 Å are higher than the RC -0.29Å by 1.2 and 3.3 kcal/mol, respectively (Fig. 4a). The potential energy surface of the S1 state is relatively flat until RC 3.23 Å. The frontier orbitals of RC -0.29 Å are similar to those of the S0 state (Fig. 5). A lone-pair orbital at the sulfur center of Cys62 (HOMO) takes vertical arrangement to the FMN ring. In RC 1.28Å, a lone-pair orbital at the sulfur center of Cys62 takes parallel arrangement to the FMN ring by rotating the thiol group (Fig. 5). The LUMO, HOMO-1 and HOMO-2 of RC 1.28 Å structure are similar to those of the S0 state. In the HOMO and HOMO-1 of RC 3.23 Å, the pS and π orbitals are mixed (pS/π) (Fig. S2b). The LUMO and HOMO-2 are similar to those of the S0 state. The DFT/MRCI excitation energies of the S1 states are shown in Table 3. In RC -0.29 Å, the electronic structure of the S1 state is the CT excitation of pS→π*. The vertical emission energy is 2.21 eV. The electronic structure of S1 does not correspond to an optically bright π→π* transition at this geometry. By the minimization of the S1 state, the pS→π* excitation is 0.57 eV lower than the excitation at the Frank-Condon (FC) region, while the π→π* excitation is almost unchanged (Fig. 7a). Thus, the excitation energy is transferred from the π→π* to the pS→π* state upon relaxation of the S1 state. The S1 electronic structure of RC 1.28 Å is also the CT excitation of pS →π* and the 11



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emission energy is 1.82 eV (Table 3). In RC 3.23 Å, the electronic structure is pS/π→π* and the emission energy is 2.13 eV. The experimental fluorescence spectrum has a peak around 2.50 eV (496 nm) and a shoulder around 2.37 eV (523 nm).7 Our S1 results of three structures underestimate the fluorescence maximum. The S2 electronic states are optically bright π→π* transition in RC -0.29 Å and 1.28 Å. These emission energies of 2.56 and 2.53 eV are closer to the experimental fluorescence maximum. The S1 and T1 states are almost degenerated at RC 1.28 Å. The T1 and T2 states are situated below the S1 states in RC -0.29 and 3.23 Å. The spin–orbit matrix elements (SOMEs) are also shown in Table 3. Since the electronic structures of S1 states are the pS→π* excitations, the SOMEs between the S1 state and the T1 and T2 states have significant values (1.19~7.79 cm-1) in the minimized structures of the S1 states. In particular, the ISC rate is enhanced near RC 1.28 Å due to the relative large SOME (4.40 cm-1) and a very small energy gap (0.05 eV) (Fig. 7a). In accordance with El-Sayed’s rule, SOMEs between n→π* and π→π* states are much larger than SOMEs between two π→π* states. Since T2 states are found to lie about 0.09 and 0.06 eV below the S1 minimum of RC -0.29 Å and 3.23 Å, the ISC between S1 and T2 states may occur. In the previous study with different models, the SOME between the S1 and T2 states was calculated to a small value (1.62 cm-1), since both the excitations are dominated by π→π* transitions.26 An enhancement of the ISC rate constant due to the vibronic spin-orbit coupling is proposed for the (ππ*)⇒(ππ*) process.26 In addition to such an effect, the ISC rate is enhanced by an external heavy atom effect. Since the pS→π* CT excitations that we obtained for the S1 states are rapidly transferred to triplet states by the ISC, these reactive species could not emit the fluorescence. The time-resolved fluorescence spectroscopy of the Avena sativa phototropin 1 LOV2 domain showed that the fluorescence quantum yield increases by cooling.47 The fluorescence might originate from the π→π* excitation of the FMN ring of nonreactive species. We have optimized the nuclear arrangements of the second excited singlet state (S2), too. The minimized structures are obtained at RC 0.08 Å and 1.57 Å. The QM(DFT/MRCI)/MM energies are plotted in Fig. 4a. The DFT/MRCI excitation energies and SOMEs are shown in Table S2. In RC 0.08 Å, the Sγ-Hγ group makes a hydrogen bond with the crystal water and the electronic state of S2 is dominated by the π/pS→π* transition. In RC 1.57Å, the Hγ atom is located above the dimethylbenzene moiety of the FMN ring and the electronic state of S2 is dominated by the pS→π* and π→π* transitions. Those vertical emission energies are 2.77 and 2.81 eV, respectively. 12


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These energies are somewhat larger than the experimental fluorescence spectrum (2.50 eV).7 The T1 and T2 are situated below the S2 state at RC 0.08 Å. The T1, T2 and T3 are situated below the S2 state at RC 1.57 Å. The SOMEs between the S2 state and the T1, T2 and T3 states have significant values in the minimized S2 structures. Since the T3 state is found to lie about 0.03 eV below the S2 state and the SOMEs between the S2 and T3 is 3.85 cm-1 in the RC 1.57 Å, the ISC between S2 and T3 state may occur. It is known that the ISC rate is enhanced in LOV as compared to FMN in solution or cysteine-less mutants.9,11 Fluorescence line narrowing (FLN) experiments showed that a weak electronic interaction between the dimethylbenzene moiety of the FMN ring and the thiol group of the conserved cysteine facilitates the rapid electronic formation of the reactive triplet state.48 Our results, namely that the (pSπ*)⇒(ππ*) process occurring in the excited singlet states of conf A is important for ISC, are consistent with the FLN results. Triplet energy surface of conf A. The minimum nuclear arrangements of the T1 states according to the reaction pathways were determined. Three local minima were found at the RC -0.06 Å, 1.28 Å and 3.58 Å. In the RC -0.06 Å, Sγ- Hγ makes a hydrogen bond with the crystal water. The Hγ atoms of RC 1.28 Å and 3.58 Å are located on the dimethylbenzene moiety and the pyrazine moiety of the FMN ring, respectively (Fig. 3). In the RC -0.06 Å, HOMO and HOMO-1 are the π and pS orbitals, respectively (Fig. S2d). On the other hand, HOMO and HOMO-1 are the pS and π orbitals in the RC 1.28 Å and 3.58 Å, respectively (Fig. 5). The QM(DFT/MRCI)/MM energies of RC -0.06 Å is 15.5 and 13.4 kcal/mol lower than the RC 1.28 Å and 3.58 Å, respectively (Fig. 4a). Unlike the S1 potential energy surface, there is a potential barrier to the progress of the reaction. Most of the triplet states, which have been populated around the RC = 1.28 Å by ISC from the singlet states, are considered to return to the RC -0.06 Å. The triplet of YtvA decays with a time constant in 2 µs.46 The energy barrier of the triplet energy surface might be one of the reasons for such a long-lived triplet state (µ sec). The adiabatic triplet QM(DFT/MRCI)/MM excitation energy of RC -0.06 Å was 2.04 eV (47.1 kcal/mol). This value is in excellent agreement with the experimental 0-0 transition of 2.05 eV (Table 1).7 The vertical excitation energies of three T1 states are shown in Table 4. The electronic structure at RC -0.06 Å was the π→π* transition, whereas those at RC1.28 Å and 3.58 Å were the CT excitation transition (pS→π*). In the latter structures, no triplet excitation with significant oscillator strength was found. In the former structure, T5 and T7 were π→π* excitations with significant oscillator strengths. The excitation energies (1.81 and 1.89 eV) are in agreement with the experimental triplet-triplet absorption 13



