Molecular Mechanism of Flavin Photoprotection by Archaeal Dodecin

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Molecular Mechanism of Flavin Photoprotection by Archaeal Dodecin: Photo-Induced Electron Transfer and Mg -Promoted Proton Transfer 2+

Maximilian Scheurer, Daria Brisker-Klaiman, and Andreas Dreuw J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b08597 • Publication Date (Web): 25 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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The Journal of Physical Chemistry

Molecular Mechanism of Flavin Photoprotection by Archaeal Dodecin: Photo-induced Electron Transfer and Mg2+-promoted Proton Transfer Maximilian Scheurer, Daria Brisker-Klaiman, and Andreas Dreuw∗ Interdisciplinary Center for Scientific Computing, Im Neuenheimer Feld 205A, 69120 Heidelberg (Germany) E-mail: [email protected] Phone: +49 (0)6221 5414735

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Abstract Photo-induced biochemical reactions are ubiquitously governed by derivatives of flavin, which is a key player in a manifold of cellular redox reactions. The photoreactivity of flavins is also one of their greatest disadvantages as the molecules are sensitive to photodegradation. To prevent this unfavorable reaction, UV-light-exposed archaea bacteria, such as Halobacterium salinarum, manage the task of protecting flavin derivatives by dodecin, a protein which stores flavins and efficiently photo-protects them. In this study, we shed light on the photo-protection mechanism, i.e. the excited state quenching mechanism by dodecin using computational methodology. Molecular dynamics (MD) simulations unraveled the hydrogen bond network in the flavin binding pocket as starting point for proton transfer upon preceding electron transfer. Using high-level ab-initio quantum chemical methods, different proton transfer channels have been investigated and an energetically feasible Mg2+ -promoted channel has been identified fully explaining previous experimental observations. This is the first extensive theoretical study of archaeal dodecin, furthering the understanding of its photocycle and manipulation.

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Introduction Dodecin, a riboflavin-binding protein (RfbP), has been identified as key player in archaeal flavin homeostatis. 1–3 Riboflavin (Rf) and its biochemical derivates, such as flavin adenine dinucleotide (FAD) and flavin adenine mononucleotide (FMN), play pivotal roles in metabolism and biosynthetic pathways, serving mainly as redox equivalents. 4 Therefore, cellular concentrations of flavin derivatives must be thoroughly controlled to prevent damage to key molecules in the cell through flavin redox activity.

a)

b)

W36 Q55 Q55

W36

c)

Figure 1: Structural overview of Halobacterium salinarum dodecin (HsDod). a) HsDod dodecamer (PDB 1MOG). The 12 protein chains are shown in cartoon representation, where the C2-symmetric tetrad in blue surrounds a single binding pocket. The rest of the protein is colored green. b) Zoom-in on the binding pocket. Two lumiflavin (Lf) molecules are bound by hydrogen bonds to Q55 (orange lines) and by π-stacking with W36. c) Hydrogen bond donors and acceptors of Lf. The hydrogen bond donor (N3) and the acceptors (N1, O2, O4 and N5) involved in the hydrogen bonds to solvent and protein are labeled. Carbon atoms are shown in gray, nitrogen in blue, oxygen in red and hydrogen in white color. In Halobacterium salinarum, dodecin (HsDod) regulates cellular flavin concentrations by sequestering or providing Rf, FAD and FMN, depending on the cellular growth state. 1 Another important function of HsDod is protecting flavins from photodegradation, which leads 3



