Time Resolved EPR Study on the Photoinduced Long-Range Charge

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Time Resolved EPR Study on Photoinduced Long-Range Charge-Separated State in Protein: Electron Tunneling Mediated by Arginine Residue in Human Serum Albumin Masaaki Fuki, Hisao Murai, Takashi Tachikawa, and Yasuhiro Kobori J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b01072 • Publication Date (Web): 26 Apr 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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The Journal of Physical Chemistry

Time Resolved EPR Study on Photoinduced LongRange Charge-Separated State in Protein: Electron Tunneling Mediated by Arginine Residue in Human Serum Albumin Masaaki Fuki§, Hisao Murai§, Takashi Tachikawa†, and Yasuhiro Kobori†,* §

Graduate School of Science and Technology, Shizuoka University, 836 Ohya Suruga-ku,

Shizoka 422-8059, Japan †

Department of Chemistry, Graduate School of Science, Kobe University, 1-1 Rokkoudai-cho,

Nada-ku, Kobe 657-8501, Japan

ABSTRACT: To elucidate how local molecular conformations play a role on electronic couplings for the long-range photoinduced charge-separated (CS) states in protein systems, we have analyzed time-resolved electron paramagnetic resonance (TREPR) spectra by polarized laser irradiations of 9,10-anthraquinone-1-sulfonate (AQ1S−) bound to human serum albumin (HSA). Analyses of the magnetophotoselection effects on the EPR spectra and a docking simulation clarified the molecular geometry and the electronic coupling of the long-range CS

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states of AQ1S•2−–tryptophan214 radical cation (W214•+) separated by 1.2 nm. The ligand of AQ1S− has been demonstrated to be bound to the drug site I in HSA. Molecular conformations of the binding region were estimated by the docking simulations, indicating that an arginine218 (R218+) residue bound to AQ1S•2− mediates the long-range electron-transfer. The energetics of triad states of AQ1S•2−–R218+–W214•+ and AQ1S−–R218•–W214•+ have been computed on the basis of the density functional molecular orbital calculations, providing the clear evidence for the long-range electronic couplings of the CS states in terms of the superexchange tunneling model through the arginine residue.

Introduction Proteins and enzymes function by recognizing substrates, catalyzing several chemical reactions at the active regions. Recently, photoinduced protein damages after the electrontransfer (ET) reactions have attracted great attentions, since the oxidized proteins by the photosensitizations can lead to the cell-deaths in cancers.1 Therefore, it is highly important to elucidate how the molecular conformations of the reactive intermediate species and of the amino acid residues play roles for generations of long-lived photoinduced charge-separated (CS) states at these binding sites in the protein-substrate complexes. Although the protein ET dynamics have been hot topics in many years2-7, only a few studies have been performed to experimentally characterize both the molecular geometries and the electronic couplings (VDA) of the reaction intermediates in protein systems.8 Time-resolved electron paramagnetic resonance (TREPR) has been a powerful method to clarify the structures of the transient radical pairs9-12 and have been applied to the photosynthetic reaction centers8,

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and to the several donor-accepter linked


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systems14-16. VDA is one of an important factors to determine the efficiency of ET reactions.17 Using the quantum mechanical theories, effects of the molecular geometries on the VDA have been predicted through the several mediators in terms of the superexchange mechanism.18, 19 In our previous study20, a protein-ligand structure and its coupling term VDA of 5.4 cm−1 were characterized for a short-range CS state at a 0.65 nm separation in a 9,10-anthraquinone-1sufonate (AQ1S−)–human serum albumin (HSA) complex (Figure 1). However, the mechanism of the distant CS state generation is unknown in the protein-ligand system. In the present study, the TREPR method is applied to determine the structure and the VDA value for a photoinduced long-range CS state at an 1.2 nm distance in the AQ1S−–HSA complex. The electronic interaction has been characterized for the distant CS state composed of 9,10-anthraquinone-1sulfonate anion radical (AQ1S•2−) and tryptophan cation radical (W214•+). It has been concluded that an arginine residue (R218+) plays a major role to mediate the electronic interaction in the protein pocket, demonstrating an importance of the protein-ligand conformational structure for the long-range ET reactions.


