Excitonic Energy Level Structure and Pigment ... - ACS Publications

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Excitonic Energy Level Structure and Pigment-Protein Interactions in the Recombinant Water-Soluble Chlorophyll Protein. II. Spectral Hole-Burning Experiments J. Pieper,*,†,# M. R€atsep,‡ I. Trostmann,§ F.-J. Schmitt,†,|| C. Theiss,|| H. Paulsen,§ H.J. Eichler,|| A. Freiberg,‡,^ and G. Renger† †

Max-Volmer-Laboratories for Biophysical Chemistry, Berlin Institute of Technology, Berlin, Germany Institute of Physics, University of Tartu, Tartu, Estonia § Institute of General Botany, Johannes Gutenberg University Mainz, Germany Institute of Optics and Atomic Physics, Berlin Institute of Technology, Germany ^ Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia

)

‡

ABSTRACT: Persistent spectral hole burning at 4.5 K has been used to investigate the excitonic energy level structure and the excited state dynamics of the recombinant class-IIa water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The hole-burned spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) at the burn wavelength, (ii) a number of vibrational ZPHs, (iii) a broad low-energy hole at ∼665 and ∼683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, and (iv) a second satellite hole at ∼658 and ∼673 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The doublet of broad satellite holes is assigned to an excitonically coupled chlorophyll dimer. The lower-energy holes at ∼665 and ∼683 nm for chlorophyll b- and chlorophyll aWSCP, respectively, represent the lower exciton states. Taking into account the parameters of electron-phonon coupling, the lower exciton state can be assigned as the fluorescence origin. The lower exciton state is populated by two processes: (i) exciton relaxation from the higher exciton state and (ii) vibrational relaxation within the lower exciton state. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm-1 for chlorophyll b- and chlorophyll a-WSCP, respectively, based on the positions of the satellite holes. The Gaussian low-energy absorption band identified by constant fluence hole burning at 4.5 K has a width of ∼150 cm-1 and peaks at 664.9 and 682.7 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The action spectrum is broader and blue-shifted compared to the fluorescent lower exciton state. This finding can be explained by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (J. Phys. Chem. B 2008, 112, 13951).

1. INTRODUCTION The spectroscopic properties of photosynthetic pigmentprotein complexes are determined by complex pigment-protein and pigment-pigment interactions. One strategy to deal with this complexity is the isolation of specific interaction mechanisms by investigating a relatively simple native system. Such a naturally abundant model system is the water-soluble chlorophyll (Chl)binding protein (WSCP) because of three important properties:1 (i) binding of only one Chl per WSCP monomer and complete lack of carotenoids, (ii) the molecular weight of the protein matrix (20 kDa) is comparable to that of the LHC family of antenna proteins, and (iii) the WSCP apoprotein can be expressed in Escherichia coli and subsequently reconstituted to WSCP tetramers of different ratios of chlorophyll a- (Chl a) and r 2011 American Chemical Society

chlorophyll b- (Chl b) molecules. Recombinant WSCP from cauliflower (class-IIa) binds two Chl molecules per tetrameric Chl-WSCP.2,3 The crystal structure has been resolved by X-ray diffraction for the WSCP-Chl complex of Lepidium virginicum (class-IIb).4 This WSCP-Chl complex is a homotetramer comprising four protein chains of 180 amino acids and four Chl binding sites. The Chls were found to form open sandwich dimers with a tilt angle of 27 in the case of Chl a binding. All Chl molecules are tightly packed in a hydrophobic cavity at the center of the complex and Received: December 2, 2010 Revised: January 27, 2011 Published: March 18, 2011 4053