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maximum of 1.91 eV (650nm).6 In the former structure where a thiol group forms a hydrogen bond with a water molecule, electrons in the spin parallel state are trapped in the FMN ring, which is experimentally observed. Attempts to obtain a T2 minimum structure similar to the starting structure of conf A were not successful, as the energy inversion continued in the TDDFT optimization. A local minimum was found at RC 1.38 Å, in which the Hγ atom is located on the dimethylbenzene ring. The QM(DFT/MRCI)/MM energy is plotted in Fig. 4a. The electronic structure was the πH-2→π*L transition. We attempted to obtain the T1 transition state for the migration of the Hγ atom to the N5 atom of the FMN ring. However, only a structure with two negative eigenvalues was found at RC 4.56 Å, because the potential energy surface was flat in the TDDFT transition search (Fig. S1a). The distances of Sγ-Hγ and Hγ-N5 were 1.50 and 1.49 Å in this transition state like structure. When this structure was passed, a stable structure of RC 6.59 Å, with Hγ completely shifted to the N5 side (Hγ-N5 1.01 Å), was obtained using UHF optimization. The T1 energy surface of QM(DFT/MRCI)/MM calculations (Fig. 4a) was very different from the QM(TDDFT)/MM energy surface (Fig. S1a). In the energy surface of QM(DFT/MRCI)/MM, there is a barrier near RC 1.28 Å as mentioned above, and the energy of RC 4.56 Å is more stable than the energy of RC 3.58 Å (Fig. 4a). Thus, there is no potential barrier for the Hγ transfer. The structure in which Hγ is bonded to N5 (RC 6.59 Å) is easily formed. A low barrier of 14.8 kcal/mol was found near the transition state in the QM(MS-CASPT2)/MM calculations.27 In the RC 6.59 Å, the sums of Mulliken charges for the Cys and lumiflavin-Hγ moieties were -0.28 and 0.28, respectively (Table 5). Those of the electrostatic potential charges were -0.33 and 0.40, respectively. Although both moieties are not perfectly neutral, the electronic structure of RC 6.59 Å is more like a neutral biradical. This result is consistent with the previous theoretical studies.21,23-25 HOMO and LUMO of this neutral biradical are an admixture between pS and π orbitals (Fig.5). Vertical excitation spectra and spin-orbit matrix elements of theT1 neutral biradical are shown in Table 6. The T1 and S0 are almost degenerate. The SOME between the T1 and S0 states has a large value. The ISC of T1→S0 easily occurs.24 Since the structure of RC 6.59 Å got over the transition state (RC 5.58 Å) of the dark S0 energy surface as shown in Fig. 4, the reaction progresses toward the adduct formation. The detection of a neutral biradical might be difficult experimentally, because the Hγ transfer and Sγ-C4a covalent bond formation easily occur.12 The vertical DFT/MRCI excitation energies of the adduct S0 state (RC 8.06 Å) are shown in Table 2. The vertical excitation of S1 state corresponds to the optically bright 14


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transition (π→π*). The vertical excitation energies of the adduct (3.19 eV) underestimate a little the experimental absorption maximum of 3.24 eV (383 nm). Summarizing, it may be stated that our QM (DFT/MRCI)/MM model well reproduces the excited state energies of YtvA-LOV since the computed adiabatic S1 and T1 energies of conf A agree well with the experimental data (Table 1). In the S1 excitation from conf A, the S1→T1 ISC occurs easily when the thiol group locates on the dimethylbenzene moiety of the FMN ring. The triplet state is trapped in the initial structure while keeping the thiol-water hydrogen bond, or is progressed to make adducts almost without a barrier (Fig. 4a, Fig. 7a). In the latter adduct formation process, a neutral bi-radical is formed temporarily and then quickly changes the electronic state via the T1→S0 ISC. The CT state plays an important role for the photoreaction of conf A. Photoexcitation Reaction Pathway of Conformation B Next, we investigate the reaction pathway of conf B. The vertical DFT/MRCI excitation energies of the starting S0 state (RC 1.37 Å) are shown in Table 2. The excitation of the S1 state corresponds to the optically bright transition (π→π*).6 The vertical excitation energy is 2.80 eV. The oscillator strength is relatively large. The energy is in agreement with the experimental absorption maximum at 2.77 eV.46 The electronic structure of the S2 state is a low-energy CT excitation (pS→π*), but the oscillator strength is negligible. A π→π* character is also found for the S3 state. The S3 excitation energy of 3.41 eV agrees very well with the experimental absorption maximum of 3.40 eV.46 We have also explored the S1 energy surface around the starting S0 structure of conf B. However, we could not find a structure close to the starting point by the TDDFT minimization. Instead, one S1 minimized structure in which Hγ is transferred toward the carbonyl oxygen of Cys62 is found at RC 1.09 Å. In this structure, the reaction does not progress. A metastable structure of RC 3.40 Å in which the Hγ is placed just above the N5 of the FMN ring is also found. The QM(DFT/MRCI)/MM energy of RC 3.40 Å is 1.9 kcal/mol more stable than that of RC 1.09 Å (Fig. 4b). At RC 3.40 Å, the electronic structures of S1 and S2 are of pS→π* and π→π* characters, respectively. Like conf A, the excitation energy exchanges (pS→π* vs π→π*) occur between the FC region and the S1 excitation at RC 3.40 Å (Fig. 7b). The S1 excitation energies are shown in Table 3. The S1 emission energy at RC 3.40 Å is 1.46 eV. The result is not compatible to the experimental fluorescence spectrum with a peak maximum at 2.50 eV (496 nm). Like conf A, the S2 state (2.47 eV) is rather closer to the experimental value. Although the S1 and T1 states are energetically close, the value of the SOME between S1 and T1 is very 15