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HSA

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−

AQ1S

Figure 1. Structure of HSA and structure formula of AQ1S− employed as the electron acceptor and the photosensitizer, respectively.

Experimental Section Anthraquinone-1-sulfonate sodium salt (Tokyo Kasei) and human serum albumin (Sigma-Aldrich, purity 97 %) were dissolved in the mixture of the aqueous phosphate buffer (pH = 7.0) and grycerol with 1:1 volume ratio. The phosphate buffer was prepared by mixing dipotassium phosphate (K2HPO4) solution and monopotassium phosphate (KH2PO4) solution dissolved in de-ionized water. The concentrations of the sample solution were 5 mM both for HSA and for AQ1S−. The freeze–pump–thaw cycle was performed to remove the oxygen


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molecules dissolved in the sample solution. The sample solution was sealed in the quartz tube with 4 mm o.d. under the vacuum. The TREPR measurements were carried out using the Bruker EMX system. A wide-band preamplifier was used to enhance the time resolution (~60 ns). The sample was irradiated by the third harmonics (355 nm) of a Nd:YAG laser (Continuum, Minilite II, fwhm ~5 ns) to excite the AQ1S− molecule. The magnetophotoselection (MPS) measurements were carried out by changing the orientation of the laser polarization with respect to external magnetic field. The depolarized light was produced by placing the depolarizer (SIGMA KOKI, DEQ 1N) between the laser output and the microwave cavity and was used for depolarized experiments. The transient EPR signals were averaged by a digital oscilloscope (Tektronix, TDS 520D) and were transferred to personal computer to obtain the TREPR data. The temperature was controlled using a nitrogen gas-flow system. Docking simulations were performed using an Autodock Vina21 software. For the docking simulations, the HSA structure was obtained from an x-ray structure of HSA–warfarin complex (PDB ID: 2BXD). Warfarin was replaced by the AQ1S− molecule as the ligand in the docking simulation. The amino acid residues of TYR150, LYS195, LYS199, PHE211, TRP214, ARG218, LEU219, ARG222, LEU238, VAL241, HIS242, ARG257, LEU260, ILE264, ILE290 and GLU292 which are originally located around the warfarin molecule were treated as flexible. The other residues were treated as rigid. The structure of the AQ1S− was optimized using the density functional theory with B3LYP/6-31+G∗∗ by the Gaussian 09 packages22. For the calculations of the adiabatic energies, the molecular orbital calculations have been performed by using the Gaussian 09 program package. The structures of the AQ1S− and the


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amino acids were optimized using B3LYP/6-31+G∗∗ level. In the energy calculations, the polarizable continuum model23, 24 was adapted.

Results and Discussion Geometries of the CS states. Figure 2 shows TREPR spectra obtained for the HSA–AQ1S− system in aqueous phosphate buffer at a delay time of 1 µs after the 355 nm laser irradiation at T = 100 K. The laser polarization (L) effects on the TREPR spectra were observed in Figure 2a)– 2c), in which a) depolarized, b) parallel, and c) perpendicular lights were employed with respect to the external magnetic field (B0). We previously reported the TREPR spectra in the present system and characterized the short-range CS states.20 The spectra showing E/E/A/A broad fine structures (E and A denote microwave emission and absorption, respectively.) from 325 to 345 mT in Figure 2 were assigned to the short-range CS states of AQ1S•2−–histidine radical cation (H242•+) in the previous study.20 At the center position of 336 mT as marked by asterisks, the sharp spectra were assigned to the long-range CS state. The spectra in Figure 2 were thus explained by sums of two different CS states composed of 1) AQ1S•2––H242•+ for the broad spectra and 2) AQ1S•2−–W214•+ for the sharp spectra, as reported previously.20 The net E polarization of the signals were explained by the triplet mechanism (TM), demonstrating that the long range CS states were produced through the excited triplet states of AQ1S− generated by the intersystem crossing to the Z sublevel of the n–π* triplet state from the π–π* singlet states of AQ1S− as reported previously.20 We reported a theoretical model25 of the electron spin polarization transfer (ESPT) by which the anisotropic dipolar coupling characters of the excited triplet states are transferred to CS states in the spin polarization. This model has been applied to


