Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic

Article pubs.acs.org/JPCB

Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic Reappraisal Anikó Udvarhelyi and Tatiana Domratcheva* Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstraße 29, 69120 Heidelberg, Germany S Supporting Information *

ABSTRACT: The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogenbond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX. Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light state.

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flavin photoreaction in BLUF, indicating that after the photoreaction flavin remains in its oxidized state and that it forms an enhanced hydrogen bond involving its C4O4 carbonyl group4,8,9 with the conserved Q63 residue10−14 (we use the residue numbering of AppA BLUF). Because the Q63 side chain directly interacts with the electron donor residue Y21,10,13 the photoinduced electron transfer from Y21 to the excited flavin results in changes of the Q63 side chain. X-ray protein crystallography plays a key role in characterizing the hydrogen bonding network in the flavin-binding pocket of BLUF. Two different crystal structures of the AppA BLUF domain were independently presented that differ in the rotamer conformation of the Q63 side chain as well as in the fold of the β5 strand and the position of the W104 residue with respect to flavin15,16 (Figure 1A). In the crystal structure by Anderson et al. (pdb 1YRX) the side chain of W104 points into the flavin-binding pocket (dubbed Win structure),15 whereas in the crystal structure by Jung et al. (pdb 2IYG) W104 is solventexposed and M106 replaces W104 in the flavin pocket (dubbed Wout structure).16 In the following, we refer to the Q63 rotamer in the Win and Wout structures by Q63A and Q63J, respectively. Apart from the W104 position, the arrangement of the Y21, Q63, and N45 conserved residues with respect to the flavin chromophore is virtually identical according to the respective

INTRODUCTION Photoreceptor proteins detect light using photosensitive chromophore molecules. Upon photon absorption, the chromophore undergoes a photoreaction, which initiates the signaling cascade in the cell. The photoreaction alters the chromophore−protein interactions that in turn modify the conformation of the protein. Such a conformational transition is referred to as a switch from the dark state to the light state of the photoreceptor. To ensure light sensitivity, the photoreaction must have a high energy barrier in the electronic ground state. Consistent with this requirement, visual photoreception for example relies on the high activation energy of the cis to trans isomerization around a double CC bond in retinal. Another recently discovered photoactivation mechanism in blue-light photoreceptors implies photoinduced intermolecular electron transfer to an oxidized flavin chromophore. Three families of flavin-binding photoreceptors, the LOV, BLUF,1 and cryptochromes, have been identified so far. The specific photoreaction of each family depends on the chemical environment of the noncovalently bound flavin. In every case, light absorption leads to flavin accepting an electron from a nearby redox active amino acid residue. The BLUF domain was first described as a light-sensory unit of the AppA protein involved in the light-dependent regulation of photosynthetic gene expression in Rhodobacter sphaeroides.2,3 Since then several other BLUF-containing proteins have been characterized. A 15-nm red-shifted UV/vis3−7 and a 20-cm−1 down-shifted IR absorption4 in the light state characterize the © 2013 American Chemical Society

Received: January 14, 2013 Revised: February 15, 2013 Published: February 19, 2013 2888