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small (0.72 cm-1). This SOME value is about 1/6 of the value at RC 1.28 Å of conf A. It seems that the ISC between S1 and T1 does not occur so often in the photoexcitation of conf B. Beyond the metastable structure of RC 3.40 Å, Hγ transfers from Sγ to the N5 of the FMN ring. The TDDFT minimization of S1 state stopped near the conical intersection (CIn) between the S1 and S0 states (RC 5.00 Å). The Hγ-N5 distance is 1.10 Å. The QM(TDDFT/B3LYP)/MM energy at the CIn was 6.4 kcal/mol higher than that at RC 3.40 Å (Fig. S1b), whereas the QM(DFT/MRCI)/MM energy at the CIn is 11.3 kcal/mol lower than that at RC 3.40 Å (Fig. 4b). Because there are almost no barriers after the vertical excitation to the S1 state, Hγ easily moves to N5 as shown in Fig. 4b. The sum of Mulliken charges for Cys and lumiflavin-Hγ moieties are -0.20 and 0.21 in the CIn, respectively (Table 5). Those of electrostatic potential charges were -0.39 and 0.51, respectively. Thus, the electronic distribution at the CIn (RC 5.00 Å) is close to a neutral bi-radical. Since the energy of CIn is considerably higher than the transition state of the dark S0 energy surface, the reaction may proceed towards the adduct formation (Fig. 4b). At the same time, since the RC of CIn is shorter than that of the transition state in the dark (5.00 Å < 5.58 Å), a significant part of the reaction returns to the initial state. A similar S1/S0 CIn has been reported in the QM/MM calculations of 1-deazaFMN incorporated in YtvA-LOV, and a radiation-free channel along a hydrogen transfer path is proposed. 49 The minimized structures of the S2 state are found at RC 1.36 and 2.87 Å. The QM(DFT/MRCI)/MM energies are plotted in Fig. 4b. The frontier orbitals of the structure of RC 1.36 Å are close to those of the starting S0 structure of conf B. The electronic structure is of pS→π* character. At RC 2.87 Å, the Hγ is placed just above the N5 of the FMN ring and the excitation from the mixed orbitals of the Sγ lone pair and the π of the FMN ring is dominant (pS/π→π*). Those vertical emission energies are 2.99 and 2.75 eV, respectively (Table S2). They overestimate the experimental fluorescence spectrum of 2.49 eV. The T1, T2 and T3 are situated below the S2 state. The SOMEs between the S2 state and the T1, T2 and T3 states have significant values. Since the T3 states of RC 1.36 and 2.87 Å are found to lie about 0.12 and 0.02 eV below the S2 states and the SOMEs are 2.89 and 4.13 cm-1, respectively, the ISC between S2 and T3 states may occur. The minimum nuclear arrangements of the T1 state have been determined. Two local minima of RC 1.32 Å and 3.54 Å are found. In the RC 1.32 Å, the thiol group forms a hydrogen bond with the carbonyl group of Cys62. In the RC 3.54 Å, the Hγ is located above N5 of the FMN ring. The QM(DFT/MRCI)/MM energy of RC 1.32 Å is lower 16


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than at RC 3.54 Å (Fig. 4b). The adiabatic triplet QM(DFT/MRCI)/MM excitation energy of RC 1.32 Å is 2.07 eV. This value is in agreement with the experimental 0-0 transition of 2.05 eV (Table 1).7 The vertical T1 excitation energies of conf B are also shown in Table 4. The electronic structure at RC 1.32 Å is of π→π* character, whereas those at RC 3.54 Å is of pS→π* character. In the former structure, T5 and T6 are π→π*+nO→π* and nO→π*+π→π* excitations with a significant oscillator strength. In the latter structures, no triplet excitation with significant oscillator strength is found. The excitation energies (1.77 and 1.78 eV) are somewhat lower than the experimental transient absorption maximum of 1.91 eV (650nm).6 The minimum nuclear arrangements of the T2 state have been determined. Two local minima of RC 1.55 and 2.93 Å are found. In the RC 1.55 Å, the thiol group forms a hydrogen bond with the carbonyl group of Cys62. In the RC 2.93 Å, the Hγ is located above N5 of the FMN ring. The QM(DFT/MRCI)/MM energy of RC 1.55 Å is lower than that of RC 2.93 Å (Fig. 4b). The adiabatic DFT/MRCI excitation energy is 2.69 eV. The electronic structures are of π→π* character. In the S1 excitation from conf B, the molecule easily reaches the S1/S0 CIn by χ2 rotation of Cys62 and Hγ transfer. Most of the reactants return to the initial ground state (Fig. 4b, Fig. 7b). Some parts may directly reach adducts. The CT state plays an important role for the photoreaction of conf B. Dual Photoexcitation Reaction Pathway X-ray crystallographic results of several LOV domains showed the presence of two rotamers of the reactive cysteine side chain (conf A and conf B) in the dark state.8 These findings brought up the question whether the two cysteine conformations affect the photoexcitation in different ways. Fourier transform infrared (FTIR) spectroscopy and MD simulations of the LOV2 domain of Adiantum Neochrome1 at low temperature distinguished three reactive conformers.50 One conformation, in which the thiol group of the conserved Cys frees from a hydrogen bonding, and another conformation, in which the thiol group forms a hydrogen bond with an adjacent group, are observed at 77 K. The former is assigned to conf A, in which Hγ is above the dimethylbenzene moiety of the FMN ring. The latter is assigned to conf B, in which the thiol groups form hydrogen bonds with the neighboring groups. A third reactive conformation is observed at 150 K and is assigned to one of conf A, in which the thiol group forms a hydrogen bond with water. There is also an unreactive component at 150 K. It was concluded that there are multiple local structures of the FMN and Cys with different reactivities. Our theoretical results are almost consistent with the results of infrared spectroscopy at low temperature, although our model is the LOV domain of YtvA. The photo-excited 17