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determine the protein–ligand structures and the exchange couplings (2J) of the CS states. The red spectra in Figure 2 were calculated as sums of the two different CS states with using the ESPT model. This implies that some proteins undergo the photoinduced ETs from H242 and that the others do the ET reactions from W214 to the ligands of

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AQ1S–*, demonstrating an

inhomogeneity in the amino acid residues at the binding pocket.

A

a) Depolarized

* b) B0 || L

E

* c) B0 ⊥ L

* 315 320 325 330 335 340 345 350 355

Magnetic Field / mT Figure 2. TREPR spectra observed for HSA(5.0 mM)–AQ1S– (5.0 mM) system at 1 µs after the 355 nm laser irradiation at 100 K with using a) the depolarized light, b) the light (L) parallel to the external magnetic field B0 and c) L perpendicular to B0. The red lines are simulated TREPR spectra using the ESPT model taking into account the MPS effects for the two different CS states.

Figure 3 shows expanded TREPR spectra at the center field regions (asterisks in Figure 2) at the delay times of 1 µs. Resolved three peaks are obtained around 334.5, 335.4 and 336.8


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mT and are affected by the laser polarizations. Analysis of the MPS effect26 has been useful to determine the direction of the principal axis of the spin dipolar coupling with respect to the transition dipole moment (M) of the molecule. When L is parallel to B0 in Figure 3b, the outer two peaks indicated by the solid arrows exhibit an intense E/A polarization effect. On the other hand, when the L is perpendicular to B0, the center peak signal is enhanced while the outer E/A polarization effect is minor in Figure 3c, resulting in a more symmetrical emissive spectrum shape. The above spectral changes by the laser polarizations are explained by the anisotropic characters in the spin dipolar interaction; the enhanced outer peak signals for L || B0 (Figure 3b) denote that the direction of M in AQ1S– is close to the inter-spin vector between the radicals in the CS state, since the splitting by the spin-spin dipolar coupling is larger when the B0 is directed to the inter-spin vector.

a) Depolarized E b) B0 || L

c) B0 ⊥ L

334

335

336

337

338

Magnetic Field / mT


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Figure 3. TREPR spectra observed for HSA(5.0 mM)–AQ1S– (5.0 mM) system at 1 µs for the expanded field regions around asterisk marks in Figure 2 with a) the depolarized light, b) the L parallel to B0 and c) the L perpendicular to B0. The red lines are simulated TREPR spectra using the ESPT model with 2J = 0.5 mT.