dx.doi.org/10.1021/jp400437x | J. Phys. Chem. B 2013, 117, 2888−2897

The Journal of Physical Chemistry B

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The assignment of the two X-ray structures to the functional states of BLUF led to a photoreaction hypothesis that explains the light-induced hydrogen bond switch by glutamine rotation.10,11,16 It is further assumed that glutamine rotation subsequently leads to a conformational switch of the β5 strand (W104−M106 switch).16,22,23 However, there is no direct experimental evidence that glutamine rotation would indeed be coupled to the W104−M106 conformational switch. Thus, how the hydrogen bond switch leads to BLUF signaling is still unclear. The properties of the hydrogen bonding networks in the dark and light states are being constantly unraveled. Studies by several groups indicated that the spectral red shifts observed upon light activation are signatures of stronger intermolecular interactions in the less dynamic light state.18,24−27 Mathes et al. showed that both the dark and light states undergo electron transfer coupled to proton transfer upon photoexcitation, yet with distinct photodynamics:18 The photosensitive dark state switches to the light state,10 whereas the photostable light state recovers after photoexcitation.24 However, the light state thermally decays to the dark state within times spanning three decades, depending on the BLUF domain sequence.4−7,28 In another recent study, Mathes et al. demonstrated that the dynamics of both photoactivation and the dark state recovery are linked to the redox potential of BLUF,29 which the authors interpreted according to the glutamine rotation mechanism. However, several inconsistencies in the Win structure containing Q63A were pointed out30−32 that question the glutamine rotation mechanism. The major issue concerns the 2.7 Å distance between the flavin carbonyl O4 and the glutamine carbonyl Oε1 atoms being clearly too short to represent an equilibrium distance between two carbonyl oxygen atoms. The corresponding distance in the Wout structure is 2.8 Å, where it describes a hydrogen bond between the flavin O4 and glutamine Nε2 atoms. To explain the short distance in the Win crystal structure, computational studies suggested a tautomeric glutamine30,32 and proposed light-induced glutamine tautomerization as the photoactivation mechanism in BLUF.30,33 To model the crystallographic Q63A rotamer in the Win structure with the short oxygen−oxygen distance as the dark state, other computational studies19,21 introduced restraints. However, the physical meaning of these restraints stabilizing the Q63A rotamer is unclear. Another important question is why most crystal structures of BLUF proteins adapt the Wout conformation,34−37 which according to the lightinduced glutamine rotation hypothesis represents the light state. Furthermore, the hydrogen bonding network postulated by the Win structure (Figure 1C) is inconsistent with the IR study of Takahashi et al. that established Y21 to be a hydrogenbond donor in both the dark and light states.38 In the most recent IR study, Iwata et al. found that the hydrogen bond between Y21 and Q63 in the light state is unusually strong:39 The Y21 OH stretching frequency is 400−600 cm−1 lower than the typical frequency of a hydrogen bond between a phenolic OH and a carbonyl group.39 This finding is inconsistent with the glutamine rotation mechanism that proposes the Wout structure as the light state where the Y21 hydroxyl group hydrogen bonds to the Q63J carbonyl. All these observations suggest that the Q63A rotamer in the Win structure cannot represent the dark and the Q63J rotamer in the Wout structure cannot represent the light state. Finally it is questionable, from the theoretical viewpoint, whether the energy barrier of

Figure 1. Comparison of the AppA-BLUF domain structures, Win pdb structure 1YRX (yellow) and Wout pdb structure 2IYG (gray), both chain A. (A) Superposition of the backbone of the two structures in cartoon representation and the side chains of conserved residues around flavin in ball and stick representation. (B) The hydrogen bonding network of the flavin-binding pocket with key distances indicated in Å. The upper and lower values correspond to the 1YRX and 2IYG structures, respectively. The 2IYG experimental electron density contoured at two sigma overlays the two structures. (C) The two hydrogen bonding networks of the two glutamine rotamers Q63A (Win) and Q63J (Wout).

electron densities (Figure 1B). However, the differently assigned glutamine rotamers define two distinct hydrogen bonding networks in the two pdb structures (Figure 1C). The decisive difference is that the Q63J rotamer forms a hydrogen bond to the flavin C4O4 carbonyl, whereas the Q63A rotamer does not. This hydrogen bond could explain the redshifted flavin absorption spectrum in the light state,11 therefore most experimental10−14,17,18 and computational19−21 studies consider the Win structure with Q63A as the dark state and the Wout structure with Q63J as the light state model of BLUF. 2889



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Figure 2. Supermolecular cluster models of BLUF. (A) Win AppA model based on the pdb structure 1YRX, chain A. (B) Win PixD model based on the pdb structure 2HFN, chain D. The black-colored atoms (all Cα and four flavin atoms) were constrained during geometry optimization.

