Competitive Mechanistic Pathways for Green-to-Red Photoconversion

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Competitive Mechanistic Pathways for Green-to-Red Photoconversion in the Fluorescent Protein Kaede: A Computational Study Xin Li,† Lung Wa Chung,† Hideaki Mizuno,‡ Atsushi Miyawaki,‡ and Keiji Morokuma*,† Fukui Institute for Fundamental Chemistry, Kyoto UniVersity, Kyoto 606-8103, Japan, and Laboratory for Cell Function and Dynamics, AdVanced Technology DeVelopment Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan ReceiVed: October 24, 2010

In Kaede, a new class of fluorescent protein, dramatic changes of photophysical and chemical properties by UV illumination have been observed in which the color of fluorescence is irreversibly altered from green to red. Unusual photoinduced peptide backbone cleavage resulting in extending π-conjugation of the chromophore takes place. Two mechanistic pathways (E1 and E2 mechanisms) involving the N-CR bond cleavage at His62 and deprotonation at Cβ by Glu212 have been proposed. Here several possible pathways are explored with explicit consideration of protein environment by ONIOM(B3LYP:AMBER) calculations. The results reject the concerted E2 pathway. Instead, the stepwise E1 and new E1cb mechanisms are suggested to occur and may compete with each other in the electronic ground state. Absorption for the green- and red-type chromophore in vacuum and within the Kaede protein matrix was studied. Introduction Wild-type green fluorescent protein (GFP) and its variants play an indispensable role in biological imaging and analysis.1,2 Recently, photoactivatable fluorescent proteins, including reversible photoswitching and irreversible photoconversion fluorescent proteins, became a new class of fluorescent proteins (FPs) and provided a remarkable advance in fluorescent protein technology.3-11 Kaede and EosFP, irreversible photoconversion fluorescent proteins,7,9-11 were cloned from stony corals by Miyawaki’s and Nienhaus’s groups, respectively,9,10 in which color of fluorescence can be irreversibly changed from green to red by irradiation with UV or violet light via the unusual peptide backbone cleavage (Scheme 1).12-14 The new fluorescent proteins with a large shift in emission caused by irradiation can serve as regional optical markers and be used for superresolution imaging. On the basis of the experimental observations (such as mutation and X-ray crystal structures),9,10,12-14 excited-state proton transfer (ESPT) from the neutral chromophore was proposed as the first step that operates in wt-GFP and its variants.1,2 However, the detailed reaction mechanism for the photoconversion is unclear and under debate, partly due to its very low quantum yield. Two mechanisms, stepwise E1 and concerted E2 pathways, have been proposed by Miyawaki and Nienhaus, respectively (Scheme 2).12-14 The recently observed cis-alkenyl product in KikGR favors the E1-type pathway.11 Recent QM/MM simulations with semiempirical QM methods proposed an uncommon pathway involving ESPT from the conserved neutral imidazole of His62 and intersystem crossings without direct involvement of the critical Glu212 (Scheme 3).15 This proposed ESPT is not conventional but should be regarded as ESPT induced by electron transfer from the neutral imidazole to the excited-state neutral chromophore to give the key species with both zwitterionic and diradical nature. However, this new * Corresponding author. E-mail: [email protected]. † Kyoto University. ‡ RIKEN.

proposed pathway could lead to other side products (e.g., c3′, see Scheme 4) and may hardly explain a few experimental observations: The role of the vital Glu212 is vague. The cationic His62, which should be stable in the lower pH condition having the higher conversion rate, should disfavor electron transfer for the proposed ESPT. Electron transfer was recently observed, mainly from Glu212, in IrisFP or other FPs, but no evidence for electron transfer from His62 yet. Instead, electron transfer from the tyrosine to the histidyl radical was observed in the photooxidized peptides.16 It may also fail to explain no photoconversion from the trans chromophore in IrisFP. This new pathway should be experimentally verified by mutation of Tyr63 by another neutral aromatic Phe or Trp, which allows electron transfer but does not allow deprotonation from the chromophore. An insight has been made for the vacuum UV photodissociation of peptide via a Rydberg excitation and/or electron detachment mechanism by DFT calculations and experiments.17 In the present work, we systematically examined several possible mechanisms of the photoconversion in Kaede starting from the proposed intermediate 1, Figure 1, by ONIOM(B3LYP: AMBER) calculations18 including explicitly 4121 atoms with a larger QM part of 108 atoms (see Computational Methods and Models).19 Our calculations on the absorption (see the last part of Results and Discussion) suggests that the strong absorption of the greenform Kaede protein is mainly of the π-π* type within the chromophore and the entire reaction depicted in Scheme 1 involves σ-bond cleavage and proton transfers outside the greenform chromophore. Therefore, the reaction is most likely to take place after the system has fallen to the electronic ground state and the excitation energy has been converted to internal energy that can help the system to surmount reaction barriers. Therefore, we confine the present study to chemical reactions in the electronic ground state, which involve peptide CR-N backbone cleavage, proton transfer from His62, deprotonation of the Cβ, and tautomerization of the released fragment of Phe61.