dx.doi.org/10.1021/jp111457t | J. Phys. Chem. B 2011, 115, 4053–4065

The Journal of Physical Chemistry B

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thus isolated from bulk solvent. This structural motif is proposed to be the origin for the high photostability of WSCP. WSCP (type-IIa) has already been intensively studied using low-temperature absorption3,5 and CD spectroscopy,5 femtosecond absorption,3 as well as time- and wavelength-correlated picosecond fluorescence spectroscopy.6 Information on excited state positions and pigment-pigment and electron-phonon coupling is available from simulations of conventional lowtemperature absorption spectra.7 The major obstacle of conventional low-temperature absorption spectroscopy of amorphous pigment-protein complexes is the presence of significant inhomogeneous broadening in the order of 100-300 cm-1, which hides almost all spectral substructure due to pigment-protein and pigment-pigment interactions even at low temperature. This drawback can be overcome by using site-selective spectroscopies like spectral hole burning (SHB) (for reviews, see refs 8-10), which utilizes quasi-monochromatic laser light to selectively bleach a particular transition frequency within an inhomogeneously broadened absorption band. As a result, SHB induces a change in the absorption spectrum, which typically appears as a resonantly burned “hole” at the burn frequency. Provided that the burn fluence is sufficiently low to prevent so-called power broadening, the hole width is directly related to the homogeneous broadening and concomitantly to the total dephasing time of the excited electronic state under study (see ref 10 and references therein). Thus, resonant SHB has been used to establish pure dephasing times T2 of the lowest excited states of, e.g., the FMO complex,11 the B777 complex from purple bacteria,12 and the CP29 antenna complex of green plants,13 but also excited state lifetimes T1 due to downward excitation energy transfer.14 In addition, SHB can also provide parameters of inhomogenous broadening, i.e., shape and width of the inhomogenous distribution function (IDF) using constant fluence (or zero-phonon action spectroscopy).15 In this technique, the IDF is determined as a distribution of spectral holes burned subsequently across an inhomogeneously broadened absorption profile using constant burn fluence. Action spectroscopy has been applied for the first time to the LH1 antenna complex of Rhodobacter sphaeroides15 but has later also been used to characterize the IDFs of the LH2 antenna complexes of Rhodopseudomonas acidophila and R. sphaeroides,16 the FMO complex,11 trimeric LHC II14 and CP2913 of green plants, as well as the CP4317 and CP4718 core antenna proteins. Furthermore, nonresonant satellite holes beside the burn frequency provide information on electron-phonon,19-23 electron-vibrational,24,25 or excitonic coupling.26 Therefore, SHB is a quite versatile tool to unravel spectral substructures in strongly inhomogeneously broadened absorption spectra of amorphous pigment-protein complexes. In the present study, we apply SHB spectroscopy for a detailed investigation of excited state positions as well as homogeneous and inhomogeneous broadening of Chl a- and Chl b-WSCP (type-IIa) in the frequency domain.

with either Chl a or Chl b, and purified as described in part I (DOI 10.1021/jp111455g). WSCP reconstituted with either Chl a or Chl b was diluted in a glass-forming buffer solution containing 300 mM imidazole, 20 mM NaP (pH = 7.5), and 70% w/w glycerol. The total Chl concentration of samples used for hole burning and fluorescence measurements was 0.200 and 0.004 mg/mL, respectively (∼200 and 4 μM). Experimental Setup. Hole-burning measurements with burn wavelengths between 638 and 688 nm were carried out using a Spectra Physics model 375 dye laser (line width of 1) is obtained by folding the one-phonon profile l1 R-times with itself, so that the form of the one-phonon profile determines the shape of the whole PSB. The dimensionless Huang-Rhys factor S is a measure for the linear electron-phonon coupling strength and characterizes the average number of phonons accompanying a particular electronic transition. The inhomogeneously broadened absorption and fluorescence spectra are obtained according to28 -

Sample Preparation. Purified Chl a and Chl b were prepared as described in Hobe et al.27 Aliquots of pigments were dried and stored at -20 C in inert (nitrogen) atmosphere. Recombinant WSCP from cauliflower (Brassica oleracea var. Botrytis) with an N-terminal hexahistidyl (His) tag was expressed, reconstituted

∑k Sk ð2nk þ 1Þ Y

Z

k

¥

R

R-r

½Sk ðnk þ 1Þ ½Sk nk ∑ ∑ ðR - rÞ!r! R¼0 r¼0

dΩ0 NðΩ0 - ωC ÞlR, r ½ω - Ω0 -

r

∑k ðR - 2rÞωk ð2Þ

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dx.doi.org/10.1021/jp111457t |J. Phys. Chem. B 2011, 115, 4053–4065


Figure 1. 4.5 K absorption (smooth black line) and typical hole-burned spectra of Chl a-WSCP obtained with different burn wavelengths. Hole spectra a-c were generated with a read resolution of 0.4 nm and burn fluences of about 2.2 (curve b) and 48 J/cm2 (curves a, c). The burn wavelengths (labeled by thick arrows) were (a) 665.1 nm, (b) 670.0 nm, and (c) 682.0 nm, respectively. Letters A and B label the broad satellite holes at ∼682 and ∼674 nm, respectively. Hole spectra are separated by a ΔA of 0.1 for ease of inspection. A thick, broken arrow marks the position of a pseudophonon sideband hole which is located ∼22 cm-1 to the red of the ZPH at 682.0 nm. The smooth red line is a Gaussian fit of the action spectrum presented in Figure 2 centered at 14648 ( 5 cm-1 (682.7 nm) with a width of 150 ( 5 cm-1. Thin black arrows indicate the position of vibrational ZPH.