coordinates 1YRX chain A, includes lumiflavin, the residues Y21, Q63, N45, L65, W104, and A46, as well as the backbone atoms of residues C20, R22, F62, W64, E66, H105, G103, and H44; in total 184 atoms (Figure 2A). To account for the ambiguous assignment of the hydrogen atom positions in the crystal structures, we assumed three forms of the glutamine side chain: the two rotamers Q63A and Q63J as well as the tautomeric form, Q63tau. We also considered the Q63A rotamer in the Win molecule of the PixD BLUF protein because this structure was determined with the highest resolution among the BLUF X-ray structures.37 The PixD model, built based on the pdb coordinates 2HFN chain D, consists of lumiflavin, residues Y11, Q53, V54, L55, N35, P36, A37, N38, and W94, as well as the backbone atoms of residues I10, S12, L52, E56, G39, N34, S95, and V93 and the water molecule WAT1005 (PixD numbering); in total 225 atoms (Figure 2B). In the PixD model we only considered the crystallographic Q53A rotamer. The HyperChem program42 was used to add the hydrogen atoms and to optimize their coordinates in the starting geometries with the Amber force field, keeping the pdb coordinates of the heavy atoms fixed. In the following description, we use the standard pdb names for amino acid atoms. Geometry Optimization. To characterize the hydrogen bonding network in BLUF, the geometries of the prepared starting models were optimized with B3LYP/6-31G**. To mimic the protein scaffold, the Cartesian coordinates of the Cα carbon atoms, the water oxygen atom in the PixD model, and the flavin carbon atoms C6, C8, C1′, and C10a were always constrained to their crystallographic values (black-colored atoms in Figure 2). We used a three-step optimization procedure to conserve the crystallographic distance between the glutamine Oε1/Nε2 and flavin O4 atoms as much as possible. First, starting from the X-ray coordinates, the geometry was optimized with constrained pdb coordinates of the glutamine Oε1/Nε2 and flavin O4 atoms to conserve the glutamine−flavin interactions postulated by the pdb structures (opt1). Then, the optimization was continued with the released glutamine and the constrained pdb coordinates of the flavin O4 atom (opt2). Finally, the geometry optimization was completed without the additional flavin−glutamine constraints (opt3). The geometries were optimized to an energy gradient of 0.0001 hartree/bohr in all three steps. We regard the finally obtained models (opt3) as models for the flavin hydrogen bonding

glutamine rotation in the BLUF pocket is high enough to ensure photosensitivity. To resolve the inconsistencies listed above, here we propose three criteria for physical models of BLUF. First, both the dark and light state models must contain an atom arrangement that represents equilibrated molecular forces and corresponds to minimum-energy structures. After optimization of the respective molecular models, at least the dark-state model must be consistent with the experimental X-ray electron density. Second, the light-state model must reproduce the 15-nm redshifted flavin absorption compared to the dark state. Third, the transition from the dark to the light state must have a high energy barrier in the electronic ground state to ensure light sensitivity. All criteria must be satisfied simultaneously. In particular, it is not sufficient for physically meaningful models of BLUF to only reproduce the spectral red shifts. Here, using these three criteria, we re-examine the crystallographic assignment of the Q63 side chain rotamer in the Win structure and reappraise the Q63A and Q63J models as proposed dark and light states, complementing previous computational studies addressing the Q63 orientation and tautomeric forms in both the Win and Wout structures.32,40,41 We investigate the Win structure since its Q63 rotamer assignment is questionable, whereas in the case of the Wout structure a consensus exists that it contains the Q63J rotamer. In fact, also the Win structure might contain the Q63J rotamer. Therefore, here we focus on the Win structure and compare our models not only to the pdb structures but also directly to the experimental X-ray electron density. Clarifying the Q63 rotamer assignment in the Win structure is of highest importance since it directly influences the interpretation of all spectroscopy data. First the glutamine orientation in the dark state and the light-induced changes of the glutamine side chain during the photocycle have to be clarified before one tries to explain BLUF signaling because these changes trigger subsequent conformational changes in the protein.

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COMPUTATIONAL DETAILS Supermolecular Cluster Models. We use supermolecular cluster models for a complete quantum-mechanical description of the hydrogen bonding network around flavin and a substantial part of the β sheet (parts of β1, β3, and β5 strands). The AppA BLUF model, built based on the pdb 2890