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to loss of their functionality. Intriguingly, HsDod is capable of quenching the photoexcited state of flavins in an ultrafast manner. 5 Differences in structure and function between bacterial and archaeal dodecin have been extensively discussed, whereby HsDod is best understood with respect to its unique structure and function. 1–3,6,7 Archaeal dodecin is a homododecameric and symmetric protein complex that stores two flavin molecules in each of its six binding pockets, which are located between the individual dodecin trimers. Thus, every dodecin tetramer fully surrounds a single C2-symmetric binding pocket. 3 An overview of the HsDod protein and binding pocket structure is presented in Figure 1. Flavin derivatives are bound to HsDod via four-fold π-stacking, forming a unique aromatic tetrad in the binding pocket. 1–3,5,6,8 Further stabilization is gained through two hydrogen bonds from N3 and O2 of the flavin species to Q55 (Fig. 1b). The excited state quenching mechanism has been experimentally studied by a number of spectroscopic methods. 5,7 A photo-induced electron transfer from the adjacent tryptophan residue (W36, Fig. 1b) to the flavin species has been suggested as initial step in the deactivation mechanism. 5 This photo-induced charge separation is well known in flavin photochemistry. 9–16 Furthermore, subsequent proton transfer from surrounding water molecules has been discussed as possible second deactivation channel forming a flavosemiquinone radical. The combination of both steps is referred to as coupled electron-proton transfer. The N5 atom of the flavin system (Fig. 1c) has been suggested as proton target by analysis of crystal structures and based on previous experiments on the basicity of photo-excited flavins. 5,17 Interestingly, proton transfer to N5 of flavin derivatives embedded in proteins was also proposed for other enzymes, yet structurally and functionally different from HsDod. 9,10 Therefore, such flavin-related proton transfer seems to be a recurring biophotochemical phenomenon. Even though a plethora of experiments have been performed in order to understand the exotic behavior and characteristics of archaeal dodecin, a theoretical description by computational means augmenting the experimental findings is still missing to date. Hence, computational methods at different levels of theory are here applied to study HsDod: first, the

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protein dynamics of the binding pocket and the underlying interactions are examined at an atomic level of detail using molecular dynamics (MD) simulations. Second, quantum chemical (QC) calculations are performed to provide a theoretical foundation for the proposed electron and proton transfer mechanism. Based on MD simulations of representative binding pocket structures, analyses of possible proton transfer channels are performed taking into account different charge and redox states of the system. Especially, our results from high-level excited state calculations employing the algebraic diagrammatic construction scheme for the polarization propagator of second order (ADC(2)) gave proof of the nature of the chargeseparated state with a negatively charged semiquinone flavin radical as proton acceptor. This charge-separated redox state, induced upon photoexcitation, exhibits a significantly lower energy barrier for proton transfer to the N5 atom of the flavin species than the electronic ground state, confirming the initial electron transfer step of the quenching mechanism also from a theoretical perspective. Excited state calculations of the molecules involved in the proton transfer reactions revealed the same spectroscopic signatures as already observed in experiment. The paper is organized as follows. In the next section, the applied computational methods and protocols are presented. In the subsequent results section, the MD results are presented, which serve as starting point for follow-up hybrid QM/MM simulations and quantum chemical calculations of possible proton transfer channels. The paper concludes with a brief summary of the main results, comparison with experimental findings and a comprehensive model of the photoprotection mechanism.

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Computational Methodology Atomic structures and Molecular Dynamics simulations The starting geometry for MD simulations was taken from the H. salinarum crystal structure (PDB 1MOG). 3 Four dodecin monomers, forming a single binding pocket, were extracted from the dodecamer (Fig. 2). The riboflavin residues were truncated to lumiflavin (Lf) molecules for simplicity. Hydrogen atoms were added to the structures using the VMD psfgen plugin and the structures were put into a water box, including 150 mM NaCl, using the VMD solvate and autoionize plugin, respectively. 18 Ions were placed randomly with ◦

a minimum distance to other ions and solvent of 5 A. In total, 149 Na+ ions and 105 Cl− ions were added. Three simulation systems of the dodecin tetrad were set up, where the composition of the binding pocket varied as shown in Tab. 1. All simulations were Table 1: Summary of performed MD simulations # system I II III

binding pocket composition description Lf dimer and Mg2+ holo-dodecin Lf dimer holo-dodecin without Mg2+ Lf monomer and Mg2+ holo-dodecin with single ligand

carried out with NAMD2 19 using the CHARMM27 force field for the protein, TIP3P water and ions. 20–22 Lumiflavin was parametrized with SwissParam. 23 The used parameter file is included as Supporting Information. During the simulations, a 2 fs integrator timestep was used. To constrain bond vibrations and water molecule geometries, RATTLE and SETTLE algorithms were applied, respectively. 24,25 The NpT ensemble system temperature was kept at 310 K and the system pressure was 1 atm. Particle Mesh Ewald (PME) 26 was used ◦

to calculate long-range electrostatics beyond a smooth 12 A cutoff. Every system was first minimized, then annealed to the target temperature for 200 ps by increasing the temperature by 1 K every 20 fs, and equilibrated for 5 ns. During these simulations, the protein backbone ◦