10.1021/jp1101779  2010 American Chemical Society Published on Web 11/17/2010

Photoconversion in the Fluorescent Protein Kaede

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SCHEME 1: UV-Induced Protein Cleavage and Green-to-Red Conversion

SCHEME 2: Proposed Mechanisms of the Photoconversion in Kaede (E1) and EosFP (E2)

Computational Methods and Models A. System Preparation and Molecular Mechanics Optimization. The initial structures of the green- and red-form proteins were obtained from the Protein Data Bank (PDB IDs: 2GW3 and 2GW4). Orientation of histidine, asparagine, and glutamine residues was examined by WhatCheck, MolProbity, and visual inspection to have better hydrogen bonding or lesser steric repulsion. Missing hydrogen atoms were added and major hydrogen bond networks were optimized by the program PDB2PQR.20a Meanwhile, protonation states of the titratable residues at pH 7 were determined by PROPKA implemented in PDB2PQR.20 In addition, the nature of histidine, i.e., protonated at the δ or/and ε nitrogen, was assigned on the basis of the local hydrogen bonding network via visual inspection. Then, the prepared structures of the two proteins were fully solvated in the truncated octahedron water box constructed from a cubic box of about 87 Å for the two proteins, and neutralized by addition of Cl- counterions via the Amber Leap module.21 All classical molecular mechanics (MM) calculations have been performed with AMBER all-atom force field and TIP3P water model.22 All available force field parameters, charges, and atom types of each amino acid have been taken from the

AMBER library. Since the atomic charges for the AMBER force field are derived from RESP charges calculated at the HF/631G(d) level, RESP atomic charges of the green and red forms of the chromophore were obtained at the same level by the Gaussian and Amber programs.23 The water molecules and counterions were first optimized, and then the entire system was further optimized to remove close contacts. Finally, the chain A and its nearby crystal water molecules of the MM-optimized structures were taken as the initial geometry of the ONIOM optimizations. B. ONIOM(QM:MM) and QM Calculations. The protein structures containing the green- and red-form chromophore prepared from the MM optimization were refined by the ONIOM(B3LYP/6-31G*:AMBER) optimization.23-26 The real system was further divided into the optimized MM region and the frozen MM region.27 The residues within about 9 Å of the chromophore were assigned to the optimized MM region and allowed to be optimized. As shown in Scheme 5, a large QM model with 108 atoms, in which the nearby charged residues (Arg66, Glu144, His194, Glu212) around the green chromophore forming a well-knitted hydrogen-bond network are included in the QM region to allow their mutual polarization,

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SCHEME 3: Proposed Pathway for EosFP Adapted from Ref 15a

a

A better representation for some structures responsible for ESPT is given in the box.

SCHEME 4: Energetic Profile (in kcal/mol) for the Rearrangement Related to Scheme 3 Calculated by the B3LYP/ 6-31G* Method in the Gas Phase with the Relative Electronic Energies and ZPE Corrected Energies (in Parentheses)

was utilized to investigate the whole green-to-red conversion mechanism. All intermediates and transition states in protein were optimized by ONIOM mechanical embedding (ME) scheme. A new quadratic coupled algorithm was used for transition state optimization.28 ONIOM harmonic vibrational frequency calculations of the whole system at the same level were further performed to characterize and verify the key transition states and intermediates and to calculate the zero point energy (ZPE) corrections. Single-point calculations were also performed on the key optimized structures by the ONIOM(B3LYP/ 6-311G*:AMBER) method. Effect of the larger basis set (6311G*) on the energetic profiles is insignificant. All energies

presented in the text are relative electronic energies at the ONIOM(B3LYP/6-31G*:Amber) level relative to 1, unless otherwise stated, without or with ZPE corrections (in parentheses). The gas phase optimization for the truncated green- and redform chromophore models was performed by the two-stateaverage SA2-CASSCF(14e,13o)/6-31G* (the green form) and SA2-CASSCF(12e,12o)/6-31G* (the red form) methods by the Molpro program, respectively.29-31 The chromophores have also been optimized by the B3LYP/6-31G* method. The vertical absorption energies on the optimized structures were then evaluated by CASPT2(14e,13o)/6-31G* and CASPT2(12e,12o)/