where N(Ω - ωC) is the inhomogeneous distribution function (IDF); hnk = [exp(pωk/kT) - 1]-1 describes the thermal occupation for phonons of mode k according to Bose statistics; and - and þ correspond to absorption and fluorescence, respectively. As in eq 1, R (with R = 1, 2, ...) denotes the total number of phonon transitions regardless of creation and annihilation processes, while r gives the number of annihilated phonons (0 e r e R). By analogy to eq 1, the profile lR,r (R > 1) is obtained by folding the one-phonon profile l1,0 |R - 2r|-times with itself. Then, for l1,0 being a Gaussian with a width of ΓG, the profile lR,r becomes a Gaussian with a width of |R - 2r|1/2ΓG. If l1,0 is a Lorentzian with a width of ΓL, the profile lR,r becomes a Lorentzian with a width of |R - 2r|ΓL.28 In this work, the onephonon profile is assumed to have Gaussian and Lorentzian shape at its low- and high-energy wings, respectively.

3. RESULTS Chl a-WSCP. The 4.5 K absorption spectrum of WSCP reconstituted with Chl a is shown as a black line in Figure 1. As discussed in the accompanying paper (part I, DOI 10.1021/ jp111455g), the main absorption band is found at 672.5 nm, while a weaker shoulder is located at about 681 nm. The 4.5 K absorption spectrum of Chl a-WSCP is quite similar to the lowtemperature spectra reported by Hughes et al.,5 Renger et al.,7 and Theiss et al.,2 who identified the main absorption peak and the shoulder toward the red with the absorption bands of the upper and lower exciton levels, respectively, of a strongly coupled homodimer of Chl a molecules. To characterize the positions, excited state lifetimes, and inhomogeneous broadening of the above exciton levels, persistent nonphotochemical hole burning (NPHB) was performed at

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Figure 2. Low-energy region of selected hole spectra of Chl a-WSCP obtained with burn wavelengths of 670.0 nm (curves a and b) and 682.0 nm (curve c), respectively, and a read resolution of 0.4 nm. The burn wavelengths are labeled by thick arrows. Curves a and c were generated with a burn fluence of about 48 J/cm2, while curve b is equivalent to curve a except for a lower burn fluence of about 0.4 J/cm2. The ZPH at 682 nm is cut off at ∼40% of its fractional depth. Letters A and B mark the broad satellite holes at ∼683.3 and ∼673.5 nm, respectively. Smooth black lines are fits to various hole features discussed in the text. Hole spectra are given arbitrary offsets for ease of inspection. Bottom: ZPH-action spectrum (black diamonds) of Chl a-WSCP obtained with a constant burn fluence of 150 mJ/cm2 and a read resolution of 0.2 nm. The smooth red line is a Gaussian fit of the action spectrum centered at 14648 ( 5 cm-1 (682.7 nm) with a width (fwhm) of 150 ( 5 cm-1.

several burn wavelengths (λB) across the absorption spectrum of Chl a-WSCP with burn fluences ranging from 0.03 to about 48 J/ cm2 at 4.5 K. Typical hole spectra of Chl a-WSCP are shown in Figure 1 for three different λB-values of 665.1 (blue line), 670.0 (red line), and 682.0 nm (green line). Hole burning within the absorption band of the upper exciton level (λB = 670.0 nm) produces four main features: (i) a narrow ZPH at 670 nm (see thick arrow in Figure 1), (ii) a number of vibrational ZPHs at lower energy (labeled by small arrows in Figure 1 and listed in Table 1), (iii) a shallow broad hole (hole A) at ∼683 nm (see also below), and (iv) a second broad satellite hole (hole B) at higher energy than hole A (∼674 nm). The broad hole A is most probably due to exciton relaxation to the energetically lower exciton level, while hole B appears to be an excitonic satellite hole building on hole A. A hole-burned spectrum obtained with λB = 665.1 nm and higher fluence (see curve a of Figure 1) is quite similar to spectrum b but exhibits more intense vibrational ZPHs in the spectral region of hole A. A hole-burned spectrum with λB located within the energetically lower exciton level is shown as curve c in Figure 1. The latter spectrum obtained with λB = 682.0 nm exhibits a different composition than spectra a and b consisting of three main features: (i) a narrow ZPH at the burn wavelength (see thick arrow in Figure 1), (ii) a broad feature located about 22 cm-1 toward the red of the ZPH that can be identified with a pseudoPSB (see broken arrow in Figure 1), and (iii) a broad satellite hole (hole B) at higher energy than the ZPH (∼674 nm). The position and width of hole B are similar in curves a, b, and c in Figure 1; i.e., its shape is virtually independent of the location of 4055