For the simulations of the TREPR spectra, we have employed the ESPT model25 to determine the structure and 2J of the CS states with taking into account the effects of the laser polarizations and of the anisotropies in the Zeeman and hyperfine interactions, as reported previously.20 The zero-field splitting (ZFS) parameters of the excited triplet state of AQ1S− were used as DT = −237 mT and ET = 5.3 mT. The initial populating ratios of the triplet sublevels of AQ1S– by the S1-T1 intersystem crossing are considered to be px = py = 0 and pz = 1 for the Tx, Ty, and Tz respectively.27 The principal axes of the ZFS interactions are set as shown in Figure 4. The parameters of the dipole-dipole interaction in the CS state of AQ1S•2−–W214•+ were determined to be Dcs = −1.7 ± 0.1 mT with applying the point dipole approximation. In the spin dipolar coupling, the principal Z’ axis is set to be linear with the inter-spin vector as shown in Figure 4. The direction of the Z’ axis was represented by the polar angle θ and the azimuthal angle φ with respect to the principal axes (X, Y, and Z) of the triplet state of AQ1S− as shown in Figure 4. The anisotropies in the g-tensor and in the hyperfine tensors were also considered for Trp•+ in the CS state as detailed in the Supporting Information (SI). In the CS states, the gtensors of AQ1S•2−, W214•+ and H242•+ are computed for the reference axes system of (X’, Y’, Z’) by rotation matrices Rg,i with using the Euler angles.20 The Euler angles (α, β and γ) of the principal axes (gX, gY and gZ) are defined with respect to the principal axes (X, Y and Z) of the zero-field splitting interaction of the triplet AQ1S− as shown in Figure 4b. In principle, the


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anisotropic hyperfine tensors in the amino acid radicals influence the EPR spectrum shapes in the CS states and are thus considered by using the rotation matrices Rg,i, as reported previously.20 Thereby, one is able to determine the molecular conformations in the distant CS states from α, β and γ together with θ and φ as detailed in SI with Figure S1 and Table S1.

Figure 4. a) Geometries of CS state of AQ1S•2−–W214•+ with respect to the principal axes of the triplet state of 3AQ1S−*. θ = 40° and φ =151° are determined as the direction of spin dipoledipole interaction of the CS state. The M denotes the direction of the transition dipole moment of AQ1S−. The δ is the angle between the M and the Z axis in YZ plane. In W214•+, two β−protons are indicated by arrows. b) Orientation of the principal axis of the g-tensor are characterized as α = 30°, β = 40° and γ = 10° with respect to the principal axes (X, Y and Z) of the zero-field splitting interaction in the triplet state of the AQ1S−.


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Concerning isotropic hyperfine coupling constants from β−protons indicated by arrows in Figure 4a, Aiso = 2.2 mT and 0.3 mT are considered from a reported study by Pogni et al.28 Magnitudes of the hyperfine coupling constants for the β-protons of the tryptophan are determined by the spatial orientation of the side chain, which are given by the McConnell equation as,29

A ( H β ) = ρCπ ( B '+ B ''cos2 ξ )

(1)

where ρCπ is the spin density on the ring carbon bonded to methylene group in the indole plane, B' and B'' are the empirical constants, in which B' is usually set to be 0, while B'' is estimated to be 5–6 mT. ξ is the dihedral angle between the pz axis of the π-orbital in the indole ring and the projected CβHβ bond as shown in Figure 5. The spin density (ρC3) was estimated to be 0.37 at the ring carbon30. The ξ values were calculated using the hyperfine coupling constants determined by the TREPR experiments and the B'' assumed to be 6.0 mT. The β-proton positions were determined to be located at ξ1 = 5.4° and ξ2 = 111.6° with respect to the pz axis of indole plane, respectively. The orientations of β-protons that well explain the EPR spectra (Figure 3) are depicted in Figure 5.


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pz axis 5.4°

Hβ1

111.6° Indole Plane C3

Hβ2 R Figure 5. The orientations of the β-protons in W214 radical cation with respect to the indole plane.

The molecular geometry of the photoinduced CS state for AQ1S•2−–W214•+ is shown in Figure 4 determined from the spectrum calculations in Figure 3. The angles of the CS state for AQ1S•2−–W214•+ are determined to be (α, β, γ, θ, φ) = (30°, 40°, 10°, 40°, 151°). Additionally, from the fitting procedures of Figure 3b and 3c, the electronic transition dipole moment (M) of the AQ1S– is determined to be δ = 60° when M is assumed to lie in the aromatic ring (YZ plane) in AQ1S− in Figure 4. This parameter well agrees with the singlet π–π* transition dipole moment of AQ1S− around 355 nm excitations20, strongly supporting the validity of the present