SCHEME 5: Structure for the ONIOM Calculations and the QM Models (Except Link Atoms)

6-31G* methods for the green- and red-form chromophores, respectively. A level shift of 0.2 au was used in the CASPT2 calculations to avoid intruder-state problems.32 The SACCI(LevelTwo)/D95(d) method has also been used to evaluate the absorptions of the optimized chromophores. In addition, the absorptions of the green- and red-form chromophores in the proteins were further calculated by ONIOM(QM:AMBER)-EE, where the QM method is CASPT2 or SAC-CI, based on ONIOM(B3LYP:AMBER) and ONIOM(CASSCF:AMBER) optimized proteins.33

Results and Discussion E1 Pathway. At first we examine the E1 pathway, in which the peptide CR-N backbone cleavage occurs at the beginning. As shown in Figures 1 and 2, the acid-catalyzed heterolytic peptide CR-N backbone cleavage of the green-form Kaede 1 takes place concurrently with the proton transfer from His62 Nδ to Phe61 oxygen via a transition state TS1-2 with a barrier of about 18.4 (15.2 with ZPE correction, in parentheses throughout the paper) kcal/mol and gives 2 with the formation

Figure 1. Potential energy surfaces (in kcal/mol) of the E1 mechanism for Kaede calculated by the ONIOM(B3LYP/6-31G*:MM) method. The ZPE corrected energies are in parentheses, and the ONIOM(B3LYP/6-311G*:MM) computed energies with ZPE correction from the ONIOM(B3LYP/ 6-31G*:MM) method are in square brackets. Only the key QM part is shown.

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Figure 2. Acid-assisted CR-N bond cleavage in the E1 pathways for Kaede by the ONIOM(B3LYP/6-31G*:AMBER) method. Energies (ZPE corrected energies in parentheses) are in kcal/mol. Only the key QM part has been shown.

Figure 3. Two deprotonation routes for the E1 mechanism in Kaede by the ONIOM(B3LYP/6-31G*:AMBER) method. Energies (ZPE corrected energies in parentheses) are in kcal/mol. Only the key QM part has been shown.

of a neutral leaving imidic acid. This process is endothermic by about 15.5 (12.4) kcal/mol. The CR-N cleavage and proton transfer cannot take place stepwise; no stable intermediate corresponding to the sole proton transfer from His62 to Phe61 in 1 can be found, but the calculations led to 1 instead. In TS1-2 connecting 1 and 2, the breaking His62 CR-N, the breaking His62 Nδ-HN and the forming His62 HN-Phe61 O distances are 2.01, 1.55, and 1.05 Å, respectively (Figure 2). The proton from the Nδ of His62 is almost fully transferred to the amidic oxygen of Phe61, while the His62 CR-N bond is substantially broken, suggesting that this is a concerted but asynchronous TS. The ion-pair cleavage product 2, although endothermic, is stabilized substantially by the conjugation between the cationic His62 CR and the negatively charged chromophore (Figure 2).13 To complete the E1 pathway from 2 to afford the product 5, both the tautomerization of the neutral imidic acid form of one fragment to give the more stable amide form and the deproto-

nation of His62 Cβ by Glu212 have to take place. If the deprotonation occurs first, 2 leads to 3 via TS2-3 with the relative energy of 27.7 (21.5) kcal/mol (Figures 1, 3, and 4) and then water-assisted tautomerization transforms 3 to 5 via TS3′-5′ (see below for the prime) with the relative energy of 16.1 (11.8) kcal/mol. On the other hand, if the water-assisted tautomerization takes place first, 2 is converted to 4 via TS2′4′ with the barrier of 25.3 (18.2) kcal/mol (or TS2-4 with the barrier of 27.2 (22.7) kcal/mol), and then deprotonation gives 5 via TS4-5 with the barrier of 10.6 (4.4) kcal/mol (Figures 1, 3, and 4). Among the above-discussed two pathways, the latter with tautomerization followed by deprotonation seems to be slightly more favorable. The reason why the water-assisted tautomerization barrier at TS2′-4′ is lower than the deprotonation barrier at TS2-3 by 2.4 (3.3) kcal/mol can be attributed to a higher exothermicity for the tautomerization.



