pB2 Intermediate of the Photoactive Yellow Protein

ARTICLE pubs.acs.org/JPCB

pB2 Intermediate of the Photoactive Yellow Protein: Structure and Excitation Energies Ya-Wen Hsiao and Walter Thiel* Max-Planck-Institut f€ur Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470, M€ulheim an der Ruhr, Germany ABSTRACT: pB2 is the last electronically excited intermediate of the photoactive yellow protein (PYP) before it thermally reverts to the dark state. We investigate the structure of pB2 by quantum refinement and QM/MM methods and compare our results with a previously published crystal structure (1TS6). We find differences in the chromophore geometries, mostly with regard to torsion angles, that lead to a somewhat higher degree of planarity than in 1TS6. Quantum chemical calculations at the DFT/MRCI level show that these geometry changes affect the excitation energies and oscillator strengths for transitions into the two lowest singlet excited states. The DFT/MRCI results for the presently determined structures agree well with the experimental spectrum. A classical molecular dynamics simulation of free water molecules surrounding the fixed-in-space PYP indicates hydrogen bonding between water and the solvent-exposed pB2 chromophore, since one water molecule is constantly found around the phenolic OH group. Including this additional water molecule in the quantum refinement yields improved electron density maps with a good fit of previously unresolved densities. On the other hand, this extra water molecule has little influence on the chromophore structure and the calculated excitation energies. The overall best pB2 structure from the present work comes from quantum refinement with electrostatics and with an additional water molecule near the phenolic OH group of the chromophore (model 4).

’ INTRODUCTION Photoactive yellow protein (PYP) is a bacterial photoreceptor isolated from Halorhodospira halophila.1 PYP-containing microorganisms respond to blue light and navigate to a less illuminated environment; i.e., they show negative phototaxis.2 The 3-D structure of ground state PYP has been determined via X-ray crystallography:3 the secondary structure of PYP is a mixed R/β fold, with a six-strand antiparallel β-sheet in the center, and helices containing hydrophobic cores on both sides. The smaller core comprises the N-terminus that contains the Per-Arnt-Sim (PAS) sequence motif, while the larger hydrophobic core contains the chromophore-binding pocket. Figure 1 shows the chromophore in the protein environment at the pB2 stage, and Figure 2 depicts the chromophore itself, which is a p-coumaric acid with a thioester bond to Cys69. In the dark state, the chromophore is a phenolate anion4,5 stabilized by hydrogen bonds with Tyr42, Glu46, and Thr50 and by the positive charge of Arg52.3,6 PYP has been viewed as the structural prototype for a large family of signaling proteins containing the PAS domain amino acid sequence motif. It has also been the key model system for studying the signal transduction mechanism both experimentally and theoretically due to its photochemical properties, the availability of high resolution crystal structures, and its small size: 14 kDa from 125 amino acids and the chromophore. When exposed to blue light at room temperature, PYP absorbs photons of wavelength 446 nm (2.78 eV).1 The absorption initiates a trans-cis isomerization at the vinyl double bond (C2dC3) of the chromophore, and the system enters a photocycle consisting of a series of photo-intermediates before it eventually reverts to the dark ground state. To summarize the many investigations of the intermediates, the cycle involves the sequence pG f pR f pB f pG, r 2011 American Chemical Society

where pG, pR, and pB stand for the trans-form ground state, the cisform intermediate with a deprotonated chromophore as a phenolate anion, and the blue-shifted photo-intermediate which is the putative signaling state with the protonated chromophore,7 respectively. In the pB state, the protein environment undergoes more structural changes, especially an unfolding of the two N-terminal helices8,9 which is believed to mediate the PYP signaling activity.10 In the chromophore of the pB state, the phenolate oxygen is protonated, and the phenolic ring moves away from Tyr42 and Glu46 (toward an opening of the binding pocket) and forms a hydrogen bond with Arg52, which at this stage also swings out of the way of the now solvent-exposed chromophore. The characteristic light absorption of the pB intermediate is blue-shifted to 355 nm (3.49 eV).5 On the basis of time-resolved crystallographic data spanning the range from 1 ns to 1 s, Ihee et al.11 proposed a chemical mechanism characterized by five distinct intermediates. pB2 is the last intermediate detected before the dark state. Its crystal structure11 (PDB code 1TS6) indicates hydrogen bonds between the phenolic oxygen atom and Arg52 and between the carbonyl oxygen atom and the Cys69 backbone. We have examined the difference electron density map of this structure and found it to be rather crowded. In addition, the low-lying vertical excitation energies calculated for the 1TS6 geometry at the DFT/MRCI level (density functional theory with multireference configuration interaction)12 agreed with the experimental spectrum not quite as well as expected.12,13 These findings prompted us to reinvestigate the structure of pB2. Received: November 2, 2010 Revised: January 12, 2011 Published: February 14, 2011 2097

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Figure 1. pB2 chromophore and the thioester-linked Cys69 in the protein environment (based on PDB structure 1TS6).