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network in the Win BLUF conformation. Heavy atom RMSD values with respect to the crystallographic coordinates along the three-step optimization procedure were computed with a custom-written perl script. We compare the optimized geometries with the experimental electron densities computed with the ARP/wARP program43 using the structure factors of the respective pdb models by performing 25 cycles of “map improvement by atoms update and refinement”. Q63 Dihedral-Angle Energy Scans. To estimate the energy barriers between different glutamine rotamers, we computed relaxed energy scans along the glutamine and glutamine−flavin dihedral angle coordinates. As the scans were computed not in Cartesian but in intrinsic coordinates, protein scaffold constraints were applied as constraints of distances between all pairs of the atoms constrained in the previous geometry optimization procedure (black-colored atoms in Figure 2). To produce scans (every 10° of the chosen dihedral angle), geometry optimization was carried out until an energy gradient of 0.0003 hartree/bohr with a fixed value of the dihedral angle. Complete 360° scans in both clockwise and counter clockwise directions were obtained and analyzed. The energy scans were computed with the B3LYP/631G** method. Excitation Spectra. At the optimized structures, the excitation spectrum was computed with the TD-B3LYP/ccpVDZ method for the ten lowest lying excited states. The HOMO−LUMO transition of the flavin is of interest in this work as that corresponds to the absorption maximum around 450 nm (2.76 eV). Among the ten computed states, the state corresponding to the HOMO−LUMO flavin excitation was identified according to the contributions of the respective molecular orbitals. This state is the lowest energy excited state characterized by a considerable oscillator strength. We consider the excitation energy of this state as a measure for the position of the flavin absorbance maximum consistent with previous studies on blue light photoreceptors.33,44 We used the Firefly program45 which is partially based on the GAMESS US source code46 for all quantum chemical calculations.

Table 1. Four Selected Distances in Å Involving Glutamine Heteroatoms of the Optimized AppA Models, Along the Three-Step Optimization Procedure: opt1 (Constrained Glutamine Oε1 or Nε2 to Flavin O4 Distance), opt2 (Constrained Flavin O4 Atom), and opt3 (Fully Optimized without Flavin-Q63 Constraints) Q63A model

opt1

opt2

opt3

Y21-OH to Q63-Nε2 Q63-Nε2 to fl-N5 Q63-Oε1 to fl-O4 Q63-Oε1 to W104-Nε1 Q63J model

3.0 3.2 2.7 3.1 opt1

3.1 3.0 3.7 2.8 opt2

3.1 3.0 3.6 2.8 opt3

Y21-OH to Q63-Oε1 Q63-Oε1 to fl-N5 Q63-Nε2 to fl-O4 Q63-Nε2 to W104-Nε1 Q63tau model

2.8 3.1 2.7 3.4 opt1

2.8 3.1 2.8 3.5 opt2

2.8 3.1 2.8 3.4 opt3

2.8 3.1 2.8 2.9

2.8 3.1 2.7 2.9

Y21-OH to Q63-Nε2 Q63-Nε2 to fl-N5 Q63-Oε1 to fl-O4 Q63-Oε1 to W104-Nε1

position of the two oxygen atoms must be constrained. As a consequence of the distance constraint, the energy of opt1Q63A is 9.5 kcal/mol higher than that of opt3-Q63A (Figure 4E), demonstrating the unphysical oxygen−oxygen interactions. In contrast, the energy of the opt1-Q63J model is almost identical to the opt3-Q63J model, indicating that the intermolecular forces between Q63 and flavin are equilibrated in the starting structure. The same is true for Q63tau. Thus, in the fully optimized opt3 models, the Q63A rotamer is incompatible with the experimental electron density in contrast to the Q63J and Q63tau models. Therefore, either the Q63J or Q63tau form but not the Q63A rotamer should be used to model the X-ray density of the Win structure. We conclude that Q63A does not satisfy our first criterion, but Q63J and Q63tau do. With the Q63J rotamer, the crystallographic Win and Wout hydrogen bonding networks would be the same (differing only in the W104 position), and with the Q63tau form they would differ because of the chemically distinct Q63tau. We emphasize that in either case the glutamine rotation mechanism cannot be proposed on the basis of the BLUF X-ray structures. We computed the flavin excitation energy of the AppA models with and without constraints to estimate the flavin absorption maximum (Figure 4E). In the case of the constrained opt1-models, we find that the excitation energy in the Q63A model is significantly blue-shifted with respect to the Q63J model. After releasing the distance constraint (opt2), the difference in the predicted absorption almost vanishes. In the final opt3 models, we obtain a small difference of 0.03 eV (4.5 nm). These calculations demonstrate that there is a caveat when working with constrained or restrained models since constraints/restraints influence the absorption maximum and may introduce artifacts. Our physical criteria related to the protein photoactivation function help to minimize artifacts of computational models. According to the first criterion, only the fully optimized opt3 Q63A, Q63J, and Q63tau models correspond to equilibrated BLUF models, and only opt3Q63J and opt3-Q63tau consistent with the electron density can represent the dark state (Figure 4). The flavin absorption energy in the opt3-Q63tau model (Figure 4C) is 2.75 eV and 14 nm red-shifted with respect to that of the Q63J model (Figure