Figure 4. Tautomerization in Kaede calculated by the ONIOM(B3LYP/6-31G*:AMBER) method. Energies (ZPE corrected energies in parentheses) are in kcal/mol. Only the key QM part has been shown.

When we optimized a reactant starting from TS2′-4′, the obtained structure 2′ was higher in energy than 2 by 1.2 kcal/ mol (Figure 4). This path can be compared with the path through TS2-4 with the barrier 1.9 (4.5) kcal/mol higher than TS2′-4′, which directly connects from 2 (see Figures 1 and 4). Apparently, in 2 and 2′, there are notably different locations and orientations of one QM water molecule that participates in the tautomerization reaction. In 2 this water molecule is singly hydrogen-bonded to the Phe61 fragment and also to another MM water and Gln38 (not shown in the figures), while in 2′ this water molecule forms two hydrogen bonds with HN and HO of Phe61, which facilitates tautomerization with a relatively low energy (Figure 4).34 A similar situation was also found for 3 from TS2-3 and 3′ from TS3′-5′. There are also differences between 4 from TS4-5 and 4′ from TS2′-4′ as well as 5 from TS4-5 and 5′ from TS3′-5′. Considering the high mobility of water molecules in a protein environment and the relatively small energy differences between these different structures, we did not explicitly determine the pathway connecting them. We rather assume that the conversion between these structures takes place with ease, and concentrate more on the reaction steps requiring substantial activation energies. Upon reaching the deprotonated complexes 3 and 5, Glu212 becomes protonated and neutral, and by a rotation of about 90° loses a hydrogen bond with His194 and forms a new hydrogen bond with the imidazolinone nitrogen of the chromophore. The side chain of His194 also rotates to form another hydrogen bond with the neighboring backbone of Asp193. In this connection, the rotation of Glu212 and His194 has previously been found in the trans chromophore of IrisFP, a mutant of EosFP.7

An alternative and not-yet-proposed acid-catalyzed peptide backbone cleavage process has been found to give directly the above-discussed stable intermediate 4 via TS1-4, where the proton from the Nδ is directly transferred to the N of His62 without the involvement of tautomerization of the imidic acid (Figures 1 and 2). The computed energy of TS1-4 for this sixmembered-ring cleavage pathway is 24.7 (20.0) kcal/mol, which is very close in energy to the above-discussed stepwise pathway via TS1-2 and TS2′-4′. Although the breaking His62 CR-N bond length of 2.06 Å is similar, TS1-4 with the transferring HN-Nδ distance of only 1.11 Å is in an earlier stage of proton transfer than TS1-2 (Figure 2). The entire potential energy surfaces for the green-to-red photoconversion via the proposed E1 mechanism are shown in Figure 1. Overall, the C-N bond cleavage and tautomerization steps are suggested to have higher barriers than the deprotonation step. The most favorable E1 route is 1 f TS1-2 f 2 f TS24′ f 4 f TS4-5 f 5. However, alternative pathways (such as 1 f TS1-4 f 4) within the E1 mechanism are rather close in energy and may compete with this pathway. E2 Pathway. As to the proposed E2-type β-elimination mechanism for EosFP,14 after many trials, the concerted E2type transition state involving the simultaneous CR-N bond cleavage and deprotonation by Glu212 could not be found for Kaede (and possible for EosFP), but the optimization all led to the E1 or E1cb (see below) pathway. To estimate energetic profile for the E2 pathway, 2D relaxed scan calculations with respect to the two key distances (Hβ-Cβ and CR-N) with all other geometrical parameters optimized were performed. As indicated in Figure 5, for the E2-type reaction, the reaction path

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Figure 5. Potential energy surfaces (in kcal/mol) along the two key reaction coordinates (in angstrom, other coordinates optimized) in Kaede calculated by the ONIOM(B3LYP/6-31G*:MM) method.

has a high-energy potential hill of about 33.8 kcal/mol and is not likely to occur. E1cb Pathway. As mentioned above, in search of the E2 path, a new E1cb mechanism initiating with deprotonation by Glu212 followed by the peptide bond cleavage was found to be lower in energy (Scheme 2, as well as Figure 6). In the E1cb mechanism, the deprotonation transition state TS1-6 has a relatively low barrier of about 14.3 (11.8) kcal/mol to afford intermediate 6. The proximity of the cationic imidazole should increase the acidity of CβHβ and stabilize the deprotonation product 6. Compared to the deprotonation transition states TS2-3 and TS4-5 in the E1 mechanism, TS1-6 is a later transition state with Cβ-Hβ and Hβ-OGlu bond distances of 1.42 and 1.23 Å, respectively. This is consistent with the fact the deprotonation steps in the E1 mechanism are exothermic, while that in the