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both the QM and MM parts are subject to the X-ray data restraints. The resulting structures from quantum refinement are then used to calculate the excitation energies of pB2 in order to assess their sensitivity toward geometrical variations. A second issue is the source of the proton that is taken up by the chromophore upon pB formation. Experimental observations point to two possibilities: the proton may come directly from the aqueous medium15-17 or from Glu46.7,18 Mutation experiments show that the formation rate of the pB intermediate is unaffected by changing Glu46 to Gln or Ala, while other experimental results indicate that the deprotonation of Glu46 and the protonation of the chromophore are tightly coupled. In this context, it is of interest to see whether there are nearby water molecules that offer opportunities for the chromophore to abstract or to donate a proton. Such water molecules may also be involved in the conversion of pB2 to the dark state. We have performed molecular dynamics (MD) simulations to find out whether any of the water molecules surrounding the PYP become associated with the solvent-exposed chromophore. Even if Glu46 is the proton donor for the formation of the pB intermediate, it could still be water that mediates the proton transfer during the process of deprotonation and reverting to the dark state; the distance between the Glu46 carboxylate group and the phenolate oxygen of the chromophore is nearly 8 Å in the published pB2 crystal structure.11

’ COMPUTATIONAL DETAILS

Figure 2. pB2 chromophore and the thioester-linked Cys69 structure. Atom labels follow the 1TS6 nomenclature.

Protein intermediates often have special structural features, and it is thus not straightforward to perform a routine X-ray crystallographic refinement based on molecular mechanics (MM), because this may demand unique force field parameters that are not readily available. On the other hand, quantum mechanics (QM) is normally reliable in describing any stationary state structure. We thus decided to apply a crystallographic quantum refinement,14 in which the experimental raw data are supplemented with theoretical QM/MM information during the refinement process. In this approach, the pB2 chromophore is treated by QM and the remainder of the protein by MM, while

Quantum Refinement, QM/MM, and MD. The original protein coordinates and the structure factors were obtained from the Protein Data Bank19 (PDB code 1TS6, resolution 1.6 Å). The initial PDB coordinates refined by Ihee et al. with the use of SHELXL-9720 were further optimized by applying the Crystallography & NMR System (CNS)21 with fixed B factors and occupancies. This led to very slight changes in the coordinates and to a slight reduction in the difference between Rfree factors obtained from CNS and SHELXL-97. The resulting structure was then used as a starting point for the subsequent quantum refinement and QM/ MM studies. Missing hydrogen atoms (needed for QM/MM) were added by using the HBUILD module in CHARMM22 using standard protonation state conventions which led to a total charge of -6. Subsequent inspection of the two histidine residues showed that His3 is located near the N-terminus with its side chain pointing away from the protein toward the solvent, while His108 is also situated near an opening of the protein (with its NE2 atom close to the OD1 atom of Asn89, at an ideal site for protonation). Both histidines were thus protonated at NE2 and ND1 (Hsp assignment). The remaining four negative charges were compensated by protonating surface residues Glu81, Asp10, Asp19, and Asp34 such that the total system was neutral. The positions of added hydrogen atoms were then relaxed at the MM level with 10 000 steps of steepest descent algorithm followed by an ABNR (augment basis NewtonRaphson) minimization which converged before reaching the maximum number of allowed steps (10 000). The QM region in the QM/MM calculations consisted of the chromophore (HC4), the side chains of the Tyr42, Glu46, Thr50, Arg52, Cys69, and Phe96 residues, and the backbone of Tyr98 (atoms CA, C, O, and N). Tyr42 and Glu46 were included in the QM region because they play an important role in the PYP photocycle through their interactions with the chromophore, while the other residues were selected for the QM part because they are located close to the chromophore. The covalent bonds across the QM-MM boundary were capped with hydrogen link atoms. 2098

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The Journal of Physical Chemistry B There are nine such junctions (between CA and CB for Tyr42, Thr50, Cys69, and Phe96; between CB and CG for Glu46; between CD and CG for Arg52; and three more involving the backbone atoms of Tyr98). Consistent with experimental findings for the pB intermediate of the PYP photocycle,7,17,18 Glu46 was taken to be deprotonated and hence negatively charged. The QM region was neutral overall because of the presence of Arg52 (containing a guanidinium cation). Turbomole23 and DL_POLY24 were used to calculate the energies and gradients of the QM and MM parts, respectively. ChemShell25,26 was employed to handle the coordinate, energy, and gradient data communication between the QM, MM, and refinement programs, and to perform the geometry optimizations through the HDLC optimizer.27 All QM calculations were done using density functional theory (DFT) with the BP86 functional28,29 and the 6-31G* basis set.30 This combination has proven to be efficient and reliable as long as geometry is the main concern; it normally reproduces bond lengths of organic molecules to within 0.02 Å, which is better than medium-resolution protein crystal structures where the average error in bond lengths is about 0.1 Å. The BP86/6-31G* combination has been successfully applied in several studies by Ryde et al.14,31-35 The MM force field parameters for proteins were taken from CHARMM22.36 Quantum refinement in the form of an X-ray-data-restrainedQM/MM minimization has recently been implemented in the ChemShell package.37 In this procedure, CNS is applied to assist in the refinement; it calculates the X-ray structure factor amplitudes F from each new protein structure and then evaluates the maximum likelihood with amplitudes (MLF) to obtain a hypothetical energy Exref and the corresponding gradient. Exref is proportional to (|Fo(hkl)| - Æ|Fo(hkl)|æ)2, where the expected value of Æ|Fo(hkl)|æ is derived from the observed Fo and the calculated Fc values.38 The energy function of quantum refinement takes the following form: ð1Þ Etotal ¼ EQ M = MM þ wxref Exref where wxref is determined by a grid scan to identify the structure with the lowest Rfree factor which is defined in analogy to the R factor: P jjFo ðhklÞj - jFc ðhklÞjj hkl P ð2Þ R ¼ jFo ðhklÞj hkl