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S1 structure in which Hγ is just above the dimethylbenzene moiety of the FMN ring (conf A S1 RC 1.28 Å) can be easily transferred to the triplet state by ISC at 77 K (Fig. 2). Since there is no barrier, the triplet excited structures can reliably lead to the formation of adducts via neutral bi-radical species as shown in Fig. 4a. As for conf B, our results are a little complicated. We found two S1 minimized structures by TDDFT optimizations. One is the structure in which Hγ has transferred to the carbonyl O of Cys62 (conf B S1 RC 1.09 Å). The second one is the structure in which Hγ is just above N5 of the FMN ring (conf B S1 RC 3.40 Å) . Furthermore, a structure close to CIn where Hγ moved to the N5 side is obtained (conf B S1 RC 5.00 Å, Hγ-N5 1.15 Å). Among the three structures, the second one is the most stable in the QM(TDDFT)/MM calculations (Fig S1b). However, the third CIn structure is the most stable in the QM(DFT/MRCI)/MM calculations as shown in Fig. 3b. Accordingly, direct formation of adducts from the S1 excited state is possible through the channel of S1/S0 CIn (Fig. 2). At the same time, a considerable fraction is thought to return to the initial state through this CIn channel. This returned fraction can be considered as unreactive species. Since the SOME between S1 and T1 is small in the second structure, the formation of triplet states by ISC is not so much expected in conf B. The conformation, where the thiol group forms a hydrogen bond with a water molecule and reacts at 150 K (conf A S1 RC -0.29 Å), can easily transfer to the triplet state by the ISC. Since it is necessary to overcome the barrier of 15.5 kcal/mol in order to proceed the reaction by the rotation of the Cys side chain, a higher temperature will be required for the reaction of the conformation. The MD simulations at room temperature show that the interconversions of the χ1 and χ2 dihedral angles of conserved Cys occur on the nanosecond and picosecond timescale, respectively.50 Since the lifetime of the singlet excited state is a few nanoseconds, the conformation of the excited state depends on the ground state with a structure of conf A or conf B. In FLN spectroscopy of Avena sativa LOV2 at room temperature, an Sγ-Hγ conformer free of H-bonding (conf A) is found as a reactive conformer.48 It is speculated that the enhancement of the ISC rate is induced through weak electron donation of the thiol group for π-electrons on the dimethylbenzene moiety of the FMN ring.48 In our S0 minimized structures of conf A , the orbital levels of HOMO and HOMO-1 are very close and the orbitals are of pS and π character, respectively. In the metastable structure free of H-bonding (conf A RC 1.97 Å) , the orbitals of HOMO and HOMO-1 are admixtures of a lone pair orbital of sulfur parallel to the FMN ring and the π orbital delocalized on the FMN ring (Fig. S2a). Since the relaxed S1 states of conf A side (RC -0.29 Å, RC 1.18 Å and RC 3.23 Å) are pS→π * 18


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or mixed (pS/π)→π * charge transfer characters (Table 3), direct spin-orbit coupling ISC is considered to occur. Thus an external heavy-atom effect of the thiol group is significant for the triplet formation at room temperature. Spectroscopic studies of Crphot-LOV2 showed that the quantum yield of the adduct formation is higher than that of triplet formation.9,10 This phenomenon is unique to Crphot-LOV2, but it shows that the formation of a photo-adduct can proceed directly via a singlet excited state in addition to the triplet pathway. In the Crphot-LOV2 case, the adduct is formed through the S1/S0 CIn channel near conf B. In the other LOV domains including YtvA, the S1 excited states return to the initial ground state through the CIn channel. The singlet excited states from the structure of conf A transfer to the triplet states by the ISC, and the triplet states can safely reach the adduct via the second ISC. On the other hand, the singlet excited states from the structure of conf B do not transfer efficiently to the triplet states by ISC and reach the nearby S1/S0 conical intersection. In the case of YtvA LOV, most singlet states return to the initial ground state structure. In the case of Crphot-LOV2 in which the quantum yield of adduct formation is higher than that of triplet formation, most of the singlet states are considered to advance to the ground state adduct formation. In YtvA LOV, it is inferred that the triplet excitation pathway of efficient conf A is chosen by bypassing the singlet excitation pathway of the less efficient conf B. Our QM (DFT / MRCI) / MM calculation model well reproduced the observed LOV excited energies, because the calculation accuracy was high and the size of the structure was appropriate. We believe that the deduced dual photochemical reaction pathway here is highly reliable.

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CONCLUSIONS The results of a high-level QM/MM study of the primary photochemical reaction in the LOV domain of the blue-light photo-sensor YtvA of Bacillus subtilis are report here. Starting from the two conformations of the reactive cysteine side chain found in the X-ray crystallographic structure (conf A and conf B), we determined the reaction pathway of photoexcitation. In conf A (70% occupancy), the Sγ atom is orienting toward the dimethylbenzene moiety of the FMN ring. In conf B (30% occupancy), the Sγ atom is orienting toward the pyrazine moiety of the FMN ring. We have found that there is a dual reaction pathway dependent on the conformers in the flavin-based photoreceptor LOV domain. One of the dual pathways proceeds via the triplet excited state. The electronic structure of the S1 state is the CT excitation of pS→π* when starting from conf A. In the S1 excited states, the side chain rotation of the cysteine occurs easily and the Hγ comes just above the dimethylbenzene moiety. The S1 and T1 states are almost degenerated or the T1 states are situated below the S1 states. The SOMEs between the S1 state and the T1 state have significant values. Therefore, the S1 states decay non-radiatively to the triplet states by intersystem crossing (ISC). Since the triplet reaction surface has a low energy barrier here, most of the excited-state population is trapped temporarily in triplet state. Then, the Hγ transfers to the N5 atom of the FMN ring without a further barrier, and a neutral bi-radical is formed. The triplet states cross over to the singlet states by the second ISC and the flavin-cysteinyl adducts are efficiently formed. The other path proceeds via the singlet excited state. The singlet states excited from conf B are located near the S1/S0 conical intersection (CIn). Although the S1 and T1 states are almost degenerate, the value of SOME between S1 and T1 is very small here. The Hγ atom moves toward the N5 atom side and the singlet excited state undergoes a CIn. Most parts return to the initial ground states through the CIn. The rest may directly reach the adduct state. Thus, the photo-excitation of LOV has a dual reaction pathway. Although the Sγ atom of conf A is away from the C4a atom of the FMN ring, the formation of the adduct efficiently proceeds by the triplet excitation pathway. On the other hand, the Sγ atom of conf B is close to the C4a atom, but there is little reaction toward formation of adduct. The presence of rotamers of reactive cysteine side chain in the dark state can be said to have important significance for the photoexcitation reaction of LOV. In YtvA-LOV, the triplet excitation pathway, in which ISC occurred on the dimethylbenzene side of the FMN ring, was chosen as the main reaction in order to avoid the less efficient singlet excitation pathway. 20


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ACKNOWLEDGEMENT: This work was supported by the Grant-in-Aid for Scientific Research (C) of Japan Society for the Promotion of Science, and a Kinjo Gakuin University Special Research Subsidy. SUPPORTING INFORMATION AVAILABLE: Extended information on the reaction profiles, full list of the optimized structures and the frontier orbitals, and the vertical excitation spectra at S2 states.