Li et al. E1cb mechanism is endothermic. The reaction barrier of the subsequent concerted but asynchronous CR-N bond cleavage via TS6-3′′ giving intermediate 3′′ is 20.2 (17.8) kcal/mol, with respect to 1. The cleaving CR-N bond distance is 2.00 Å in TS6-3′′, which is similar to that in TS1-2 for the E1 mechanism. However, different from TS1-2, the O-HN and HN-Nδ bond distances (1.62 and 1.06 Å, respectively) in TS6-3′′ suggest a still early stage of the proton transfer from the His62. The strong interaction between the anionic Cβ derived from deprotonation and the cationic imidazole presumably lowers the acidity of the cationic imidazole and thus proton transfer becomes harder in the E1cb mechanism. The acid-catalyzed peptide backbone cleavage concerted with the proton directly transferred to the N of His62, which is similar to the case for TS1-4 in the E1 mechanism, could not be found, possibly due to unfavorable disruption of the conjugation interaction between the anionic Cβ and the cationic imidazole as well as the lower acidity of the imidazole NδH of His62 in the transition state. As in the E1 mechanism, in the E1cb mechanism the intermediate 3′′ can transform to the more stable isomer 3 and then proceed with the water-assisted tautomerization to finally form a more stable complex 5. A tautomerization route from intermediate 3′′ via TS3′′-5′′ was also found and directly gave intermediate 5′′ with the computed barrier of about 20.8 (17.7) kcal/mol (Figures 6-7); this is 4.7 kcal/mol higher in energy than TS3′-5′ connecting different conformations. This again shows that the water-assisted tautomerization is favorable. Similar to the case of the E1 mechanism, the reaction barrier for the CR-N bond cleavage step is higher than that for the deprotonation step in the E1cb mechanism. Intermediate 5′′ can further isomerize to form a more stable intermediate 5 by rotation of Glu212. To roughly estimate energies of possible key processes in the lowest excited singlet S1 (π f π*) state of the chromophore, ONIOM(TD B3LYP/6-31G*:Amber) single-point calculations

Figure 6. E1cb mechanism in Kaede calculated by ONIOM(B3LYP/6-31G*:AMBER). Energies (ZPE corrected energies in parentheses) are in kcal/mol. Only the key QM part has been shown.



Figure 7. Potential energy surfaces (in kcal/mol) of the E1cb mechanism for Kaede calculated by the ONIOM(B3LYP/6-31G*:MM) method. The ZPE corrected energies are in parentheses, and the ONIOM(B3LYP/6-311G*:MM) computed energies with ZPE correction from the ONIOM(B3LYP/ 6-31G*:MM) method are in square brackets.

TABLE 1: Absorptions (in eV) of the Green- and Red-Form Chromophores in the Gas Phase and Proteins green-form (expt 2.45) red-form (expt 2.17)

QM for energy//QM for geometry

gas phase ∆Egasa

protein (geometric) ∆EQM,modelb

protein (total) ∆EONIOM-EEc

SAC-CI//B3LYP SAC-CI//CASSCF CASPT2//CASSCF SAC-CI//B3LYP SAC-CI//CASSCF CASPT2//CASSCF

2.50 2.55 2.68 1.62 1.76 2.29

2.30(-0.20) 2.31(-0.24) 2.66(-0.02) 1.63(+0.01) 1.55(-0.21) 2.17(-0.12)

2.34(+0.04) 2.33(+0.02) 2.68(+0.02) 2.14(+0.51) 2.00(+0.45) 2.72(+0.55)

a The calculated energy difference between S0 and S1 in the gas phase. b The calculated energy difference between S0 and S1 in the gas phase for the QM model part taken from the ONIOM optimized geometry. The geometrical effect on the change of absorption, ∆EQM,model//ONIOM ∆Egas//gas, is shown in parentheses. c The electronic effect on the change of absorption, ∆EONIOM//ONIOM - ∆EQM,model//ONIOM, is shown in parentheses.