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RESULTS AND DISCUSSION Q63 Rotamer in the Win Structure. The conservative protocol of our three-step optimization procedure assures to find the local minimum (the final opt3 models) closest to the initial AppA and PixD Win pdb structures. In the opt3-Q63J and opt3-Q63tau models we find the glutamine side chain close to its crystallographic position, whereas in the opt3-Q63A model both Y21 and Q63 move away from the crystal coordinates (Table 1 and Figures 3 and 4A−C). During geometry optimization, the largest geometry changes occur between the opt1-Q63A and opt2-Q63A models. The final heavy-atom RMSD values of the fully optimized Q63J, Q63tau, and Q63A models are 0.32, 0.29, and 0.53 Å, respectively (Figure 3). The Q63 carbonyl oxygen and Y21 hydroxyl oxygen atoms in Q63A undergo the largest displacements. The distance between the glutamine Oε1 and flavin O4 atoms increases from 2.7 Å in the pdb structure to 3.7 Å in the optimized AppA model (Figure 4A) and from 2.7 to 4.1 Å in the PixD model (Figure 4D). The Q63A side chain no longer stays parallel to flavin but is rather perpendicular and rotates out of the experimental electron density (Figures 4A and 4D). To keep the Q63A rotamer in the experimental density, i.e., to preserve the unphysical oxygen−oxygen distance that is inconsistent with interactions of two carbonyl groups, the 2891



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models should describe all the important steric and electrostatic interactions in the vicinity of Q63. Test calculations with even larger cluster models including also parts of the β2 strand and of the α helices resulted in very similar energy minima. Second, the important geometry changes pertain to Y21 and Q63 because of local interactions and not because of the extended environment. A comparison to the QM/MM study by Hsiao et al.21 shows that in the Win structure the QM/MM-optimized glutamine side chain of both the Q63A and Q63J rotamers shifts in an identical way21 as in our cluster models (Figure 3). Hsiao et al. reported “quantum-refined” AppA BLUF crystal structures obtained by QM/MM calculations with crystallographic restraints and compared these to fully optimized QM/ MM models.21 They considered models with both the Q63A and Q63J rotamers and computed their excitation energies. The model with the Q63A rotamer was found to have a blue-shifted flavin absorption with respect to the 180° rotated glutamine rotamer, Q63J. As a consequence, Hsiao et al. assigned the Q63A and Q63J rotamers to the dark and light states, respectively, thereby supporting the glutamine rotation as the light-activation mechanism of BLUF. To find the origin of the apparent contradiction in their and our computational work, here we analyze the QM part of three of their QM/MM models according to our three criteria of the BLUF dark and light state models. The three models are (i) the “quantum-refined” 1YRXref model (comparable to our opt1-Q63A models); (ii) 1YRXQM/MM structure without restraints (equivalent to our opt3-Q63A model), and (iii) the 1YRXQflip structure with the Q63J rotamer (equivalent to our opt3-Q63J model). The superposition of the QM part of the QM/MMoptimized models by Hsiao et al. with the 1YRX electron density (Figure 5) shows a picture fully consistent with the results of our cluster models: The Q63A rotamer is only compatible with the electron density when the geometry is restrained but not when its geometry is fully equilibrated. The energy difference between the restrained 1YRXref and fully equilibrated 1YRXQM/MM models is 15 kcal/mol because of the restraints, which is even higher than in the case of our cluster models. The distance after the unrestrained QM/MM optimization increases to 3.6 Å (because of the repulsion between the two carbonyl oxygen atoms), which is very similar to the geometry change in our cluster model. This repulsion by no means represents a “weak hydrogen bond” as Hsiao et al. referred to it.21 Just as in the case of our cluster models, the Q63A side chain undergoes the most significant geometry change upon relaxation in contrast to its 180° rotated counterpart, Q63 J. The latter is compatible with the experimental electron density (Figure 5C). Unfortunately, Hsiao et al. did not perform constrained optimization (“quantum-refinement”) of the 1YRXQflip model containing Q63J to further confirm the incorrect assignment of the Q63A orientation in the 1YRX crystal structure. Hsiao et al. reported the major structural rearrangement of Q63A; nonetheless, on the basis of the small overall RMSD values of 0.3 Å (and the blue-shifted flavin absorption) they assigned the restrained optimized 1YRXref model with Q63A to the BLUF dark state.21 However, overall RMSD values are not suitable to judge the agreement of a specific side chain conformation with the experimental electron density. Instead, atom-specific displacements should be looked at (Figure 3), from which it is apparent that in both our cluster and the QM/ MM models21 Q63A deviates significantly from the starting pdb structure in contrast to the Q63J rotamer. Most importantly,