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fitting method to determine the geometries of the CS states. From Dcs = −1.7 ± 0.1 mT, the center-to-center distance (rcc) in the distant radical pair is determined to be rcc = 1.18 ± 0.02 nm with applying the point dipole approximation31 for the distant CS state. In Table S2 in SI, the EPR parameters are summarized for the two different CS states to reproduce the TREPR spectra in Figure 2 and 3. Susceptibilities of the angle parameters on the EPR spectrum have been examined as detailed in SI. On the conformation parameters of (α, β, γ) in W214, susceptibilities of these parameters are very weak on the TREPR spectra (Figure S3 in SI), as also seen by the large errors in the Table S2. Thus, one would find other parameter sets in (α, β, γ) to explain the EPR spectra. However, the computed TREPR spectra are highly sensitive to the angles of θ and

φ since the spin polarization by the triplet ESPT is highly sensitive to the direction of the interspin vector Z’ with respect to the principal axes (X, Y, Z) of the ZFS interaction. In principle, the spin polarization pattern produced by the triplet dipolar-couplings is the E/A or A/E effect. Such pattern is not distinguished from the E/A or A/E effect created by the isotropic J-coupling. However, since the spin dipolar couplings are anisotropic, the MPS experiments will discriminate the anisotropic E/A or A/E effect by the triplet ESPT. This is because the MPS will select the directions of the applied magnetic field in the molecular axis system of (X,Y,Z). Furthermore, when the δ angle is fixed, there still exist four possible combinations of (θ, φ) = (40°, 29°), (40°, 151°), (-140°, -29°) and (-140°, -151°) to reproduce the MPS data originating from the symmetrical equivalence.

From the x-ray structure (Figure 6) and the docking

simulation in which R218+ is ligating to the substrate, (θ, φ) = (40°, 151°) is uniquely characterized (vide infra). Instead of the 2J value of 0.5 mT utilized to completely fit the MPS data in Figure 3, we have computed the TREPR spectra for different 2J values of 0.0 mT and 1.0 mT, as shown in Figure 6. Although one finds alternate sets of (θ, φ) to reproduce the entire


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shape of the spectrum in the depolarized experiment (Figure 3a), these angle combinations will generate deviated spectra both in the parallel and perpendicular conditions, as shown in Figure 6. This assures the uniqueness of the set of (θ, φ, J) = (40°, 151°, 0.5 mT) obtained from the present MPS measurements. In the case of 2J = 0.6 mT, the deviations between the calculations and the experiments were minor, while the deviations became more prominent for 2J = 0.7 mT. Thus, the error in the 2J value is evaluated to be c.a. ± 0.1 mT as described in Table S2.

Exp. 2J = 0.0 mT, θ = 45°, φ = 130° a) Depolarized

Exp. 2J = 1.0 mT, θ = 40°, φ = 170° d) Depolarized

E

E

b) B0 || L

e) B0 || L

c) B0 ⊥ L

f) B0 ⊥ L

334

335

336

337

338

Magnetic Field / mT

334

335

336

337

338

Magnetic Field / mT

Figure 6. Computed TREPR spectra (red lines) assuming 2J = 0.0 mT (left) and 1.0 mT (right) to fit the depolarized light conditions (blue lines) in a) and d), respectively. For 2J = 0.0 mT, (θ, φ) = (45°, 130°) has been obtained to reproduce the spectrum in a). However, the above set of the parameters does not reproduce the EPR spectra for the polarized light conditions in b) and c). For the excess value of 2J = 1.0 mT, (θ, φ) = (40°, 170°) has been obtained to reproduce the spectrum in d). However, this parameter set does not again reproduce the blue lines in e) and f).