−2

atoms were constrained with a force constant of k = 1.0 · kcal · mol−1 · A . Finally, a production run without constraints was performed for 500 ns in total. To improve sampling, 6


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two separate simulations with different initial velocity seeds were started for every production run. Trajectory analyses were performed within VMD and hydrogen bond analyses were executed with PyContact (https://pycontact.github.io). Hydrogen bonds were defined ◦

such that the donor-acceptor distance was below 3.5 A, and the donor-hydrogen-acceptor angle was larger than 120◦ . Cluster analyses were performed using the Bio3D package for R 27,28 as reported by Goh et al. 29

Hybrid Quantum Mechanics/Molecular Mechanics simulations To simulate the UV/VIS-spectrum of Lf in the protein environment, a snapshot from system I with the abundant N5/O4 hydrogen bond pattern was extracted from the trajectory and incorporated into a hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) simulation. The QM region was chosen to include a single Lf molecule, together with five coordinating water molecules, the Mg2+ ion and D41. The QM/MM bonds were chosen to be in the D41 backbone between Cα and the carbonyl carbon as well as the nitrogen atom and the N-terminal carbonyl carbon atom connected to D41. To prevent over-polarization at the QM/MM boundary, the redistributed charge and dipole (RCD) scheme 30 was applied to shift the charges in proximity to the QM/MM bonds. Point charges of the MM region were included via electrostatic embedding. Simulations were executed with NAMD2 on the MM side with the new QM/MM interface (http://www.ks.uiuc.edu/Research/qmmm) to Gaussian 09 31 for QM calculations. The QM calculations employed the long-range corrected CAM-B3LYP functional 32 with the 631G(d) basis set. The CAM-B3LYP functional was chosen after comparing its performance for a single Lf to ab-initio ADC(2) (data not illustrated). The QM/MM simulation was run for 12ps. From the last 6ps of the QM/MM run, 250 random snapshots were extracted and for each snapshot, the vertical excitation spectrum was calculated with TD-DFT using CAM-B3LYP/6-31G(d) as implemented in Gaussian 09. The vertical excitation spectrum of every snapshot was convoluted with Gaussian functions 7



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of 0.16 eV FWHM (full width at half maximum) scaled by the respective oscillator strengths. All the individual spectra were added up to a single spectrum, normalized to the maximum peak height. The used equations can be found in the Supporting Information.

Quantum chemical calculations A structure from the MD trajectory with the abundant N5/O4 hydrogen bond pattern was extracted and truncated to a single Lf, five water molecules, the Mg2+ ion and the corresponding D41 and W36 residues. In the following, the system is referred to as LfW36. The structure taken from MD serves as a starting point for subsequent geometry optimization and exhibits the critical hydrogen bonds for a putative proton transfer. The Cα atoms of the amino acids were replaced by a methyl group to avoid collapse of the atoms in the follow-up calculations, i.e. all backbone atoms were removed, and Cα was saturated with 3 hydrogen atoms. This task has been achieved with the VMD molefacture plugin. The system was first optimized at the CAM-B3LYP/6-31G(d) 32 level of theory in Gaussian 09. The resulting atomic coordinates are presented in Figure S1. Vertical excitations of the system were calculated using ADC(2). 33,34 To lower the prohibitive amount of memory needed for this calculation, the virtual space was restricted by 30%, legitimate for description of π-π ∗ excitations. 35 Four excited states were calculated with restricted virtual space (RVS)-ADC(2)/cc-pVDZ using Q-Chem 4.3. 36 In these calculations, 30% of the energetically highest virtual orbitals were neglected. For benchmarking, TD-DFT excitation energies were calculated with CAM-B3LYP, B3LYP, BHHLYP and BLYP in Q-Chem 4.3 and with CAM-B3LYP in Gaussian 09. Excited state analyses were performed using the libwfa features of Q-Chem. 37,38 Densities, orbitals and molecular structures were rendered with VMD. For the investigation of the proton transfer reaction path, the system has been further simplified by cutting out the W36 residue. In order to obtain a precise reactant structure, the geometry of the excised system was optimized in Gaussian 09 31 with CAMB3LYP/6-31G(d) 32 in a singlet and doublet state with a charge of +1 and 0, respectively. For 8