were performed (Table S2, Supporting Information). It is found that the energetic of transition state for the E1 mechanism is significantly reduced and becomes more favorable in the electronic excited state; for instance, the reaction barrier for TS1-2 is reduced to 5.0 kcal/mol. However, this barrier is above the vertical excitation energy of about 56 kcal/mol (2.45 eV) required to reach the S1 state, and it is substantially higher than the energy needed for the reaction in the electronic ground state discussed above. Since the most favorable pathways for the E1cb and E1 mechanisms are similar in energy (Figures 1 and 7), these two pathways are suggested to be competitive in the electronic ground state. The present results suggest that the energetics for the reaction in the electronic ground state is well within the reach of excitation energy plus thermal energy, and modification and expansion of the chromophore in Kaede is likely to occur on the electronic ground state. After the above-mentioned bond cleavage and deprotonation, tautomerization of the His62 imidazole, which then re-forms a hydrogen bond with the broken carboxamide, might be facilitated by nearby water molecules.35 Also, the neutral Glu212 may deliver its proton to the bulk solvent (or the chromophore) and then re-form the salt-bridge interaction with His194 to give the observed stable red-form Kaede. However, the details of these two processes are beyond the scope of this work.

Absorption. The vertical absorption energies of the green- and red-form chromophores in the gas phase and in Kaede are summarized in Table 1.36-38 As expected,19 the ONIOM(SAC-CI(level-2)/D95(d): AMBER)-EE//ONIOM(B3LYP/6-31G*:AMBER) method gives a good agreement with the experimental values. For the greenform chromophore in Kaede, the geometric effect by the protein plays the dominant role in the red shift in the absorption. On the other hand, in the red form of Kaede the electronic effect (electrostatic interaction and polarization) by the protein is the major role in the blue shift. As shown in Figures S3-S5 (Supporting Information), the π f π* excitation of the greenform chromophore involves intramolecular charge transfer (ICT) mainly from the phenol moiety to the bridge carbon, whereas the π f π* excitation of the red-form chromophore mainly involves ICT from the phenol and imidazolinone moieties to the newly formed alkenyl moiety. The presence of the newly formed alkenyl bond might also be useful to design a new type of FPs, if photoinduced addition/elimination to the alkenyl bond could happen.39 Conclusion In this work, the ONIOM(QM:MM) method has been employed to study the unusual peptide bond cleavage reaction in the green-to-red photoconversion of the fluorescent protein

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Kaede. Three reaction pathways (E1, E1cb, and E2 mechanisms) involving the cleavage of the peptide bond between the amide N and CR at His62, as well as deprotonation of the Cβ by Glu212, have been investigated. The photoconversion reaction is proposed to take place in the electronic ground state and the stepwise E1 mechanism and the new E1cb mechanism are likely to compete. In addition, a direct E1 pathway via TS1-4 with fewer steps, which is calculated to be slightly higher in energy than the most favorable pathway for the E1 and E1cb mechanisms, might also operate. If the reaction takes place in the electronic excited state S1, the E1 mechanism may be most favorable. The proposed concerted E2 β-elimination mechanism is much higher in energy and is unlikely to occur. Our proposed pathways for Kaede may also operate in the other irreversible photoconversion fluorescent proteins. Acknowledgment. L.W.C. acknowledges the Fukui Institute Fellowship. This work is in part supported by Japan Science and Technology Agency (JST) with a Core Research for Evolutional Science and Technology (CREST) grant in the Area of High Performance Computing for Multiscale and Multiphysics Phenomena. The computational resource at Research Center of Computer Science (RCCS) at the Institute for Molecular Science (IMS) is also acknowledged. Supporting Information Available: Figures S1-S5 (tautomerization pathways, active space conformations, Mulliken charge analysis), Tables S1 and S2 (H bond distances, energies), and Cartesian coordinates of the QM-optimized structures. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Tsien, R. Y. Annu. ReV. Biochem. 1998, 67, 509–544. (2) Zimmer, M. Chem. ReV. 2002, 102, 759–781. (3) Shaner, N. C.; Patterson, G. H.; Davidson, M. W. 2007, 120, 4247– 4260. (4) Dronpa: Ando, R.; Mizuno, H.; Miyawaki, A. Science 2004, 306, 1370–1373. (5) asFP595: Andresen, M.; Wahl, M. C.; Stiel, A. C.; Gra¨ter, F.; Scha¨fer, L. V.; Trowitzsch, S.; Weber, G.; Eggeling, C.; Grubmu¨ller, H.; Hell, S. W.; Jakobs, S. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 13070– 13074. (6) mTFP0.7: Henderson, N. J.; Ai, H.-W.; Campbell, R. E.; Remington, S. J. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 6672–6677. (7) IrisFP: Adam, V.; Lelimousin, M.; Boehme, S.; Desfonds, G.; Nienhaus, K.; Field, M. J.; Wiedenmann, J.; McSweeney, S.; Nienhaus, G. U.; Bourgeois, D. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 18343– 18348. (8) Photoactivatable: Patterson, G. H.; Lippincott-Schwartz, J. Science 2002, 297, 1873–1877. (9) Kaede: Ando, R.; Hama, H.; Yamamoto-Hino, M.; Mizuno, H.; Miyawaki, A. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 12651–12656. (10) EosFP: Wiedenmann, J.; Ivanchenko, S.; Oswald, F.; Schmitt, F.; Ro¨cker, C.; Salih, A.; Spindler, K. D.; Nienhaus, G. U. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15905–15910. (11) KikGR: Tsutsui, H.; Shimizu, H.; Mizuno, H.; Nukina, N.; Kuruta, T.; Miyawaki, A. Chem. Biol. 2009, 16, 1140–1147. (12) Mizuno, H.; Mal, T. K.; Tong, K. I.; Ando, R.; Furuta, T.; Ikura, M.; Miyawaki, A. Mol. Cell 2003, 12, 1051–1058. (13) Hayashi, I.; Mizuno, H.; Tong, K. I.; Furuta, T.; Tanaka, F.; Yoshimura, M.; Miyawaki, A.; Ikura, M. J. Mol. Biol. 2007, 372, 918– 926. (14) Nienhaus, G. U.; Wiedenmann, J.; Nar, H. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 9156–9159. (15) Lelimousin, M.; Adam, V.; Nienhaus, G. U.; Bourgeois, D.; Field, M. J. J. Am. Chem. Soc. 2009, 131, 16814–16823. (16) Morozova, O. B.; Yurkovskaya, A. Y. Angew. Chem., Int. Ed. 2010, 49, 7996–7999. (17) Parthasarathi, R.; He, Y.; Reilly, J. P.; Raghavachari, K. J. Am. Chem. Soc. 2010, 132, 1606–1610.