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Figure 7 shows an X-ray structure of a HSA–warfarin complex (PDB ID: 2BXD). From the X-ray structure, the warfarin molecule docks at the drug site I which is referred to as the subdomain IIA of HSA. The binding site is within the core of the domain.32 H242 and W214 are located around the warfarin molecule in the binding pocket of HSA. The rcc are 0.6 and 1.2 nm for warfarin–H242 and for warfarin–W214 in Figure 7, respectively. The geometries of warfarin in HSA are (θ, φ) = (19°, 0°) and (θ, φ) = (52°, 119°) for warfarin–H242 and for warfarin–W214, respectively in Figure 7. The TREPR conformations are (θ, φ) = (−7°, −10°) and (θ, φ) = (40°, 151°) for AQ1S•2––H242•+ and for AQ1S•2––W214•+, respectively. Similarities in the angle and distance parameters between the warfarin–HSA and the AQ1S−–HSA strongly indicate that the AQ1S− also docks at the drug site I of HSA as warfarin does.

Figure 7. X-ray structure of a HSA–warfarin complex at the binding region (PDB ID: 2BXD).


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Characterizations of the electronic couplings. From the computational simulations of the TREPR spectra to completely fit the whole experimental data in Figure 2 and 3 (red lines), it was essential to consider the exchange coupling parameters (2J) of 4.1 mT and of 0.5 mT for the AQ1S•2––H242•+ and for the AQ1S•2−–W214•+ systems, respectively, as described above. The positive 2J values were explained by the charge-transfer (CT) interaction (JCT) model33, by which the singlet-triplet energy gap is generated by the electronic coupling perturbation through the recombined singlet characters. According to the model of JCT, the exchange coupling is approximated, as follows:

2J CT =

V

2

(2)

∆ECR

where V and ∆ECR are the electronic coupling matrix element and the vertical energy gap for the CR process, respectively. If one assumes two different electronic tunneling routes in the CS state, the electronic coupling VDA should be expressed by a sum of the two couplings of V1 and V2. Depending upon the signs of V1 and V2, the VDA term can be constructive or destructive. However, fast molecular librations often dynamically average the multiple-pathways as described in Ref. 41. In this case, an ensemble-average square D-A coupling expressed by |V|2 = is effective for the exchange coupling in eq.(2). In the present study, one is thus able to evaluate the square root of the average of V =

V 2DA from J. 2J and ∆ECR are 4.1 mT and 1.0

eV for AQ1S•2––H242•+ and 0.5 mT and 1.46 eV for AQ1S•2−–W214•+, respectively. Therefore,

V = 5.4 cm−1 and V = 2.3 cm−1 have been determined for AQ1S•2––H242•+ and AQ1S•2−– W214•+, respectively.


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In our previous study20, V = 5.4 cm−1 for the AQ1S•2––H242•+ system has been explained in terms of the through-space interaction; due to the orthogonal relationship between the singly occupied molecular orbitals (SOMOs) in AQ1S•2– and H242•+, the electronic coupling is very weak, contributing to the prevention of energy-wasting charge recombination, even at a contact edge-to-edge separation (0.35 nm) between AQ1S•2– and H242•+. In the AQ1S•2−–W214•+ system in Figure 4, since the ET separation is substantially larger (rcc = 1.2 nm), the electronic coupling can be smaller. However, V = 2.3 cm−1 is evidently larger than the through-space interaction for the 1.2 nm separation34, since the orbital overlap between the SOMOs significantly decays with the increase in the through-space separation. This large electronic coupling for the AQ1S•2−–W214•+ thus needs to be explained in terms of the bridge-mediated, superexchange coupling at the binding site of HSA. Gray et al. demonstrated that the aliphatic polypeptide chains mediate the long-range electron tunnelings for the Ru-modified proteins.35-37 In the present system, AQ1S− binds to H242 at the drug site I to produce the two different CS species as described above. Thus, one is able to identify the possible tunneling route from the amino acid sequence to oxidize W214 though H242–V241–K240–···–A215–. However, since such a tunneling route extends more than 8 nm from H242 to W214 though the polypeptide chain, it is impossible to induce the nanosecond charge-separation. Therefore, the other electronic mediators for the 1.2 nm-separated CS state are required to explain the electronic coupling of 2.3 cm−1. Connection between the long-range electronic coupling and the docking structure. To evaluate the CS state structure and the mechanism of the electronic coupling, we have performed the docking simulations by using the Autodock Vina program. Figure 8 shows one of the protein