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these systems, relaxed potential energy surface (PES) scans were computed along the reaction coordinate (r.c.) of a putative proton transfer reaction. For the proton transfer reaction, the electrostatic interactions, which are corrected by the CAM-B3LYP functional, largely dominate dispersion. Hence, the dispersion energy of the transferred proton to the reaction path is minor and we did not employ dispersion correction in the optimizations. The reaction coordinate has been defined as the difference between the distances of the transferred proton P to the target atom T dPT and the distance between P and the donor oxygen atom O dOP , so that r.c. = dOP − dPT . During a scan with 10 individual steps, the proton was moved from the donor to the target atom by constraining dPT and dOP . Because only the system with a doublet state where N5 was the proton acceptor yielded a favorable energy profile in the relaxed PES scans, its reaction profile was further investigated performing a transition state (TS) search. To find the TS, the synchronous transit-guided quasi-Newton (STQN) method in Gaussian 09 was used. 39,40 Further confirmation of the TS structure was achieved through vibrational frequency analysis. The precise energy profile of the reaction was explored by calculating the intrinsic reaction coordinate (IRC) as implemented in Gaussian 09. 41,42 For the reactant, transition state and product structures, 25 excited states were calculated with TD-DFT/CAM-B3LYP/6-31G(d) in Gaussian 09. The spectra were fitted as previously described.

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Results and Discussion To study the dynamics of the HsDod binding pocket, hydrogen bond networks and structural changes due to varying binding pocket compositions, MD simulations were carried out. An ensemble of three different systems (Tab. 1) was set up to tackle the following questions: How does the presence or absence of Mg2+ , a lewis acid, in the binding pocket affect the stability of the Lf binding and the hydrogen bond patterns? How is the orientation of the Lf ligands changed due to Mg2+ ? Can a single Lf molecule establish a stable conformation of the binding pocket? In addition, the excited electronic states of the protein-flavin system are investigated with high-level quantum chemical methods. We further reproduce the absorption spectrum of Lf bound to HsDod by means of QM/MM simulations. From the hydrogen bond network of the Lf ligand to surrounding solvent molecules, we spot putative water molecules as proton donors and perform potential energy surface scans and transition state searches to gain insight into the energetically most favorable proton transfer path. Finally, evidence of the proton transfer reaction path is given by comparison of the spectroscopic properties of the molecules involved in the excited state proton transfer to experimental results.

Structural dynamics of HsDod with and without Mg2+ In the HsDod binding pocket, a Mg2+ ion coordinates the amino acid D41 as well as five water molecules that form hydrogen bonds with the incorporated flavin species. 3 To identify the effect of Mg2+ on the stability of the binding pocket and flavin binding, MD simulations of HsDod with and without Mg2+ (system I and system II, Tab. 1) have been performed. The root-mean-square deviations (RMSD) of the protein backbone over simulation time are presented in Figure S2 for every system. As a matter of fact, clustering of the protein-Lf structures resulted in two marginally different ligand orientations for the holo-dodecin with Mg2+ (system I), which are very similar to the crystal structure (Fig. 2). On the contrary,