The Rfree factor is obtained from a set of randomly chosen reflections (10.3% in this case) which are set aside from the beginning and hence not included in the refinement. It provides a cross-validation in order to avoid overfitting of the diffraction data.39 The quantum refinement is considered converged when the maximum gradient component is less than 0.004 au (≈20 kJ mol-1 Å-1). As in standard crystallographic protocols, hydrogen atoms are not considered in the present quantum refinement. Traditionally, crystallographic refinement is done without including electrostatic interactions due to the incompleteness of the diffraction data for determining the full structure and the concern that the use of electrostatics may lead to biased models.38 In our implementation,37 we can perform quantum refinements both without and with electrostatics. In the former case, the electrostatic interactions are simply turned off at all stages of the QM/MM and MM calculations. When electrostatic interactions are included, we adhere to our standard setup for

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QM/MM calculations with electrostatic embedding. Briefly, the protein was solvated in a sphere of TIP3P water molecules40 with a radius of 30 Å, centered at CB of Val120. To retain the shape of the surrounding water droplet and to avoid evaporation of surface water molecules, a spherical quartic boundary potential was applied acting on the corresponding oxygen atoms. The water molecules were equilibrated at the MM level using CHARMM (program version 31b1), and the system underwent cycles of rehydration and constant-temperature MD as described previously:41 after initial placement of the water sphere, overlapping water molecules were deleted, and the remaining ones were energy minimized for 1000 cycles followed by a 15 ps constant-temperature (300 K) classical MD simulation. Subsequently, a new water sphere was superimposed on the system with overlapping atoms deleted, and was again subjected to a 15 ps MD run. This procedure was repeated 12 times, with the equilibration time being increased to 30 ps in the last three cycles. During these classical setup calculations, the heavy-atom coordinates of the protein were not allowed to change. In the subsequent quantum refinement and the QM/MM calculations with electrostatic embedding, no cutoff was applied for the electrostatic QM/MM interactions. The evaluation of the target function in the refinement did not include the added solvating water molecules. Further technical details on the ChemShell implementation of quantum refinement are available in the original report.37 The final structure from the QM/MM setup outlined above was also used as a starting point for a 10 ns constant temperature (300 K) classical MD run of the water molecules around the protein, to check for further hydrogen bonding interactions between water molecules and the chromophore. The time step was chosen to be 1 fs, and a total of 10 000 snapshots were collected, i.e., one snapshot every 1000 steps. The program VMD42 was used to visualize the MD trajectories and to extract coordinate information. To enable the calculation of spectroscopic properties of the pB2 intermediate at the published X-ray geometry (PDB code 1TS6),11 we carried out a simplified setup: the missing hydrogen atoms were added and relaxed using the program CHARMM, and then further optimized at our standard QM/MM level (QM = BP86/6-31G*, MM = CHARMM22, same QM region as defined above), with all heavy atoms being frozen. The whole protein was solvated to be compatible with calculations using electrostatic embedding. DFT/MRCI. DFT/MRCI12 was employed to calculate the excitation energies of the chromophore. In this approach, dynamic electron correlation is taken into account by DFT, while static correlation effects are captured by a limited CI expansion. The configuration state functions in the MRCI expansion are built from Kohn-Sham orbitals, and the diagonal elements of the Hamiltonian are constructed from the corresponding Hartree-Fock-based expression and a DFT-specific correction term. Using the standard values for the five empirical DFT/MRCI parameters, the typical error in vertical excitation energies is around 0.2 eV compared to experimental values12 and to high-level ab initio reference data.13 For each structure obtained from quantum refinement or QM/MM optimization, DFT/MRCI calculations were performed with two approximate representations of the environment around the chromophore: either fixed MM point charges from the rest of the protein and the surrounding water sphere (denoted as m1) or the COSMO continuum model43 with the dielectric constant ε = 78 (called m2). The QM region for the MRCI calculation included the chromophore, the side chains of 2099

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Table 1. Experimental and Calculated (DFT/MRCI)a Lowest Absorptions (eV), with Oscillator Strengths in Parentheses, of PYP Model Systems (See Text) PCA(g)molecule experiment49 DFT/MRCI

phenolate PCA(aq) PCT(g)- PCT(aq)

carboxylate

4.35

2.70

3.68

4.70

2.88 2.97

4.16

2.64

3.77

(0.65)

(1.13)

(0.77)

(1.41)

(0.82)

The calculated absorptions correspond to intense π f π* transitions. In the case of PCT(aq), there is an even lower n f π* transition at 3.69 eV (oscillator strength 0.11).