Figure 3. Heavy atom RMSD values and atom-specific displacements of the AppA cluster models Q63A (black), Q63J (red), and Q63tau (blue) as a function of the geometry optimization step, determined with respect to the starting 1YRX crystal structure. The colored background rectangles in the RMSD plot indicate the three-step optimization procedure of the corresponding models: gray, Q63A model (opt1 and opt3); light red, Q63J (opt1 and opt3); and light blue, Q63tau (opt1 and opt3). As a comparison, we also indicate the corresponding RMSDs and displacement values for the comparable models by Hsiao et al.:21 1YRXQM/MM corresponds to the QM/MMoptimized model with the Q63A rotamer (our opt3-Q63A model), 1YRXQflip to the 180° rotated glutamine (our opt3-Q63J model), and 1YRXref to the “quantum-refined” model (our opt1-Q63A).

4B). Thus, Q63tau reproduces the absorption properties of the light state. Supermolecular Cluster versus QM/MM Models. An important question is how well our cluster models can represent BLUF states since parts of the protein and the solvent are missing from our model. First, our rather large 2892



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Figure 4. (A−C) Part of the DFT/B3LYP/6-31G**-optimized Win AppA-BLUF cluster models overlaid with the experimental X-ray electron density of the pdb structure 1YRX, chain A, contoured at two sigma. The full cluster models are shown in Figure 2. (D) Part of the Win PixD-BLUF cluster model overlaid with the experimental X-ray electron density of the pdb structure 2HFN, chain D, contoured at three sigma. (E) Relative B3LYP/cc-pVDZ ground state energies (black empty squares) of the AppA-BLUF models optimized with and without constraints computed with respect to the energy of the fully optimized opt3-Q63J model, and TD-B3LYP/cc-pVDZ flavin excitation energies (blue filled squares). The red arrow indicates the high energy of the crystallographic Q63A rotamer.

Figure 5. QM part of the QM/MM-optimized geometries by Hsiao et al.21 superposed with the X-ray electron density of the pdb structure 1YRX, contoured at two sigma. Selected interatomic distances are given in Å. The relative energies with respect to the 1YRXQflip model are indicated in kcal/mol. The TD-B3LYP/cc-pVDZ excitation energies in eV define the position of the flavin absorption maximum.

the Q63A-Oε1 atom displacement in 1YRXref is larger than the displacement of the Nε2 atom at the same position in 1YRXQflip (Q63J model), thus clearly demonstrating that Q63J is preferred over Q63A. An important finding of Hsiao et al. was the blue-shifted absorption maximum of the 1YRXQM/MM models containing the Q63A rotamer. Here we computed the TD-B3LYP/cc-pVDZ flavin excitation energies for the QM part of their models to compare them to our results. Our computed values (Figure 5) are systematically blue-shifted by 0.16 eV as compared to the energies reported by Hsiao et al., computed with the DFT/ MRCI/TZVP method.21 The flavin absorption in the 1YRXref

structure with crystal restraints is blue-shifted compared to the respective fully optimized 1YRXQM/MM model. We also reproduced the blue shift between the 1YRXQM/MM and 1YRXflip structures. In our respective cluster models, this difference is smaller (Figure 4E) because of the slightly blueshifted flavin absorption in our Q63J model. As we found in our dihedral angle scans (see below), Q63 rotation occurs with a small activation energy; hence, both rotamer conformations of Q63 may exist in the BLUF dark state, and thus their excitation energies average. Unfortunately, Hsiao et al. did not consider a tautomeric form of Q63. They noted, however, that the bond distances in 2893