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REFERENCES (1) Losi, A. Flavin-based Blue-Light Photosensors: A Photobiophysics Update. Photochem. Photobiol. 2007, 83, 1283–1300. (2) Losi, A.; Gärtner, W. Old Chromophores, New Photoactivation Paradigms, Trendy Applications: Flavins in Blue Lights-Sensing Photoreceptors. Photochem. Photobiol. 2011, 87, 491–510. (3) Conrad, K. S.; Manahan, C. C.; Crane, B. R. Photochemistry of Flavoprotein Light Sensors. Nat Chem Biol. 2014, 10, 801–809. (4) Pudasaini, A.; El-Arab, K. K.; Zoltowski, B. D. Lov-based Optogenetic Devices: Light-driven Modules to Impart Photoregulated Control of Cellular Signaling. Front. Mol. Biosci. 2015, 2:18. (5) Christie, J. M.; Blackwood, L.;, Petersen, J.; Sullivan, S. Plant Flavoprotein Photoreceptors. Plant Cell Physiol. 2015, 56,401-413. (6) Losi, A; Polverini, E; Quest, B.; Gärtner, W. First Evidence for Phototropin-Related Blue-Light Receptors in Prokaryotes. Biophys. J. 2002, 82, 2627–2634. (7) Losi, A; Quest, B; Gärtner, W. Listening to the Blue: the Time-Resolved Thermodynamics of the Bacterial Blue-Light Receptor YtvA and its Isolated LOV Domain. Photochem. Photobiol. Sci. 2003, 2, 759-766. (8) Möglich, A.; Moffat, K. Structural Basis for Light-Dependent Signaling in the Dimeric LOV Domain of the Photosensor YtvA. J. Mol. Biol. 2007, 373, 112–126. (9) Holzer, W.; Penzkofer, A.;Fuhrmann, M.; Hegemann, P. Spectroscopic Characterization of Flavin Mononucleotide Bound to the LOV1 Domain of Phot1 from Chlamydomonas reinhardtii. Photochem. Photobiol. 2002, 75, 479-487. (10) Holzer, W.; Penzkofer, A.; Hegemann, P., J. Luminesc. 2005, 112, 444–448, Photopysical and Photochemical Excitation and Relaxation Dynamics of LOV Domains of Phot from Chlamydomonas reinhardtii. (11) Kennis, J. T. M.; Crosson, S.; Gauden, M.; van Stokkum, I. H. M.; Moffat, K.; van Grondelle, R. Primary Reactions of the LOV2 Domain of Phototropin, a Plant Blue-Light Photoreceptor. Biochemistry, 2003, 42, 3385–3392. (12) Kutta, R. J.; Magerl, K.; Kensy, U.; Dick, B. Photochem. A search for Radical Intermediates in the Photocycle of LOV Domains. Photobiol. Sci. 2015, 14, 288-299. (13) Kay, C. W. M.; Schleicher, E.; Kuppig, A.; Hofner, H.; Rüdiger, W.; Schleicher, M.; Fischer, M.; Bacher, A.; Weber, S.; Richter, G. Blue Light Perception in Plants Detection and Characterization of a Light-Induced Neutral Flavin Radical in a C450A Mutant of Phototropin. J. Biol. Chem. 2003, 278, 10973-10982. 22


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(14) Schleicher, E.; Kowalczyk, R. M.; Kay, C. W. M.; Hegemann, P.; Bacher, A.; Fischer, M.; Bittl, R.; Richter, G.; Weber, S. On the Reaction Mechanism of Adduct Formation in LOV Domains of the Plant Blue-Light Receptor Phototropin. J. Am. Chem. Soc. 2004, 126, 11067–11076. (15) Alexandre, M. T. A.; Domratcheva, T.; Bonetti, C.; van Wilderen, L. J. G. W.; van Grondelle, R.; Groot, M.-L.; Hellingwerf, K. J.; Kennis, J. T. M. Primary Reactions of the LOV2 Domain of Phototropin Studied with Ultrafast Mid-Infrared Spectroscopy and Quantum Chemistry. Biophys. J. 2009, 97 227–237. (16) Bauer, C; Rabl, C.-R.; Heberle, J.; Kottke, T. Indication for a Radical Intermediate Preceding the Signaling State in the LOV Domain Photocycle. Photochem. Photobiol, 2011, 87, 548–553. (17) Pfeifer, A.; Majerus, T.; Zikihara, K.; Matsuoka, D.; Tokutomi, S.; Heberle, J.; Kottke, T. Time-Resolved Fourier Transform Infrared Study on Photoadduct Formation and Secondary Structural Changes within the Phototropin LOV Domain. Biophys. J. 2009, 96 1462-1470. (18) Zayner, J. P.; Sosnick, T. R. Factors That Control the Chemistry of the LOV Domain Photocycle. PLOS ONE 2014, 9, e87074. (19) Lee, R.; Gam, J.; Moon, J.; Lee, S.-G.; Suh, Y.-G.; Lee, B.-J.; Lee, J. A Critical Element of the Light-Induced Quaternary Structural Changes in YtvA-LOV. Protein Science 2015, 24,1997–2007. (20) Heintz, U.; Schlichting, I. Blue Light-Induced LOV Domain Dimerization Enhances the Affinity of Aureochrome 1a for its Target DNA Sequence. eLIFE 2016, 5, e11860. (21) Neiß, C.; Saalfrank, P. Ab Initio Quantum Chemical Investigation of the First Steps of the Photocycle of Phototropin: A Model Study.Photochem. Photobiol. 2003, 77, 101–109. (22) Fedorov, R.; Schlichting, I.; Hartmann, E.; Domratcheva, T.; Fuhrmann, M.; Hegemann, P. Crystal Structures and Molecular Mechanism of a Light-Induced Signaling Switch: The Phot-LOV1 Domain from Chlamydomonas reinhardtii. Biophys. J. 2003, 84, 2474–2482. (23) Domratcheva, T.; Fedorov, R.; Schlichting, I. Analysis of the Primary Photocycle Reactions Occurring in the Light, Oxygen, and Voltage Blue-Light Receptor by Multiconfigurational Quantum-Chemical Methods. J. Chem. Theory Comput. 2006, 2, 1565–1574. (24) Zenichowski, K.; Gothe, M.; Saalfrank, P. Exciting Flavins: Absorption Spectra and Spin–Orbit Coupling in Light–Oxygen–Voltage (LOV) Domains. J. Photochem. 23