a

residues Cys69 and Arg52, and the backbone of Phe98, for all models 1-8 considered (see below); an additional water molecule near O40 was included for models 2, 4, 6, and 8. Molecular orbitals (MOs) were generated with Turbomole using the BH-LYP functional28,44,45 and the TZVP basis set.46,47 The active space for the MRCI treatment was built from double excitations of 10 electrons in 10 frontier MOs. The CI space was kept moderate by selecting only configurations with an estimated energy below a cutoff δEsel above the highest reference-space energy. We adopted the DFT/MRCI parameters recently developed in the Grimme group48 in combination with a tight selection criterion δEsel for enhanced computational efficiency. An initial cutoff of 0.6 hartree was applied in the selection, which was increased to 0.8 hartree from the second cycle onward. The reference configurations were determined iteratively: configurations with a squared coefficient greater than 0.003 were included in the reference space for the next CI calculation, and the iterations were stopped when the lowest 10 roots with singlet multiplicity were converged to 10-6 hartree. Despite the fact that DFT/MRCI has been established as a reliable tool for predicting UV-vis spectra,13,41 we decided to test the method on model systems that closely resemble the chromophore of PYP. Experimental electronic absorption energies are available49 for the model compounds trans-thiophenyl-p-coumaric acid (PCT) and trans-p-coumaric acid (PCA) as well as the corresponding deprotonated species PCT- and PCA-. The measured absorption maxima of the gas phase anions PCA- and PCT- are 430 and 460 nm (2.88 and 2.70 eV), while those of the acids PCA and PCT in neutral aqueous solution are 285 and 337 nm (4.35 and 3.68 eV), respectively.49 We optimized the geometries of these species at the B3LYP44,50,51/TZVP level. In the case of PCA, there are two acidic protons, so that PCA- may exist as phenolate or carboxylate. According to B3LYP/TZVP, the former is more stable by 15.5 kcal/mol, whereas the opposite has been assumed in the interpretation of the experimental work.49 Standard DFT/MRCI calculations at the optimized B3LYP/TZVP geometries were carried out for PCA- and PCT- in the gas phase, and for PCA and PCT in aqueous solution using the COSMO continuum model with ε = 78. The results for the lowest singlet transitions are compared with the experimental data in Table 1. The observed band maxima are in excellent agreement with the computed excitation energies of the intense π f π* transitions for PCA, PCT, PCA- (phenolate), and PCT-, with deviations less than 0.2 eV. By contrast, there is a huge discrepancy (1.82 eV) with the computed value for PCA- (carboxylate) so that one may safely conclude that the phenolate form is indeed favored for PCA- (as expected from the B3LYP/TZVP relative energies). In summary, the current test calculations confirm that DFT/MRCI is indeed a very reliable tool for studying the electronic excitations in PYP-like systems.

Figure 3. MD simulation of the motion of water molecules around fixed PYP: radial distribution function around chromophore atom O40 .

Figure 4. MD simulation of the motion of water molecules around fixed PYP: radial distribution function around chromophore atom O1.

’ RESULTS AND DISCUSSION Classical MD Simulation of Water around Fixed Protein. During the simulation, 10 000 snapshots were collected and the coordinates of the water oxygen atoms of each snapshot were used to analyze their spatial proximity to the two chromophore oxygen atoms, O40 (phenolate) and O1 (carbonyl). Figures 3 and 4 show the radial distribution of water molecules around these oxygen atoms. In Figure 3, there is a distinct peak which represents the first shell of water between 2 and 4 Å away from O40 . On the other hand, Figure 4 has no such distinct peak: the O1 oxygen atom is less accessible, since it is already hydrogen bonded to the backbone amide of Cys69. The peak at 2-4 Å from O40 suggests well-localized neighboring water within this range: integrating the radial distribution function up to 3 Å yields approximately one water molecule, and extending the integration up to 4 Å gives three water molecules. In the quantum refinement and the QM/MM calculations, we will thus consider also models with one additional water molecule close to O40 (which is not present in the 1TS6 structure). For the sake of completeness, we 2100

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Table 2. Rfree Factors and Associated wxref Weights from Quantum Refinement for Models 1-4

Table 4. Chromophore Bond Lengths (Å) and Angles (deg) with Larger Deviations between 1TS6 and Models 1-8 (See Text)

wxref

1

2

3

4

0.2

0.2174

0.2160

0.2178

0.2164

0.4 0.6

0.2167 0.2166

0.2151 0.2157

0.2152 0.2155

0.2148 0.2144

0.8

0.2171

0.2159

0.2156

0.2143

0.2172

0.2160

0.2156

0.2143

0.9 1.0

0

0

C4 —O4 C3dC2 C2—C1 C1—SG C10 —C3dC2 C3dC2—C1 C2—C1—O1 O1—C1—SG

0.2140

1TS6

1

2

3

4

5

6

7

8

1.34 1.29 1.38 1.76 135 127 141 112

1.43 1.34 1.46 1.79 127 137 137 121

1.40 1.35 1.47 1.79 137 136 136 121

1.39 1.35 1.45 1.78 128 135 137 120

1.42 1.33 1.43 1.78 127 136 138 121

1.40 1.37 1.47 1.82 130 130 129 122

1.40 1.37 1.47 1.82 130 130 129 121

1.38 1.37 1.47 1.82 131 131 130 121

1.37 1.37 1.46 1.83 130 130 130 121

Table 3. Selected Heavy Atom Distances Involving Surrounding Residues (Å) 0

O4 -Y42 OH O40 -E46 OE2 O40 -R52 NE O1-C69 N Y42 OH-T50 OG1 O40 -OH2a

1TS6

1

2

3

4

5

6

7

8

5.04 7.81 2.56 2.89 2.80

5.50 7.90 2.70 2.92 2.74

5.52 7.92 2.67 2.93 2.73 2.66

5.44 7.84 2.77 2.92 2.75

5.48 7.85 2.80 2.91 2.76 2.72

5.87 6.93 2.72 3.22 2.90

5.88 7.26 2.67 3.23 2.82 2.63

5.74 6.91 2.82 2.96 3.15

5.62 6.79 2.89 2.97 3.09 2.75

a Distance between O40 and the additional water included in models 2, 4, 6, and 8.