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holo-dodecin without Mg2+ (system II) resulted in two distinct clusters, where one corresponds to the crystal structure. However, in the second cluster the Lf dimer is rotated by 90◦ in the binding pocket (Fig. 2). Of note, Lf molecules rotate by 90◦ in the plane of the aromatic tetrad, breaking the C2-symmetry of the binding site. From a physiological point of view this rotation might not be feasible if flavin derivatives, such as FAD or FMN, possess an aliphatic ribityl chain. The holo-dodecin system showed both binding pocket and protein backbone stability throughout the simulation, and the key interactions of the crystal structure are also observed in the simulation. Considering the higher fluctuations of the Lf dimer in the Mg2+ -free system with respect to the native structure, the simulations clearly demonstrate a stabilizing effect of Mg2+ ions on the Lf dimer in HsDod. !"#$%&'()

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Figure 2: Structural changes in the binding pocket due to Mg2+ absence. Cluster analysis in the presence of Mg2+ reveals two similar Lf positions in the binding pocket (left). Both correspond to the native binding pattern in the protein. In the absence of Mg2+ , cluster analysis reveals two very different structures (right). While one of the structures (upper right) corresponds to the native positioning, in the second configuration (lower right) the Lf dimer is rotated by 90◦ .

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Hydrogen bond network of Lumiflavin in HsDod To further understand the observed structural fluctuations, we investigated the hydrogen bond pattern of the Lf molecules to solvent (water) molecules and the Q55 amino acid. As expected, the most abundant hydrogen bond pattern in system I from Lf to the protein is established between N3 and O2 and the terminal amide group of Q55 (Fig. 3). This interaction has already been described in the crystal structure, 1 and here it is corroborated that this double hydrogen bond is also stable over a long simulation time. In system II, when no Mg2+ is present, the native hydrogen bonds to Q55 still occur with high occupancy, however, the percentage is overall reduced by 34% and 45% for N3 and O2, respectively. Instead of the native hydrogen bonds, the rotated Lf dimer forms hydrogen bonds to Q55 with the atoms O4 and N1, as can be seen in the second cluster structure of system II (Fig. 2). Thus, the Lf dimer does not exit the binding pocket during the simulation, even if no Mg2+ was incorporated. This is consistent with experimental data, where a mutant of HsDod, incapable of Mg2+ -binding, did still stably bind flavin derivatives. 5 System III, incorporating only a single Lf molecule, also exhibits a decrease in hydrogen bond percentage to the protein due to higher flexibility of the binding pocket. The protein backbone around the binding pocket did not fully equilibrate in system III (data not illustrated). HsDod binds flavin derivatives with extremely high affinity, 6 which might explain the observed instability of an artificial, singly occupied binding pocket 43 in our MD simulation.

Hydrogen bond network of Lumiflavin to solvent water molecules Solvent molecules in proximity to the flavin species are key for the excited state quenching mechanism of HsDod as kinetic studies revealed an isotopic effect upon solvent deuteration. 5,7 Water molecules coordinating Mg2+ in the HsDod binding pocket have been suggested as possible proton donors upon photoexcitation. 5 In the MD simulation of HsDod (system I), five water molecules were consistently bound to both Mg2+ ions throughout the whole 12