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Figure 6. (A), (D), and (G) DFT/B3LYP/6-31G** energy scans for the Q63 dihedral angles and the Q63-flavin dihedral angle, starting from the Q63A model (minimum I) or the Q63J model (minimum V) in clockwise (cw) and counter-clockwise (ccw) directions in 10° steps. The black lines represent the dihedral angle potential energy that connects respective minima. The TD-B3LYP/cc-pVDZ excitation energies for the minimumenergy structures are indicated in eV. (B) and (C) The structure of the flavin binding pocket at the energy minimum II and of molecule 8 of the NMR ensemble (pdb 2BUN), respectively. (E), (F), (H), and (J) The structure of the flavin binding pocket at the indicated energy minima.

the glutamine side chain in all of their models were typical for amids and incompatible with an imid form of glutamine, thus arguing against the latter.21 However, this result is an obvious consequence of their choice to add hydrogen atoms assuming only the amid glutamine because geometry optimization always results in an energy-minimum structure consistent with the selected protonation pattern. Therefore, the results of Hsiao et al. cannot be used to evaluate the tautomeric form of Q63 in BLUF. Dynamic Aspects: Glutamine Rotation in the Dark State. The energy difference between the fully optimized Q63J and Q63A models is negligible (Figure 4E). To estimate the energy barriers of glutamine rotation, we computed energy scans by changing the glutamine Cβ−Cγ−Cδ−Oε1 dihedral angle to rotate the amide group and also by changing the glutamine CδOε1 flavin C4O4 dihedral, to control hydrogen-bonded interactions of flavin and glutamine. We

found several minima of different Q63 rotamers that are close in energy and separated by low-energy barriers (Figure 6). The Q63 dihedral angle scan starting from the opt3-Q63A model (minimum I) in both the clockwise (cw) and counterclockwise (ccw) directions revealed two other minima lower in energy than Q63A, separated by low-energy barriers (Figure 6A−C). The ccw scan starting from the opt3-Q63J model also revealed two more minima, but with higher energy (Figure 6D−F). In this case we cannot use the full 360° scans to analyze the Q63 rotamers in BLUF as we run into a model artifact: In the cw direction after 7 steps we found a minimum where W104 forms a hydrogen bond with the backbone carbonyl of W64. Along the ccw scan, a similar hydrogen bond is formed after 18 steps. Since the hydrogen bond between W104 and the backbone carbonyl most likely cannot form in BLUF (β2 is missing in our model), we exclude the respective minima from our analysis and only show minima relevant for BLUF. 2894



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those around the C2O2 carbonyl group of flavin) and a comparative study of various BLUF proteins would be of high interest. Furthermore, most BLUFs are part of a multidomain protein, and protein−protein interactions most likely influence the conformational dynamics of BLUF.