will also test models with three water molecules positioned around the O40 atom. Quantum Refinement and QM/MM Calculations. We have determined the structure of the pB2 intermediate using eight different approaches, by performing quantum refinements (models 1-4) and QM/MM geometry optimizations without X-ray restraints (models 5-8) for the whole protein. Further distinctions are the absence or presence of an additional water molecule near O40 and the embedding scheme (without or with electrostatics). The models are characterized as follows: Model 1: quantum refinement, without extra water, without electrostatics Model 2: quantum refinement, with extra water, without electrostatics Model 3: quantum refinement, without extra water, with electrostatics Model 4: quantum refinement, with extra water, with electrostatics Model 5: QM/MM optimization, without extra water, without electrostatics Model 6: QM/MM optimization, with extra water, without electrostatics Model 7: QM/MM optimization, without extra water, with electrostatics Model 8: QM/MM optimization, with extra water, with electrostatics Table 2 lists the Rfree values and the corresponding wxref weights for the quantum refinements (models 1-4). The structure with the lowest Rfree value from each model was used for subsequent analysis. The following three tables compare selected geometrical variables from the published X-ray structure (PDB code 1TS6) with those obtained from models 1-8. Generally speaking, the results from quantum refinement (models 1-4) are quite similar to each other and seem to form one group, while those from the QM/MM optimizations (models 5-8) are usually somewhat more diverse but also share many similarities and thus seem to form another group.

Figure 5. Overlay of pB2 chromophore structures: 1TS6 (green) and model 4 from quantum refinement (purple).

Both sets of results show some significant differences from the published X-ray data.11 Table 3 contains selected heavy atom distances, mostly between the chromophore and neighboring residues. The first two entries give the distances between O40 of the chromophore and oxygen atoms in Tyr42 and Glu46, respectively, which exceed 5 Å and are thus too large to support hydrogen bonds. The other four entries are generally around 3 Å and are thus indicative of hydrogen bonds. Compared with 1TS6, models 14 yield longer distances between O40 of the chromophore and Tyr42, Glu46, and Arg52, while the distance between O1 of the chromophore and N of Cys69 remains almost unchanged and the distance between Tyr42 and Thr50 becomes a little shorter. These distances are rather insensitive toward the absence or presence of an extra water molecule close to O40 (models 1 vs 2 and 3 vs 4). The QM/MM results (models 5-8) generally show similar trends. Table 4 lists the bond lengths and bond angles in the chromophore that differ by more than 0.05 Å or 5, respectively, from the 1TS6 structure, in more than one model. Compared with 1TS6, all models (1-8) yield longer C40 —O40 , C3dC2, C2—C1, and C1—SG bonds. Our refinements thus give more single bond character to C40 —O40 , and the C3dC2 vinyl double bond now has a rather typical length, while it is very short in 1TS6 (1.29 Å). The C1—SG bond is shorter in 1TS6 than in the quantum refinement and QM/MM structures, by about 0.02 and 0.07 Å, respectively. The angles within the C10 —C3dC2— C1dO1 moiety also clearly differ from the 1TS6 values: the general trend in the present refinements is to give smaller C10 — C3dC2, larger C3dC2—C1, smaller C2—C1—O1, and larger O1—C1—SG angles. Table 5 contains geometrical parameters related to the planarity of the chromophore, as well as torsion angles that differ by more than 5 from the 1TS6 structure in more than one model. 2101

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Table 5. RMS Deviation DPa (Å) from Planarity and Selected Torsion Angles A-B-C-D and Angles θ between Planes (deg) in the Chromophore 1TS6 DP CA—CB—SG—C1 CB—SG—C1—C2

2

3

4

5

6

7

8

0.44

0.42

0.41

0.41

0.42

0.32

0.30

0.33

0.36

-79 180

-80 176

-80 175

-79 176

-79 177

-85 171

-87 173

-76 174

-75 171

CB—SG—C1dO1

0

-5

-5

-4

-4

-7

-6

-5

-7

C1—C2dC3—C10

-24

-11

-12

-15

-12

-12

-11

-13

-16

C3—C10 —C20 —C30

179

-173

-173

-173

-172

-176

-179

-179

-175

C3—C10 —C60 —C50

-177

175

175

175

175

176

179

180

177

C30 —C40 —C50 —C60

6

6

5

4

8

-1

-1

-2

1

-29

-37

-34

-32

-39

-25

-22

-20

-21

θ(phenol plane) θ(phenol-vinyl planes)

0 150

6 141

6 146

7 149

7 143

11 158

14 160

18 161

12 160

θ(vinyl-CCdO planes)

150

160

158

156

160

159

161

153

152

53

50

48

48

51

36

31

34

41

C2dC3—C10 —C20

θ(CCdO-phenol planes) a

1

Calculated using atoms from the sulfur of Cys69 to all of residue 169.

Figure 6. Overlay of pB2 structures: QM region of models 1-4 (left) and 5-8 (right). Color code: orange, models 1 and 5; green, models 2 and 6; pink, models 3 and 7; purple, models 4 and 8.