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results, we conclude that presence of Mg2+ in the binding pocket stabilizes hydrogen bonds from water molecules to Lf. The constitutive presence of water molecules as proton sources makes an excited state proton transfer from solvent molecules robust. Because the Lf monomer in system III is generally more solvent-accessible than the Lf dimer in other systems (Tab. S1 in the Supporting Information), hydrogen bonds to solvent molecules are constantly present in our simulations. For N1, the percentage was 14% and 19% higher than for systems I and II, respectively, and photo-protection may still be functional also for singly occupied binding pockets, if the initial electron transfer step still works. Furthermore, a hydrogen bond from water molecules to the O2 atom of Lf is rather stable, as can also be seen in Figure 3. This atom is solvent-accessible and hydrogen bonding is not affected by absence or presence of Mg2+ . The O2 atom is already permanently involved in a hydrogen bond with the Q55 residue of HsDod. Most importantly, the O2- and N1-bound water molecules do not coordinate Mg2+ . In addition, the hydrogen bonds to N1 occur at low percentage. As Mg2+ is a Lewis acid, the hydroxide ion resulting from a proton transfer to Lf would be drastically stabilized. On the contrary, these hydroxide ions would reside in plain solvent when undergoing a proton transfer to either N1 or O2, making N1 and O2 unfavorable candidates for proton acceptors. On the contrary, the water molecules interacting with N5 or O4 are directly bound to Mg2+ . From the hydrogen bond pattern observed for holododecin, we thus suggest that either N5 or O4 act as proton acceptors upon photoexcitation. The properties of the underlying reaction paths are further investigated by QC calculations. Summarizing the systems under study in MD simulations, Mg2+ ions play a pivotal role in stabilizing the binding pocket geometry, both on the protein and the flavin site. The stabilization of the hydrogen bond to N5 and O4 also leads to flavin stabilization in the binding pocket, which is crucial for a robust quenching mechanism. However, absence of Mg2+ does not fully diminish these hydrogen bonds. The results of the hydrogen bond analysis hint strongly towards water molecules, stabilized by Mg2+ , to take the role as proton

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donors upon photoexcitation.

Role of charge-transfer excited states Charge-transfer (CT) excitations have proven difficult for TD-DFT when standard exchangecorrelation functionals with low amounts of Hartree-Fock exchange are employed. 44,45 As we expect low-lying charge-transfer excitations for the Lf-W36 system in the HsDod protein, as suggested by previous spectroscopic studies, 5,7 we employed both TD-DFT and ab-initio ADC(2) calculations with a double-ζ basis set as we benchmarked several DFT functionals against ADC(2) for excited states in isolated Lf-tryptophan systems (Tab. S2). In this benchmark, the CAM-B3LYP 32 functional outperformed e.g. B3LYP and BLYP. Table 2: RVS-ADC(2) Excitation energiesA # E [eV] E exp. [eV]B 1 2.57 2.79

λ [nm] ω 482 0.084

2

2.65

-

468

3

3.29

-

377

4

3.32

-

373

amplitudes HOMO → LUMO [40.2%] HOMO-2 → LUMO [36.2%] HOMO-1 → LUMO [3.6%] 0.092 HOMO → LUMO [35.6%] HOMO-2 → LUMO [39.2%] HOMO-1 → LUMO [4.2%] 0.002 HOMO-1 → LUMO [54.2%] HOMO-8 → LUMO [22.8%] HOMO → LUMO [6.2%] 0.001 HOMO-8 → LUMO [51%] HOMO-1 → LUMO [22.0%] HOMO-11 → LUMO [4.8%]

A

These calculations employed the cc-pVDZ basis set while discarding 30% of the highest energy virtual orbital space. B Experimental values are obtained from Staudt et al. 5 ADC(2) calculations are summarized in Table 2. The ADC(2) calculations with a restricted virtual space (RVS), in which the 30% highest virutal orbitals have been discarded, yielded a low-lying charge-transfer excitation (Tab. 2), as can be recognized from the attachment and detachment densities of the respective excitations (Fig. 4). The first two excited states calculated with ADC(2) show identical characteristics and belong to the same state, 15



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which is observable as the S0 → S1 transition in experiment 5 and thus most important for the photoprotection mechanism. TD-DFT calculations also yielded a CT excitation as lowest excited state in Lf-W36. The attachment and detachment densities thereof are depicted in Figure S3. From the excited state calculations, it is clear that an electron is transferred from W36 to Lf upon photoexcitation, yielding a flavosemiquinone radical. In the following, to investigate the fate of the generated flavosemiquinone radical, the system can be simplified by excluding W36 from the calculations and consider the flavosemiquinone radical only. The spin-density of the radical is located at the N5 atom of the flavin ring (data not illustrated) and further hints at N5 as most likely acceptor in a proton transfer reaction.