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structures around the drug site I of HSA obtained by the docking simulation. The Autodock Vina program will generate various calculated model structures on each docking simulation. The docking simulations were performed for several times by varying input parameters of the exhaustiveness and of the size in the search space. In each run, we were able to obtain the structure shown in Figure 8, although this structure was not the most stable one in the binding affinities. It is readily seen that the geometries of AQ1S− and W214 in Figure 8 are comparable to the CS geometry in Figure 4. One also finds that an arginine residue (R218+) is located between AQ1S− and W214 in Figure 8. This arginine location in close proximity to the –SO3− substituent of AQ1S− is highly reasonable38 since the arginine residue is positively charged in aqueous buffer at pH 7 because of pKa = 12.5.39 The arginine ligation to an anionic substituent (– CO−) in warfarin is also known at the drug site I of HSA.40 This strongly supports the validity of the docking structure of Figure 8 which well explains the geometry (Figure 4) of the distant CS state, as determined by the present TREPR analysis.


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Figure 8. The structure of HSA–AQ1S− complex obtained by the docking simulation. The edgeto-edge distances (dotted lines) between AQ1S– and R218+ and between R218+ and W214 are 3.2 Å and 3.6 Å, respectively.

Furthermore, the R218+ ligation to AQ1S− strongly indicates that this residue plays a role to directly mediate the electronic coupling in the distant CS state. The residue-mediated coupling has also been considered though a tryptophan on the electron tunneling from a pheophytin radical anion (HA−) to a ubiquinone (QA) in one of the sequential charge-separation steps in the bacterial photosynthetic reaction center.41 According to the McConnell superexchange model,42, 43

the electronic coupling in the bridge-mediated system is simplified as follows,

V =

hDb ∆ε

hbA

(3)

where hDb, hbA and ∆ε are the coupling between the electron donor and the bridging molecule, the coupling between the bridge and the electron acceptor, and the vertical energy gap to reduce (or oxidize) the bridge molecule from the CS state. In order to check whether the arginine is a valid mediator for the superexchange coupling in the present system, the DFT calculations (B3LYP/6-31+G**) were performed to obtain the state energies for triads of W214•+–R218+– AQ1S•2− and W214•+–R218•–AQ1S−, as shown in Figure 9 with applying the polarizable continuum model (PCM). The dielectric constant of εeff = 3 is applied on the DFT calculations to compute the adiabatic energies of W214•+ (−18672.44 eV), R218+ (−16518.79 eV), AQ1S•2− (−35707.64 eV), R218• (−16521.35 eV), and AQ1S− (−35705.57 eV) by the geometry optimizations for the individual species assuming a hydrophobic environment in the protein