The rms deviation from planarity (DP) is calculated including all the atoms from the sulfur of Cys69 to the phenolate oxygen using the program GEOMCALC,52 in analogy to previous work.11 The DP values from models 1-4 are similar to that of the 1TS6 structure (slightly smaller), whereas models 5-8 clearly have smaller DP values. This is probably related to the known DFT trend to overemphasize the resonance effects in conjugated systems of this kind, which is mitigated by the X-ray restraints in models 1-4. The last four rows in Table 5 provide angles between normal vectors of various planes. The first of these is the angle between the normal vectors of the phenolic ring plane of the 1TS6

structure and that of the refined structure. Clearly, all present calculations show that the phenolic ring rotates from the 1TS6 structure by 6-7 in the quantum refinements, and by twice as much in the QM/MM optimizations. The second of these rows contains the angle between the phenolic plane and the vinyl plane (defined by atoms C10 , C3, and C2) and thus indicates the extent of resonance between the aromatic ring and the double bond. Compared with 1TS6, the angles from the quantum refinements and the QM/MM calculations differ by 1-9 and 8-11, respectively, in opposite directions (less and more planar). The last two of these rows refer to the angle between the vinyl plane and the CCdO plane (C2, C1, O1) and between the phenolic ring and the CCdO plane, 2102

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Table 6. Real Space R Factors for Residues in the QM Region

a

Figure 7. Difference electron density (Fo - Fc) around the chromophore contoured at þ3σ (blue) and -3σ (green) for mixtures of the pB2 and pG structures with occupancies11 of 0.65 and 0.35, respectively: top, 1TS6 and pG; middle, model 3 and pG; bottom, model 4 and pG.

respectively. The present results for these angles are generally slightly closer to planarity than in 1TS6, especially in the QM/MM case (models 5-8), but the differences are generally rather moderate. Figure 5 shows the 1TS6 structure (green) overlaid with the refined structure (purple) from model 4. In the latter, the ring face turns slightly more toward the protein side, and so does the vinyl double bond, while the rest of the chromophore is almost perfectly superimposed. The dihedral angle C1—C2dC3—C10 is slightly less negative in our refined structures than in 1TS6 (Table 5), which

residue

1TS6

model 3

model 4

Tyr42

0.180

0.165

0.164

Glu46

0.223

0.163

0.159

Thr50

0.195

0.210

0.207

Arg52

0.371

0.341

0.335

Cys69

0.153

0.141

0.134

Phe96

0.198

0.199

0.192

Tyr98

0.202

0.188

0.184

HC4 water

0.300

0.294

0.297 0.643

avga

0.228

0.213

0.209

Average taken without the additional water molecule in model 4.

indicates that the plane of the vinyl double bond rotates mainly by raising the C2 atom and lowering the C1 atom. Overlays of the chromophore structures from models 1-4 and 5-8 are shown in Figure 6. There is only little structural diversity among models 1-4, which are confined by the X-ray data restraints. Exceptions are the side chain of Arg52, where models 1-2 and 3-4 seem distinct, and the orientation of the hydroxy group of the chromophore, for which model 3 deviates from the rest. The differences are more pronounced in the pure QM/MM models (5-8). Apparently, the results from quantum refinement are more robust than those from QM/MM calculations, presumably because of the presence of X-ray restraints which make the procedure less modelsensitive. In this sense, quantum refinement is not only an improvement over the conventional crystallographic refinement using MM force fields, but it also provides more realistic structures than a purely theoretical QM/MM treatment. The quality of the structures obtained from quantum refinement can be visualized by the electron density maps generated from them as models. Difference electron density maps were calculated using CNS and visualized using the program Coot.53 The top panel in Figure 7 shows the difference electron density (Fo - Fc) at (3σ level around the chromophore of the original 1TS6 structure. It can be seen that the electron densities around the pB2 phenolic ring mix with the densities from Arg52 of the pG conformation. The phenolic rings, both in pG and pB2, are clouded with residual densities which sprawl over to the C10 atom from the pB2 conformation and almost connect to the residual densities from the vinyl part of the pG conformer. The difference density plot (from model 3) in the middle panel of Figure 7 exhibits a clear reduction of unresolved electron densities by the quantum refinement: the unresolved densities around the phenolic rings and Arg52 of the pG conformation have recessed. Further comparison with the quantum refinement structure obtained with an added water molecule near O40 of the pB2 conformation (from model 4) shows that the densities around O40 and Arg52 of the pG conformation are almost resolved (bottom panel of Figure 7), accompanied by a great general reduction of residual densities in this area. By contrast, tests with a model containing three extra water molecules near O40 give a difference electron density map with negative densities around at least one of those water oxygens (not shown). In summary, the difference density map for model 4 (bottom panel of Figure 7) demonstrates that the quantum refinement with one extra water molecule provides a much improved match with the raw X-ray data. The real space R factors of the QM residues in Table 6 confirm that models 3 and 4 both are an improvement over the original 1TS6 model. 2103

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Table 7. Lowest DFT/MRCI Excitation Energies (eV), Oscillator Strengths (in Parentheses), and Normalized Weights of the Leading Configurations for 1TS6 and the Quantum Refinement Models nonelectrostatic

electrostatic

þ1w method

configuration

m1

1TS6

1

þ3w

þ1w

2

3

4

3.14 (0.07)

3.43 (0.26)

3.39 (0.43)

3.43 (0.36)

3.30 (0.42)

3.35 (0.47)

πfπ*

0.20

0.48

0.68

0.60

0.67

0.73

nbþπfπ*

0.48 3.68 (0.65)

0.32 3.78 (0.43)

0.18 3.75 (0.26)

0.23 3.77 (0.31)

0.18 3.70 (0.28)

0.14 3.79 (0.22)

π f π*

0.70

0.42

0.23

0.28

0.23

0.18

nb þ π f π*

0.13

0.36

0.52

0.43

0.52

0.57

3.16 (0.05)

3.50 (0.19)

3.46 (0.26)