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!#

!$

!%

Figure 4: Detachment (blue) and attachment (red) densities of S1 to S4 of the Lf-W36 system calculated at RVS-ADC(2). The two lowest excitations reveal very similar attachment and detachment densities. As both of them show an electron density decrease on the W36 fragment of the system, they can be considered to be charge-transfer excitations. The attachment densities for these states exhibit a major increase in electron density at the N5 atom of the Lf fragment, subsequently leading to a flavine semiquinone anion radical upon photoexcitation.

Absorption spectrum of HsDod-bound Lumiflavin Hybrid quantum mechanical and molecular mechanical (QM/MM) simulations were employed to provide more accurate input structures for the subsequent simulation of the UV/VIS spectrum of Lf in the protein environment. As the adjacent W36 residue can

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obtained spectrum demonstrates how QM/MM simulations can be employed to easily study the influence of a protein environment onto the optical properties of a chromophore. Including point charges representing the environment explicitly through electrostatic embedding seems sufficient to compute a qualitatively correct spectrum.

Importance of different proton transfer channels From the hydrogen bond pattern around Lf in the holo-dodecin MD simulation, we concluded Mg2+ to stabilize water molecules bound to O4 and N5, which have frequently been suggested as proton acceptors in flavins. Therefore, the potential energy surfaces have been computed along the proton transfer reaction to both N5 and O4 for singlet and doublet multiplicity. The latter one corresponds to a flavosemiquinone radical created by the previously identified photo-initiated electron transfer from W36 to Lf. Relaxed surface scans have been performed to provide initial insight into the energetics of different putative proton transfer channels. The relaxed surface scans revealed only the N5 proton transfer path in an anionic doublet state to exhibit an energy profile with two minima and a maximum on the PES. All other PES strongly increase along the reaction coordinate (Fig. 6), and possess no proton-transfer minimum. These initial calculations of the potential proton transfer channels further emphasize a charge-separation upon photoexcitation leading to a doublet state of the Lf subsystem to be a prerequisite for proton transfer. For the singlet state (Fig. 6), proton transfer is energetically not feasible. Since for both doublet and singlet multiplicity states of Lf, when the proton is transferred to O4, the PES steeply increases by more than 60 kJ/mol, it can further be recognized that O4 is not a possible target atom for the proton transfer. However, this computational model is not sufficient to fully characterize the reaction path of the proton transfer upon photoexcitation.

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Figure 6: Relaxed surface scans for putative proton acceptors and redox states. The potential energy surfaces for the presented proton transfer reactions unravel differences in energetics for the respective proton acceptors and redox states. Interestingly, all systems showed a steep increase in energy with the reaction coordinate, except for the N5 system in doublet state, where a rather flat surface with two minima was obtained.

Energetic and electronic properties of the N5 proton transfer reaction To further investigate the proton transfer pathway to N5, transition state searches and IRC calculations have been performed in the doublet multiplicity state (Fig. 7), i.e. for the flavosemiquinone radical anion. Thereby, a transition state for the proton transfer reaction with an energy barrier of ∆E ‡ ≈ 9 kJ/mol (activation energy) was found. This is an extremely flat surface with a small barrier and a proton-transferred minimum only 8 kJ/mol above the reactant (reaction energy). Upon photoexcitation and subsequent electron transfer, sufficient excess energy is certainly available to make the proton transfer feasible. The energy differences are, however, so small that they may very well change slightly when higher level of theory or improved environment models are employed. However, based on the previously executed scans for different target atoms and multiplicity states, it is safe