Li et al. (18) Dapprich, S.; Koma´romi, I.; Byun, S.; Morokuma, K.; Frisch, M. J. THEOCHEM 1999, 461-462, 1–21. (19) We recently proposed one feasible pathway via photo-isomerization of the imidazolinone ring coupled with ESPT for Dronpa: Li, X.; Chung, L. W.; Mizuno, H.; Miyawaki, A.; Morokuma, K. J. Phys. Chem. B 2010, 114, 1114–1126 and Li, X.; Chung, L. W.; Mizuno, H.; Miyawaki, A.; Morokuma, K. J. Phys. Chem. Lett. 2010, 1, 3328. (20) (a) Dolinsky, T. J.; Nielsen, J. E.; McCammon, J. A.; Baker, N. A. Nucleic Acids Res. 2004, 32, W665. (b) Li, H.; Robertson, A. D.; Jensen, J. H. Proteins: Struct., Funct., Bioinf. 2005, 61, 704. (21) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Merz, K. M.; Wang, B.; Pearlman, D. A.; Crowley, M.; Brozell, S.; Tsui, V.; Gohlke, H.; Mongan, J.; Hornak, V.; Cui, G.; Beroza, P.; Schafmeister, C.; Caldwell, J. W.; Ross, W. S.; Kollman, P. A. AMBER 9; University of California: San Francisco, CA, U.S., 2006. (22) (a) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179. (b) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Comput. Phys. 1983, 79, 926. (23) (a) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian03 DeVelopment Version, G.01 and G.03; Gaussian, Inc.: Wallingford, CT, U.S., 2007. (24) (a) Becke, A. D. J. Chem. Phys. 1986, 84, 4524. (b) Perdew, J. P. Phys. ReV. B 1986, 33, 8822. (25) (a) Maseras, F.; Morokuma, K. J. Comput. Chem. 1995, 16, 1170. (b) Humbel, S.; Sieber, S.; Morokuma, K. J. Chem. Phys. 1996, 105, 1959. (c) Matsubara, T.; Sieber, S.; Morokuma, K. Int. J. Quantum Chem. 1996, 60, 1101. (d) Svensson, M.; Humbel, S.; Froese, R. D. J.; Matsubara, T.; Sieber, S.; Morokuma, K. J. Phys. Chem. 1996, 100, 19357. (e) Svensson, M.; Humbel, S.; Morokuma, K. J. Chem. Phys. 1996, 105, 3654. (f) Vreven, T.; Morokuma, K. J. Comput. Chem. 2000, 21, 1419. (g) Vreven, T.; Byun, K. S.; Koma´romi, I.; Dapprich, S.; Montgomery, J. A., Jr.; Morokuma, K.; Frisch, M. J. J. Chem. Theory Comput. 2006, 2, 815. (26) A new quadratic coupled algorithm was used in all ONIOM calculations: Vreven, T.; Frisch, M. J.; Kudin, K. N.; Schlegel, H. B.; Morokuma, K. Mol. Phys. 2006, 104, 701. (27) Altun, A.; Shaik, S.; Thiel, W. J. Comput. Chem. 2006, 27, 1324. (28) Vreven, T.; Frisch, M. J.; Kudin, K. N.; Schlegel, H. B.; Morokuma, K. Mol. Phys. 2006, 104, 701. (29) (a) Werner, H.-J.; Knowles, P. J. J. Chem. Phys. 1985, 82, 5053. (b) Knowles, P. J.; Werner, H.-J. Chem. Phys. Lett. 1985, 115, 259. (30) Werner, H.