3.48 (0.17)

3.37 (0.27)

3.45 (0.28)

π f π*

0.16

0.39

0.47

0.34

0.48

0.49

nb þ π f π*

0.39

0.39

0.33

0.41

0.32

0.32

3.78 (0.70)

3.89 (0.53)

3.83 (0.45)

3.86 (0.54)

3.75 (0.47)

3.85 (0.46)

0.72 0.06

0.50 0.28

0.43 0.36

0.55 0.24

0.42 0.35

0.41 0.37

m2

π f π* nb þ π f π*

Electronic Excitations. The DFT/MRCI results are collected in Tables 7 and 8. They have been obtained from singlepoint calculations at the geometries from 1TS6 and from models 1-8. In the discussion, we focus on the m1 data that reflect the situation in the solvated protein (with the environment represented by MM point charges) but also make comparisons with the m2 results that refer to aqueous solution (COSMO treatment with ε = 78). For the original 1TS6 structure of pB2, the first DFT/MRCI (m1) excitation energy with substantial oscillator strength (0.65) is 3.68 eV (see Table 7). The corresponding transition is mainly of π f π* type. The π MO extends over the phenolic ring and the vinyl double bond. Compared to the experimental value of 3.49 eV, the calculated energy is too high, contrary to the general trend of DFT/MRCI to underestimate excitation energies by typically 0.1-0.2 eV.13 Furthermore, the overestimate of 0.19 eV exceeds the average deviation (less than 0.1 eV) found for the current model systems (Table 1). There is another lower excitation (3.14 eV, m1) with low oscillator strength (0.07) which mainly originates from a MO formed by the nonbonding orbitals of the carbonyl group and the sulfur atom, with an additional small admixture of the π orbital from the phenolic ring; we label this MO as “nb þ π”. For this lower excited state, the normalized weights of the leading configurations (nb þ π) f π* and π f π* are 0.48 and 0.20, respectively. Table 7 also contains the DFT/MRCI results for the structures obtained by quantum refinement (models 1-4). In contrast to the 1TS6 model, both low-energy transitions now have substantial contributions from the π f π* and (nb þ π) f π* configurations, with the former being dominant in the lowestenergy state for all models. This first singlet transition (at 3.303.43 eV, m1) carries most of the oscillator strength in models 24 (0.36-0.47), while the second one (at 3.70-3.79 eV) is somewhat weaker (0.22-0.31). The energy difference between these two excited states is clearly smaller than that obtained from the 1TS6 model. Overall, these results are consistent with the experimental finding of a broad absorption band in pB2 with a maximum at 355 nm. Going from 1TS6 to the quantum refinement structures thus causes a blue shift in the first singlet transition and a

significant redistribution of oscillator strength, which leads to better agreement with experiment. The presence of an additional water molecule near O40 has only a minor nonuniform effect on the calculated excitation energies (less than 0.1 eV, see models 1-2 and 3-4, Table 7) but tends to further shift some intensity to the first transition by increasing the weight of the π f π* contribution. These effects seem too small to provide conclusive spectroscopic evidence for or against the presence of such an extra water molecule in the pB2 structure. This is not surprising in view of the fact that the MOs involved in the first two transitions have little contributions from the phenolate oxygen atom O40 so that an additional water molecule near O40 should have little influence. The m2 results in Table 7 are qualitatively compatible with the m1 data, showing similar trends when going from 1TS6 to the refined structures from models 1-4. The first transition is again blue-shifted from 3.16 to 3.37-3.50 eV and gains oscillator strength, while the second one remains around 3.8 eV and loses intensity. Table 8 contains the DFT/MRCI results for the QM/MM structures (models 5-8). Compared with 1TS6, qualitatively similar changes are found as in the case of the quantum refinement structures (Table 7). The first excitation energy is again increased, and the mixing of the π f π* and nb þ π f π* configurations is again enhanced in both transitions, with a concomitant redistribution of oscillator strength. The energy difference between the two first excited states is again reduced, even more so than in models 1-4. Generally speaking, the spectroscopic results for the QM/MM structures (models 5-8) show some more diversity than those for the quantum refinement structures (models 1-4), especially when comparing m1 and m2 data. This is probably due to their more pronounced geometrical diversity. Table 9 lists the relevant MO energies and their differences. Going from 1TS6 to models 1-8, the orbital energy is lowered slightly for the π* MO and more strongly for the nb þ π MO. This causes a small decrease of the energy gap between the π and π* MOs and a larger increase of the gap between the nb þ π and π* MOs. These shifts rationalize the changes in the character of the two excited states discussed above: as a consequence of the 2104

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Table 8. Lowest DFT/MRCI Excitation Energies (eV), Oscillator Strengths (in Parentheses), and Normalized Weights of the Leading Configurations for the QM/MM Models nonelectrostatic

electrostatic

þ1w configuration m1

5

þ3w

þ1w

6

7

8

3.34 (0.20)

3.32 (0.24)

3.26 (0.43)

3.33 (0.36)

3.21 (0.48)

π f π*

0.42

0.46

0.67

0.63

0.76

nb þ π f π*

0.34 3.68 (0.47)

0.30 3.64 (0.45)

0.17 3.57 (0.30)

0.20 3.66 (0.24)

0.11 3.61 (0.18)

π f π*

0.48

0.43

0.23

0.21

0.14

nb þ π f π*

0.27

0.27

0.41

0.33

0.52

3.40 (0.13)

3.40 (0.15)