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to assume that the identified proton transfer pathway corresponds to the one observed in experiment. Due to the shallow proton-transferred minimum, this species is only transient and will quickly undergo back-transfer. When the structure of the proton-transferred minimum of the system with W36 included is optimized in the S1 state, while the relative orientation of Lf and W36 is constrained, the excitation energy becomes very small. Most likely, a conical intersection between S0 and S1 is present, which however can generally not be unambiguously identified using DFTbased methods, and multi-reference methods are required. Nevertheless, since the observed geometric changes are small, the presence of a conical intersection mediating the efficient excited-state deactivation of the proton-transferred Lf-W36 complex is very likely. It has been previously shown that a local minimum along of a path between two potential energy surfaces is usually the shoulder of a conical intersection. 46 Thus, we assume that this is also the case here, yielding a plausible deactivation channel of the excited state. Once the electronic ground state is reached, thermal proton and electron back-transfer restores the initial state, and Lf is back to the electronic ground state without degradation. To further corroborate this pathway, the changes in the absorption spectrum along the path have been computed. Indeed, in the computed absorption spectra of the reactant, transition state and product, clear differences are visible (Fig. 7), which should be monitored during the proton transfer. Intriguingly, the peak with lowest energy in the absorption spectrum is red-shifted following the reaction path, with a decrease in absorption at 440 nm and an increase in absorption at 499 nm. This spectroscopic signature has previously also been observed in time-resolved experiments, 5 and strongly supports our findings. Together with the excited-state analysis of the charge-transfer excitation and the molecular species involved in the subsequent proton transfer reaction, a consistent computational model of the photoprotection mechanism of Lf by HsDod has been established. We have shown proton transfer to N5 upon photoexcitation to be favored among other imaginable proton transfer pathways.

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Conclusion HsDod is an important protein involved in archaeal flavin homeostasis, since it stores flavins and protects them from photodegradation by ultrafast excited state quenching. The presented study of HsDod is an example of how structural biology, spectroscopy and multi-level theoretical methods can be combined in order to understand photochemical properties and mechanisms of biomolecules. The excited state quenching mechanism has been illuminated from multiple perspectives, starting at the MD level, where key hydrogen bonds were elucidated to establish the starting point for follow-up quantum chemical calculations. Furthermore, MD studies revealed the impact of Mg2+ in the binding pocket, which is found to be beneficial for a robust quenching mechanism by stabilizing hydrogen bonds of solvent molecules to the flavin species. Energetics of putative proton transfer reaction pathways were explored on the level of quantum chemistry, and one pathway in agreement with experimental data was found most favorable among all other pathways. Studying the photoexcitation initiating the proton transfer, CT excitations transferring an electron from the adjacent tryptophan residue to the flavin molecule have been identified as lowest excited electronic states, generating a flavosemiquinone radical. High-level excited state calculations using the algebraic diagrammatic construction scheme of the polarization propagator emphasize the importance to correctly account for CT excitations in biophotochemical systems. Computation of the energy profile of the proton transfer to the N5 atom of the flavosemiquinone radical anion showed a low energy barrier, demonstrating the feasibility of this reaction. Computed absorption spectra along this reaction path nicely agree with experimental time-resolved spectra. Based on our findings, we propose the first comprehensive mechanism of active flavin photoprotection by archaeal dodecin, summarized in Figure 8. In brief, the CT excitation leads to Mg2+ -promoted proton transfer, and subsequent excited-state deactivation through a conical intersection is likely to occur. Electron and proton back-transfer to the electronic ground state restore the initial situation. Thereby flavins are protected from photodegrada22


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Figure 8: Schematic illustration of the identified photoprotection mechanism. The initial charge-transfer excitation leads to charge separation between Lf and Trp. Subsequent ultrafast proton transfer to the N5 atom of Lf provides a channel for deactivation, most likely through a conical intersection. Thus, the ground state is reached and Lf is still intact. tion. In the future, computational investigations on several W36 mutants with different redox potentials are necessary to theoretically unravel dodecin photocycle manipulation as well. 7 With our findings, we hope to trigger follow-up experiments on this fascinating protein and its intriguing photochemical properties.

Acknowledgement AD and DBK gratefully acknowledge financial support by the German Research Foundation through grant DFG no. DR 428/9-1. The authors also acknowledge support by the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant no. INST 40/467-1 FUGG and INST 35/1134-1 FUGG. The authors thank Dr. Tim Stauch for fruitful discussions.

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Supporting Information Available The following file is available free of charge. • Supporting_Information.pdf : Supporting figures and tables, atomic coordinates for excited state calculations. • Lumiflavin_Parameters.txt : CHARMM force field parameters for lumiflavin, as obtained from SwissParam.

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Molecular Mechanism of Flavin Photoprotection by Archaeal Dodecin

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