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Photobiol. A: Chem. 2007, 190, 290–300. (25) Dittrich, M.; Freddolino, P. L.; Schulten, K. When Light Falls in LOV: A Quantum Mechanical/Molecular Mechanical Study of Photoexcitation in Phot-LOV1 of Chlamydomonas reinhardtii. J. Phys. Chem. B 2005, 109, 13006–13013. (26) Salzmann, S.; Silva-Junior, M. R.; Thiel, W.; Marian, C. M. Influence of the LOV Domain on Low-Lying Excited States of Flavin: A Combined Quantum-Mechanics/Molecular-Mechanics Investigation. J. Phys. Chem. B 2009, 113, 15610–15618. (27) Chang, X.-P.; Gao, Y.-J.; Fang, W.-H.; Cui, G.; Thiel, W. Quantum Mechanics/Molecular Mechanics Study on the Photoreactions of Dark- and Light-Adapted States of a Blue-Light YtvA LOV Photoreceptor. Angew. Chem. Int. Ed. 2017, 56, 9341-9345. (28) Freddolino, P. L.; Gardner, K. H.; Schulten, K. Signaling Mechanisms of LOV Domains: New Insights from Molecular Dynamics Studies. Photochem. Photobiol. Sci. 2013, 12, 1158-1170. (29) Sherwood, P.; de Vries, A. H.; Guest, M. F.; Schreckenbach, G.; Catlow, C.R. A.; French, S. A.; Sokol, A. A. ; Bromley, S. T.; Thiel, W.; Turner, A. J. et al. QUASI: A General Purpose Implementation of the QM/MM Approach and its Application to Problems in Catalysis. J. Mol. Structure: THEOCHEM 2003, 632, 1-28. (30) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926-935. (31) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. CHARMM: A Program for Macromolecular Energy, Minimization, and Dynamics Calculations. J. Comput. Chem. 1983, 4, 187–217. (32) Smith, W.; Forester, T. R. DL_POLY_2.0: A General-Purpose Parallel Molecular Dynamics Simulation Package. J. Mol. Graphics 1996, 14, 136–141. (33) de Vries, A. H.; Sherwood, P.; Collins, S. J.; Rigby, A. M.; Rigutto, M.; Kramer, G. J. Zeolite Structure and Reactivity by Combined Quantum-Chemical−Classical Calculations. J. Phys. Chem. B 1999, 103, 6133-6141. (34) Bauernschmitt, R.; Ahlrichs, R. Treatment of Electronic Excitations within the Adiabatic Approximation of Time Dependent Density Functional Theory. Chem. Phys. Lett. 1996, 256, 454–464. (35) Becke, A. D. A New mixing of Hartree–Fock and Local Density-Functional Theories. J. Chem. Phys. 1993, 98, 1372–1377. (36) Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. Ab initio Calculation 24


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of Vibrational Absorption and Circular Dichroism Spectra Using Density Functional Force Fields. J. Phys. Chem. 1994, 98, 11623–11627. (37) Billeter, S. R.; Turner, A. J.; Thiel, W. Linear Scaling Geometry Optimisation and Transition State Search in Hybrid Delocalised Internal Coordinates. Phys. Chem. Chem. Phys. 2000, 2, 2177-2186. (38) Dreuw, A.; Weisman, J. L.; Head-Gordon, M. Long-Range Charge-Transfer Excited States in Time-Dependent Density Functional Theory Require Non-Local Exchange. J. Chem. Phys. 2003, 119, 2943- 2946. (39) Grimme, S.; Waletzke, M. A Combination of Kohn–Sham Density Functional Theory and Multi-Reference Configuration Interaction Methods. J. Chem. Phys. 1999, 111, 5645–5655. (40) Ahlrichs, R.; Furche, F.; Hättig, C.; Klopper, W.; Sierka, M.; Weigend, F.; TURBOMOLE V6.3 2011, a development of University of Karlsruhe GmbH, 1989-2007, TURBOMOLE GmbH, since 2007; available from http://www.turbomole.com. (41) Kleinschmidt, M.; Tatchen, J.; Marian, C. M. Spin-Orbit Coupling of DFT/MRCI Wavefunctions: Method, Test Calculations, and Application to Thiophene. J. Comput. Chem. 2002, 23, 824–833. (42) Kleinschmidt, M.; Marian, C. M. Efficient Generation of Matrix Elements for One-Electron Spin–Orbit Operators. Chem. Phys. 2005, 311, 71–79, (43) Heß, B. A.; Marian, C. M.; Wahlgren, U.; Gropen, O. A Mean-Field Spin-Orbit Method Applicable to Correlated Wavefunctions. Chem. Phys. Lett. 1996, 251, 365– 371. (44) Bocola, M.; Schwaneberg, U.; Jaeger, K.-E.; Krauss, U. Light-Induced Structural Changes in a Short Light, Oxygen, Voltage (LOV) Protein Revealed by Molecular Dynamics Simulations－Implications for the Understanding of LOV Photoactivation. Front. Mol. Biosci. 2015, 2:55. (45) Raffelberg, S.; Mansurova, M.; Gärtner, W.; Losi, A. Modulation of the Photocycle of a LOV Domain Phtoreceptor by the Hydrogen-Bonding Network. J. Am. Chem. Soc. 2011, 133, 5346-5356. (46) Song, S.-H.; Madsen, D.; van der Steen, J. B.; Pullman, R.; Freer, L. H.; Hellingwerf, K. J.; Larsen, D.S. Primary Photochemistry of the Dark- and Light-Adapted States of the YtvA Protein from Bacillus subtillis. Biochemistry 2013, 52, 7951-7963. (47) van Stokkum, I. H. M.; Gauden, M.; Crosson, S.; van Grondelle, R.; Moffat, K.; Kennis, J. T. M. The Primary Photophysics of the Avena sativa Phototropin 1 LOV2 25



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Domain Observed with Time-Resolved Emission Spectroscopy. Photochem. Photobiol. 2011, 87, 534-541. (48) Alexandre, M. T. A.; van Grondelle, R.; Hellingwerf, K. J.; Robert, B.; Kennis, J. T. M. Perturbation of the Ground-State Electronic Structure of FMN by the Conserved Cysteine in Phototropin LOV2 domains. Phys. Chem. Chem. Phys. 2008,10, 6693-6702. (49) Silva-Junior, M. R.; Mansurova, M.; Gärtner, W.; Thiel, W. Photophysics of Structurally Modified Flavin Derivatives in the Blue-Light Photoreceptor YtvA: A Combined Experimental and Theoretical Study. ChemBioChem. 2013, 14, 1648-1661. (50) Sato, Y.; Nabeno, M. ; Iwata, T. ; Tokutomi, S. ; Sakurai, M.; Kandori, H. Heterogeneous Environment of the S-H Group of Cys966 Near the Flavin Chromophore in the LOV2 Domain of Adiantum Neochrome1. Biochemistry 2007, 46 , 10258–10265.