-J.; Knowles, P. J.; Lindh, R.; Manby, F. R.; Schu¨tz, M.; Celani, P.; Korona, T.; Rauhut, G.; Amos, R. D.; Bernhardsson, A.; Berning, A.; Cooper, D. L.; Deegan, M. J. O.; Dobbyn, A. J.; Eckert, F.; Hampel, C.; Hetzer, G.; Lloyd, A. W.; McNicholas, S. J.; Meyer, W.; Mura, M. E.; Nicklass, A.; Palmieri, P.; Pitzer, R.; Schumann, U.; Stoll, H.; Stone, A. J.; Tarroni, R.; Thorsteinsson, T. MOLPRO 2006.1, a package of ab initio programs. (31) The molecular orbitals and the occupancies of the active space as well as major electronic configurations in the ground state for the chromophore in the green and red forms of Kaede are shown in Figures S3 and S4 of the Supporting Information. (32) (a) Celani, P.; Werner, H.-J. J. Chem. Phys. 2000, 112, 5546. (b) Roos, B. O.; Andersson, K. Chem. Phys. Lett. 1995, 245, 215. (c) Andersson, K.; Malmqvist, P.-Å.; Roos, B. O. J. Chem. Phys. 1992, 96, 1218. (33) (a) Celani, P.; Werner, H.-J. J. Chem. Phys. 2000, 112, 5546. (b) Nakatsuji, H. Chem. Phys. Lett. 1978, 59, 362. (c) Nakatsuji, H. Chem. Phys. Lett. 1989, 67, 329. (d) Nakatsuji, H. Chem. Phys. Lett. 1989, 67, 334. (34) Tautomerization of the simplest imidic acid without the water catalyst has a very high barrier of 30.7 kcal/mol with ZPE correction, which is 24.0 kcal/mol higher in energy than the water-assisted tautomerization pathway (Figure S1, Supporting Information). (35) Tautomerization of the imidazole moiety of His62 with two and three water molecules as the catalyst, respectively, has a high barrier of 34.4 and 18.1 kcal/mol with ZPE correction. Therefore, more water molecules can assist the tautomerization (Figure S2, Supporting Information).

Photoconversion in the Fluorescent Protein Kaede (36) The SAC-CI computed absorption energy to S2 for the red-form chromophore is 3.19 eV (DFT geometry) and 3.29 eV (CASSCF geometry) in the gas phase, while it is calculated to be 3.53 eV (DFT geometry) and 3.48 eV (CASSCF geometry) in the protein. (37) Nifosı´, R.; Amat, P.; Tozzini, V. Systematic study of the absorption energy for different chromophores. J. Comput. Chem. 2007, 28, 2366–2377.

J. Phys. Chem. B, Vol. 114, No. 49, 2010 16675 (38) Yampolsky, I. V.; Kislukhin, A. A.; Amatov, T. T.; Shcherbo, D.; Potapov, V. K.; Lukyanov, S.; Lukyanov, K. Absorption of the red-form chromophore and its analogs in solution. Bioorg. Chem. 2008, 36, 96. (39) Dempsey, G. T.; Bates, M.; Kowtoniuk, W. E.; Liu, D. R.; Tsien, R. Y.; Zhuang, X. J. Am. Chem. Soc. 2009, 131, 18192–18193.

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