The glutamine CδOε1 flavin C4O4 dihedral angle scan starting from the opt3-Q63A model revealed a low-energy barrier between the Q63A and Q63J rotamers (Figure 6G−J). In this case again, we can only use parts of the scan because of the W104−backbone hydrogen bond formation. However, the part of the energy scan meaningful for BLUF unveils a lowenergy path between the Q63A and Q63J (minimum IV) rotamers, with almost equal energies. Minimum IV differs from the opt3-Q63J model by the orientation of the Y21 hydroxyl group because we started the scan from the opt3-Q63A model. The orientation of the Y21 hydroxyl group with respect to Q63 represents another degree of freedom that could be included in future studies. In fact, when changing a given dihedral angle, all the dihedral angles of glutamine and not only those of the headgroup change, indicating that there are many important degrees of freedom to consider. We computed the flavin excitation energies at the identified local minima (Figure 6) and found differences of 0.02−0.04 eV (3−6 nm). Spectral differences of this order for minima of close energies average out when protein dynamics are taken into account. All of these minima together should be regarded as the dark state model of BLUF. This picture is consistent with the results of the MD study by Khrenova et al. that identified four conformational substates with different Q63 rotamers in the BLUF Wout structure with the flavin excitation energy varying by 0.03 eV.32 In contrast, the excitation energy of the opt3Q63tau model is significantly red-shifted by 0.09−0.12 eV compared to the amid forms. The energy barrier of glutamine tautomerization is certainly much higher than the energy of thermal fluctuations because it requires the rearrangement of covalent bonds. In contrast to the rotation energy barrier, the tautomerization energy barrier ensures photosensitivity. Rotations of both the amid Q63 and imid Q63tau side chains are implicated in protein dynamics of the dark and light states, respectively. Rotation of the imid Q63tau has been a subject to other computational studies.32,33,41 The results of our calculations suggest a rather dynamic flavin-binding pocket in the dark state with several glutamine rotamers present. This finding is in agreement with the NMR study of the AppA-BLUF domain that revealed intermediate exchange broadening of the Q63 signals and suggested several Q63 rotamers.47 However, none of the 20 NMR structures of the Win conformation contain the crystallographic Q63A rotamer. The hydrogen bonding network around flavin in all NMR structures is distinct from the Win crystal structure. The Y21 OH group points toward Q63 in all NMR molecules and not away from it, as it is assumed in the crystal structure. Therefore, the NMR solution structure cannot support the crystallographic Q63A rotamer, as other studies implied.13,19−21 Interestingly, one of the 20 NMR structures resembles the lowenergy minimum II found here (Figure 6B,C). Further NMR studies on other BLUF proteins revealed a rather complex picture of protein dynamics. For instance, BlrB BLUF appeared highly dynamic,48 similar to AppA BLUF, whereas BlrP1 BLUF turned out to be more stable.49 Whether these differences are implicated in the light-sensing properties of BLUF is still unknown and must be elucidated in upcoming studies. In the literature we find only limited molecular dynamics analysis of the flavin-binding pocket19,20,32 and to the best of our knowledge no studies on the energy barriers separating conformational substates. A systematic study of the various dihedral degrees of freedom of the hydrogen bonding network residues (not only Q63 and Y21 but also S23, H85, S41, and

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CONCLUSION We investigated the Q63 rotamer assignment in the Win X-ray structure of the BLUF photoreceptor that has been fueling the debate on the BLUF light activation mechanism for several years. We demonstrated that from the three forms of the conserved glutamine only Q63J and Q63tau but not Q63A are compatible with the experimental electron density of the Win structure. Thus, the Q63A rotamer as originally proposed in the X-ray structure cannot serve as an experimental basis for the BLUF dark state model. We emphasize that the NMR structures do not support the crystallographic Q63A assignment in the Win crystal structures because they contain different Y21 and Q63 rotamers. Q63A rotamers (and rotamers of other residues) should be considered as subpopulations of the dark state in solution. Here we demonstrated that the energy barrier between the Q63A and Q63J rotamers is low. We also showed that the computed red shift of flavin absorption between the optimized Q63A and Q63J models is insignificant. Taking all these findings together, we conclude that glutamine rotation does not require photoactivation and is unlikely to trigger the BLUF light response. The alternative mechanistic hypothesis, the light-induced glutamine tautomerization,30,40,50 is in agreement with the three criteria that we formulated for the BLUF functional states: agreement with the X-ray electron density, spectral shifts, and the light sensitivity energy barrier requirement. The tautomerization hypothesis is also compatible with a dark state where several conformational substates of Q63 rotamers coexist.

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ASSOCIATED CONTENT

S Supporting Information *

Cartesian coordinates of fully optimized models. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are very grateful to Ilme Schlichting for valuable discussions, to Roman Fedorov (Medizinische Hochschule Hannover) and Thomas Barends for computing the X-ray electron density maps, to Chris Roome for excellent computer support, and to Andreas Winkler for comments on the manuscript. We acknowledge financial support by the Minerva Program of the Max Planck Society to T.D. and by the Boehringer Ingelheim Fonds to A.U.

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REFERENCES

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Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic

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