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Table 1 Adiabatic Singlet and Triplet QM(DFT/MRCI)/MM Energies in eV for conf A and conf B in Comparison to Experimental 0-0 Transitions conf A S1 T1

state pS→π* π→π*

RC -0.29 -0.06

conf B ∆E 2.74 2.04

RC 3.40 1.32

∆E 2.47 2.07

S0 add 8.06 0.64 8.06 0.93 S0 add flp a Absorption and fluorescence spectra from ref. 6. b Photocalorimetric date from ref 7.

exp. ∆E0-0 2.56a 2.05b 1.17b

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Table 2

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Selected Vertical Excitation Spectra in eV and Oscillator Strengths at the Optimized Ground-state Geometries of the Initial and the Adduct States in Comparison to Experimental Absorption Maxima.

state

excitation

∆E

f

conf A S0 RC 0.0 Å S1

πH-1→π*L

2.80

S2 S3 S4 T1 T2 T3

pSH→π*L nNH-8 + nN H-6→π*L πH-2→π*L πH-1→π*L πH-2→π*L nNH-8 + nNH-6→π*L

3.03 3.38 3.51 2.17 2.58 3.01

∆E

f

∆E

conf A S0 RC 1.97 Å

f

Exp. λmax a [eV] (nm)

conf B S0 RC 1.37 Å

0.23

(pS/π)H→π*L

2.72

0.25

πH→π*L

2.80

0.28

0.12 0.01 0.17

(pS/π)H-1→π*L πH-2→π*L nN H-8→π*L (pS/π)H - (pS/π)H-1→π*L πH-2→π*L nN H-8→π*L

3.19 3.38 3.41 2.03 2.59 3.02

0.12 0.11 0.05

pSH-1→π*L πH-2→π*L nNH-6 - pSH-1→π*L πH→π*L πH-2→π*L nNH-6→π*L

3.26 3.41 3.49 2.14 2.68 2.98

0.01 0.23 0.02

adduct S0 RC 8.06 Å S1 πH→π*L 3.19 0.20 S2 pSH-2→π*L 3.60 0.08 * S3 πH-1→π L 4.07 0.18 S4 πH→π*L+1 4.44 0.05 T1 πH→π*L 2.71 T2 pSH-2→π*L 3.20 T3 πH-1→π*L 3.56 a Absorption maximum of the LOV domain of YtvA taken from ref. 6 and ref. 46.

28


2.76 (449) 2.77(447) 2.94(422) 3.31 (375) 3.40(365)

3.24 (383)

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Table 3 Vertical Excitation Spectra at the Optimized S1 states in eV and the Spin-Orbit Matrix Elements in cm-1 conf A S1 RC -0.29 Å conf A S1 RC 1.28 Å conf A S1 RC 3.23 Å state excitation ∆E f excitation ∆E f excitation S0 GS 0.0 GS 0.0 GS * * S1 2.21 0.00 1.82 0.00 pSH→π L pSH →π L (pS/π)H→π*L * * * S2 2.56 0.22 2.53 0.21 πH-1→π L πH-1→π L (π/pS)H-1→π*L S3 3.13 0.29 3.10 0.29 πH-2→π*L πH-2→π*L πH-2→π*L T1 1.99 1.77 πH-1→π*L pSH →π*L (π/pS)H-1→π*L * * T2 2.12 1.99 pSH →π L πH-1→π L (pS/π)H→π*L T3 2.49 2.39 πH-2→π*L πH-2→π*L πH-2→π*L

state S0 S1

SOME 5.71 2.59 9.04 conf B S1 RC 1.09 Å excitation ∆E GS 0.0 0.96 pSH →π*L

0.00

S2

pSH-1 →π*L

1.63

0.00

πH-1→π*L

2.47

0.22

S3 T1 T2

πH-2→π*L

2.62 0.95 1.63

0.24

πH-2→π*L pSH →π*L πH-1→π*L

3.08 1.41 1.92

0.26

pSH →π*L pSH-1 →π*L

T3

πH-2→π*L

2.06

πH-2→π*L

2.35

f

SOME 4.40 7.79 28.4 conf B S1 RC 3.40 Å excitation ∆E GS 0.0 1.46 pSH →π*L

f 0.00

SOME SOME 0.94 0.72 238.99 5.27 10.79 17.88 a Fluorescence maximum of the LOV domain of YtvA taken from ref. 6 and ref. 45.

∆E 0.0 2.13 2.46 3.05 1.88 2.06 2.37

SOME 7.08 1.19 5.67 CIn S1 RC 5.00 Å excitation ∆E GS -0.19 0.00 pSH →(π/pS)*L * πH-2 pSH → (π/pS) L 1.18 (π/pS)*L 1.49 pS H-1 → (π/pS)*L * -0.21 pSH →(π/pS) L 0.51 pS H-1 → (π/pS)*L * πH-2 pSH → (π/pS) L 1.60 (π/pS)*L SOME

29


217.79 14.12 1.54

f 0.00 0.21 0.28

f 0.00

0.10 0.00

Exp. λmax a [eV] (nm)

2.50 (496) 2.49 (497)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Table 4 Vertical Excitation Energies of T1 states in eV conf A T1 RC -0.06 Å state excitation ∆E T1 0.0 πH→π*L * * 0.6 T2 πH-2→π L + pSH-1 →π L * * T3 1.21 pS H-1 →π L + πH-2→π L T4 1.27 nNH-8→π*L+ pSH-1 →π*L 1.81 T5 πH-6 →π*L+πH→π*L+1 * * T6 1.84 nOH-10→π L+ nOH-13→π L T7 1.89 πH→π*L+1+πH-6→π*L T8 2.17 πH-9→π*L conf B T1 RC 1.32 Å state excitation ∆E T1 0.0 πH →π*L * T2 0.65 πH-2→π L * * 1.08 T3 nNH-7→π L + pS H-1 →π L * T4 1.31 pSH-1 →π L T5 1.77 πH-6 →π*L+ nOH-10→π*L 1.78 T6 nOH-10→π*L+π H-6→π*L T7 1.87 πH →π*L+1 * 2.11 T8 πH-9→π L

f 0.02 0.00 0.00 0.13 0.00 0.07 0.01 f 0.02 0.00 0.00 0.09 0.07 0.04 0.01

conf A T1 RC 1.28 Å excitation pSH →π*L πH-1→π*L πH-2→π*L nNOH-12→π*L+nNH-10→π*L nNOH-12→π*L+ nOH-14→π*L πH-9→π*L+nSH-6→π*L πH-11→π*L πH-1→π*L+1 conf B T1 RC 3.54 Å excitation pSH →π*L πH-1→π*L πH-2→π*L nNH-10 →π*L+nOH-11→π*L nOH-11→π*L+ nNH-10 →π*L πH-8→π*L πH-9→π*L pSH →π*L+1