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matrix around the docking domain as detailed in SI. Additionally, the electrostatic energies between the ionic species in W214•+–R218+ (0.77 eV), R218+–AQ1S•2− (−1.10 eV), W214•+– AQ1S•2− (−0.94 eV), and W214•+–AQ1S− (−0.47 eV) were obtained for the triads using εeff = 3 and center-to-center distances of 0.62, 0.87, 1.02, and 1.02 nm, respectively. Above electrostatic energies were added to the summations of the above adiabatic energies of the individual species of W214•+–R218+–AQ1S•2− (−70898.87 eV) and of W214•+–R218•–AQ1S− (−70899.36 eV). Resultant triad energies are −70900.14 eV and −70899.83 eV for W214•+–R218+–AQ1S•2− and W214•+–R218•–AQ1S−, respectively, giving rise to ∆G = 0.31 eV as the adiabatic energy gap from the distant CS state to generate W214•+–R218•–AQ1S− in Figure 9. We also considered a total reorganization energy of λ ~ 0.3 eV as a sum of the intramolecular contribution (~ 0.2 eV) and the solvent reorganization energy (~ 0.1 eV) by the protein environment, as shown in Figure 9 to obtain the potential surfaces in the triads. From Figure 9, the vertical energy gap of ∆ε = ∆G + λ = 0.6 eV is obtained to reduce the R218+ from the CS state. This small tunneling energy gap denotes that the ligating R218+ can mediate the distant ET in Figure 8. Since R218+ is situated in close proximity both to AQ1S− and to W214 at contact distances as shown in Figure 8, a relation of hbA = hDb is assumed in the present triad system. From V = 2.3 cm−1 and ∆ε = 0.6 eV, hbA = hDb = 100 cm−1 is estimated as the transfer integrals between the contact species by using eq.(3). In a bacterial photosynthetic reaction center, the transfer integral between the accessory bacteriochlorophyll (BA) and the bacteriopheophytin (HA) is reported to be 135 cm−1 for the edge-to-edge separation of 0.35 nm.44-46 This strongly supports the evaluated transfer integrals in the present triad system. Therefore it is reasonably explained that the R218+ can mediate the long-range electronic coupling in the AQ1S•2−–W214•+ system.


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−

•

•+

AQ1S –R218 –W214

•2−

+

•+

AQ1S –R218 –W214

λ Energy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


∆G = 0.31 eV

−

+

AQ1S –R218 –W214

Reaction Coordinate Figure 9. Schematic representation of the potential energy surfaces of the triads in Figure 8 on the basis of the DFT calculations with B3LYP/6-31+G∗∗ level and with the polarizable continuum model. The dielectric constant in the binding region of the protein was assumed to be εeff = 3.0. The tunneling energy gap (∆ε = ∆G + λ) is estimated to be 0.6 eV to mediate the electron tunneling.

Conclusions We have employed the TREPR method to characterize the structure and the electronic coupling of the photoinduced distant CS states in the protein–ligand system of AQ1S−–HSA. The MPSs of the TREPR spectra have been analyzed by using the triplet precursor ESPT model. As for the


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long-range CS state of AQ1S•2−–W214•+, the molecular geometries show that AQ1S•2− is located in the drug site Ι of HSA. The validity of the TREPR structure has been obtained by the docking simulations. Additionally, R218+ is revealed to be located in close proximity to AQ1S− and to W214 in the ground state by the docking simulation. It is also concluded that the arginine residue mediates the electronic coupling of 2.3 cm−1 in the distant CS state, since the coupling term evaluated by the McConnell superexchange model is consistent with V = 2.3 cm−1 determined by the TREPR method. Therefore, the above long-lived CS-state generation should be mediated by the R218+ residue (Figure 8) ligating to the excited triplet state of 3AQ1S−*. The present study clearly demonstrates the importance of the protein structure and of the arginine ligation on the efficient generations of the long-range CS states in proteins and is highly informative to design the effective photosensitized damages leading to the cancer-treatments.

ASSOCIATED CONTENT Supporting Information. Supplemental descriptions of the fitting parameters, dependences of the angle parameters on the spectrum calculations, and details of the adiabatic energy calculations for AQ1S•2−–R218+–W214•+ and AQ1S−–R218•–W214•+ are available free of charge at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Y.K. Email: [email protected] Tel. and FAX: +81-78-803-6548 Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors declare no financial interest. ACKNOWLEDGMENT MF, HM and YK are grateful to Prof. Rika Sekine (Shizuoka Univeristy) for encouraging and supporting MF on the present research.

This work was supported by a Grant-in-Aid for

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Time Resolved EPR Study on the Photoinduced Long-Range Charge

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