3.39 (0.19)

3.37 (0.17)

3.33 (0.23)

π f π*

0.31

0.35

0.36

0.37

0.43

nb þ π f π*

0.40

0.37

0.38

0.37

0.34

3.81 (0.56)

3.78 (0.55)

3.73 (0.56)

3.73 (0.52)

3.68 (0.47)

0.57 0.18

0.53 0.19

0.53 0.23

0.53 0.24

0.47 0.30

m2

π f π* nb þ π f π*

Table 9. Molecular Orbital Energies and Their Differences (eV) for 1TS6 and Models 1-8 method m1

m2

1TS6

1

2

3

4

5

6

7

8

E(π*)

-0.86

-1.10

-0.97

-1.00

-0.94

-1.19

-1.64

-0.97

-0.87

E(π) E(nb þ π)

-6.82 -8.30

-7.04 -8.83

-6.78 -8.75

-6.74 -8.74

-6.72 -8.75

-7.08 -8.95

-7.48 -9.37

-6.76 -8.75

-6.50 -8.63

E(π*)-E(π)

5.96

5.94

5.81

5.74

5.78

5.89

5.84

5.79

5.63

E(π*)-E(nb þ π)

7.44

7.73

7.78

7.74

7.81

7.76

7.73

7.78

7.76

E(π*)

-1.32

-1.34

-1.31

-1.37

-1.30

-1.40

-1.43

-1.42

-1.41

E(π)

-7.40

-7.40

-7.30

-7.25

-7.26

-7.45

-7.44

-7.35

-7.26

E(nb þ π)

-8.68

-9.10

-9.08

-9.03

-9.03

-9.13

-9.16

-9.10

-9.07

E(π*)-E(π)

6.08

6.06

5.99

5.88

5.96

6.05

6.01

5.93

5.85

E(π*)-E(nb þ π)

7.36

7.76

7.77

7.66

7.73

7.73

7.73

7.68

7.66

changes in the MO energy gaps, the lowest excited singlet state acquires more π f π* character and thus gains intensity at the expense of the second excited state. What are the structural differences between 1TS6 and models 1-8 that are responsible for these changes in the MO energies and in the character of the lowest excited states? According to our preceding discussion, there are indeed some differences between 1TS6 and the presently determined structures which however do not seem dramatic (see data in Tables 3-5). In general, the chromophore geometries in our models 1-8 appear to be somewhat closer to planarity than 1TS6, especially in the case of the QM/MM models 5-8. Relevant geometrical indicators in this context are the torsion angle around the vinyl double bond (C1—C2dC3—C10 ) and the angles between the CCdO, vinyl, and phenol planes (see Table 5). We have performed DFT/ MRCI calculations for two constrained quantum refinement structures, with either the vinyl part or the phenolic part being fixed, and found (data not shown) that the orientation of the vinyl moiety is the key feature which causes most of the changes in the calculated excitation energies and oscillator strengths when going from 1TS6 to the presently determined structures. Given the good agreement with the experimental spectroscopic data for models 1-8, we conclude that the structure of the pB2 chromophore may be closer to planarity than indicated by the 1TS6 model, particularly in the vinyl region.

’ CONCLUSION Classical MD simulations of the motion of water molecules around the pB2 intermediate indicate that there is at least one water molecule in the vicinity of the O40 atom of the phenolic ring that is not seen in the published X-ray structure (1TS6). Quantum refinement with such an extra water molecule leads to a difference electron density map that resolves the residual electron density in this region much better than the original 1TS6 model. The additional water molecule has only little influence on the chromophore geometry or the calculated spectroscopic properties. It is conceivable, however, that it might play a role during the conversion of pB2 to the dark state, since it could be involved in the required deprotonation at the phenolic oxygen atom. Compared with 1TS6, the geometries from quantum refinement and from QM/MM calculations differ mostly in torsion angles and in angles between various planes of the chromophore moiety: the presently determined structures are closer to planarity. When overlaid with the 1TS6 model, there are notable rotations at the phenolic ring and the vinyl double bond, both of which may be spectroscopically important. The structural diversity is smaller among the quantum refinement results (models 1-4) than among the QM/MM results (models 5-8). Due to the restraints from the X-ray data, quantum refinement leads to structures that are quite independent of the chosen model (e.g., mechanical vs electrostatic 2105

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The Journal of Physical Chemistry B embedding) and thus expected to be realistic. Our preferred pB2 structure comes from quantum refinement with electrostatics and with an extra water molecule near O40 (model 4, see Figure 7): it best resolves the residual electron density and gives the lowest Rfree value in the refinement among all models tested. DFT/MRCI calculations at the structures obtained from quantum refinements or QM/MM optimizations give excitation energies that are close to the experimental values, within the error range of the method. Compared with the 1TS6 model, the energy difference between the first two excited states is reduced which is consistent with the experimental finding of a broad absorption band in pB with a maximum at 355 nm. At the presently determined geometries, there is substantial mixing of π f π* and (nb þ π) f π* configurations in these two states, with a concomitant redistribution of oscillator strength. The differences between the DFT/ MRCI results for 1TS6 and for models 1-8 can be rationalized by the shifts in the relevant MO energies which are in turn caused by differences in the chromophore geometries. It is in particular the different degree of twist around the vinyl double bond in models 18 compared with 1TS6 that seems to be responsible for the differences in the computed spectra.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

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dx.doi.org/10.1021/jp1104714 |J. Phys. Chem. B 2011, 115, 2097–2106