∆E 0.0 0.23 0.62 1.11 1.67 1.71 1.92 2.18 ∆E 0.0 0.54 0.98 1.44 1.97 2.15 2.23 2.40

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f 0.02 0.02 0.00 0.00 0.01 0.00 0.01 f 0.01 0.01 0.00 0.00 0.00 0.00 0.00

conf A T1 RC 3.58 Å excitation pSH →π*L πH-1→π*L * πH-2→π*L πH-10→π*L+nOH-11→π*L nOH-11→π*L πH-8→π*L πH-9→π*L+πH-10→π*L πH-1→π*L+1

∆E 0.0 0.02 0.49 0.92 1.43 1.64 1.73 2.08

f 0.00 0.02 0.00 0.00 0.00 0.00 0.00

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Table 5 (FL).


Sum of Mulliken Charges and ESP Fit Charges of the Cys Model Molecule and Lumiflavin

conformation state coonf A S0 S1 S1 T1 T1 T1 biradical

RC

Hγ position

Mulliken charge Cys side FL side

ESP fit charge Cys side FL side

0.0 -0.29 1.28 1.28 3.58 6.59

CysHγ/FL CysHγ/FL CysHγ/FL CysHγ/FL CysHγ/FL Cys/FLHγ

0.01 -0.01 0.02 0.04 0.03 -0.28

-0.01 -0.02 -0.02 -0.03 -0.02 0.28

-0.10 -0.11 -0.12 -0.09 -0.09 -0.33

0.19 0.19 0.23 0.21 0.21 0.40

conf B S0 S1 S1 S1 CIn

1.37 1.09 3.40 5.04

CysHγ/FL CysHγ/FL CysHγ/FL Cys/FLHγ

0.00 0.01 0.03 -0.20

0.00 -0.01 -0.03 0.21

-0.07 -0.04 -0.07 -0.39

0.19 0.18 0.21 0.51

S0 adduct

8.06

CysFLHγ

0.20

-0.20

-0.25

0.34

31



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Table 6 Vertical Excitation Spectra at the Optimized T1 Bi-radical States in eV and the Spin–Orbit Matrix Elements in cm-1 T1 birad RC 6.59 Å state excitation ∆E f GS + (pS/π)H(pS/π)H S0 0.0 →(π/pS)*L(π/pS)*L S1 0.27 0.00 pSH-1→(π/pS)*L * * S2 1.31 0.08 πH-2 (pS/π)H → (π/pS) L (π/pS) L * * S3 1.68 0.00 πH-2 pS H-1 → (π/pS) L (π/pS) L T1 0.12 (pS/π)H →(π/pS)*L T2 0.48 pS H-1→(π/pS)*L * * T3 1.97 πH-2 pS H-1 → (π/pS) L (π/pS) L SOME

47.33 240.91 0.81

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Figure 1 Core LOV domain of YtvA. Conserved Cys62 (conformers A and B), Gln123 side chain, one crystal water and the lumiflavin ring contained in the QM region are shown as thick stick models. The secondary structure of core LOV (from Aβ to Iβ) contained in the MM region is 37 displayed by a ribbon model. 38 39 40 33 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Figure 2 Photocycle of LOV domain. A dual photochemical reaction pathway via singlet and triplet excited states is shown. 38 39 40 34 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Figure 3 Selected reaction structures of conf A and conf B. QM regions are shown. Reaction coordinates (Å) and Cys62 χ1 and χ2 dihedral angles (degrees) are shown. 36 37 38 39 40 35 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48


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1 2 3 4 80 80 5 6 T2 70 70 T2 7 T1 T1 8 60 60 S2 9 ISC S2 10 50 50 S1 CIn 11 S1 S0 12 40 40 S0 13 14 30 30 ISC 15 16 20 20 17 dark ts dark ts 18 10 10 19 20 0 0 21 22 -10 -10 23 -1 0 1 2 3 4 5 6 7 8 9 -1 0 1 2 3 4 5 6 7 8 9 24 Reaction coordinates [Å Å] 25 Reaction coordinates [Å Å] 26 27 28 Figure 4 Reaction profiles of ground states and singlet and triplet excited states. a Reaction profile of conf A. The S0 profile has a transition state (dark ts). Intersystem crossings (ISC) between S1 and T1 and between T1 and S0 are shown. 29 b Reaction profile of conf B. The S0 profile has a transition state (dark ts). A conical intersection (CIn) between S1 and S0 is shown. 30 31 32 33 34 35 36 37 38 39 40 36 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48

b

∆E(QM/MM) [kcal/mol]

∆E (QM/MM) [kcal/mol]

a

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Figure 5 Frontier orbitals of selected conf A structures. Reaction coordinates are shown in Å. 37 38 39 40 37 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Fig.6 Frontier orbitals of selected conf B structures. Reaction coordinates are shown in Å. 38 39 40 38 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48

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1 2 3 4.5 4.5 S0 S1 S2 4 S0 S1 S2 5 T1 T2 path B 4.0 4.0 T1 T2 path A 6 CIn 7 pS-π* 8 3.5 3.5 9 pS-π* 10 3.0 3.0 π-π* 11 π-π* 12 ISC 2.5 13 2.5 2.61 14 2.53 2.48 2.42 pS-π* 15 pS-π* CIn 2.46 pS-π* 2.0 2.0 pS-π* 16 pS-π* 17 2.11/1.93 1.5 1.5 18 pS/π-π* 19 ISC 20 1.0 1.0 21 1.18 pS/π-π/pS* 22 0.5 0.5 23 0.62 0.62 24 25 0.0 0.0 0.08 26 0.00 27 -0.5 -0.5 28 S0 1.37 S1 3.40 S1 5.00 S0 8.06 S0 0.0 S1 -0.29 S1 1.28 T1 1.28 T1 3.58 birad 6.59 S0 8.06 29 FC reg CIn add FC reg add 30 Electronic state and RC [Å Å] Electronic state and RC [Å Å] 31 32 33 34 35 Fig. 7 Electronic excitation energies at various excited-state geometries. For better comparability, the QM/MM ground-state energy at the FC region of conf A 36 has been chosen as the common origin. 37 a Reaction pathway for conf A. 38 b Reaction pathway for conf B. 39 40 39 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48 Excitation energy [eV]

a

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Dual Photochemical Reaction Pathway in Flavin ... - ACS Publications

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