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Table 1. S0fS1 Vibrational Frequencies νj ((4 cm-1) of Chl a-WSCP and Chl b-WSCP as Determined by Spectral Hole Burning at 4.5 Ka Chl a-WSCP νj(S1fS0), cm-1 from part I

Chl b-WSCP

νj(S0fS1), cm-1

νj(S0fS1), cm-1

νj(S1fS0), cm-1

νj(S0fS1), cm-1

νj(S0fS1), cm-1

origin burningb

vibrational burningc

from part I

origin burningb


183

188

185

181

193

228

228

230

221

220

227

264

268

240 269

241 275

248 272

285

283

302

302

298

300

298

327

325

325

325

325

325

356

358

359

352

350

348

384

381

381

366

369

378

404

405

406

426 465

430 470

428

488

479

259 283

402 435 469 481

496

520

520

547 574

520

283

515

514

541

537

536

572

572

569

584

598

584

609

620

617

656 690

683

641 700

653 685

703

708 713

718

718

188

734 742

746

744

754

739

757

754

803

800

797

816 840

834

821 837

863

860

887

894 907

920

920

922

985

980

977

982

998

1002

993 1024

1026

1023

1047 1067

1055 1068

1045 1068

1104

1104

1040 1081

1108

1095

1128

1119 1157

1144

1137

1150

1184

1170

1172

1175

1227

1186

1210

1217

1227

1227

1243 1259

1240

1243 1264

1248 1273

1285

1281

1288

1294

1304

1314

1305

1329

1326

1354

1348

1375

1379

1360

1326 1344 1372

4056



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Table 1. Continued Chl a-WSCP νj(S1fS0), cm-1 from part I

Chl b-WSCP

νj(S0fS1), cm-1

νj(S0fS1), cm-1

νj(S1fS0), cm-1

νj(S0fS1), cm-1

νj(S0fS1), cm-1

origin burningb


from part I

origin burningb


1390 1441

1392 1420

1393 1443

1390 1419

1490

1492

1484

1491

1532

1523

1522

1528

1556

1541

1550

1557

1591

1583

1573

1594

1620

1628

1616

1649

1654

1650

1681

1675

a

In the case of Chl a-WSCP, the burn wavelengths were 682 and 665 nm for origin excited and vibrational hole burning, respectively. For Chl b-WSCP, the burn wavelengths were 665 and 654 nm for origin excited and vibrational hole burning, respectively. The data are compared to the S1fS0 vibrational frequencies obtained by delta-FLN in the accompanying paper (part I, DOI 10.1021/jp111455g). b The term origin burning denotes vibrational holes that appear as satellite holes due to resonant hole-burning within an electronic 0-0 transition. c The term vibrational burning refers to vibrational holes that appear due to non-resonant hole-burning within the vibrational satellite region building on electronic 0-0 transitions.

λB in the upper and lower exciton level of Chl a-WSCP. The latter finding further corroborates the assignment of hole B as an excitonic satellite hole. Furthermore, the broad low-energy hole A assigned to exciton relaxation disappears because the burn wavelength is located within the (terminal) lower exciton level. In addition, curve c of Figure 1 reveals a rich structure of vibrational ZPHs at the high-energy side of the burn frequency, which are labeled by small arrows. Therefore, these multiple holes correspond to S0fS1 vibrational frequencies in the wavenumber range between 180 and 1665 cm-1 building on electronic transitions burned resonantly at 682 nm. That is, the vibrational ZPHs observed in curve c are produced by origin hole burning within 0-0 transitions at 682 nm. In contrast, curves a and b in Figure 1 exhibit some vibrational ZPHs at the low-energy side of the burn frequencies, which are mainly located in the spectral region of hole A. Thus, the latter set of holes corresponds to electronic transitions burned nonresonantly via the mechanism of vibrational hole burning within the manifold of their S0fS1 vibrational frequencies. The vibrational frequencies resolved in this experiment are similar to those obtained before for Chl a in solution29 and for the CP29 antenna complex.30 All individual frequencies are listed in Table 1 and compared to the corresponding S1fS0 vibrational frequencies determined by delta-FLN spectroscopy for Chl a-WSCP in part I (DOI 10.1021/jp111455g). All together, the hole-burning experiments presented here and the delta-FLN investigations of part I provide a complete characterization of the vibrational substructure in both the ground and excited state of an excitonically coupled Chl homodimer bound by WSCP. Figure 2 shows a magnification of the low-energy region of hole-burned spectra obtained with λB of 670.0 nm at different burn fluences (curves a and b). The broad hole A is only visible in spectrum b of Figure 2 (red line) obtained with a relatively low burn fluence of 0.4 J/cm2. A Gaussian fit of hole A (see black line in Figure 2) yields a peak position of 14636 ( 5 cm-1 (683.2 nm) and a width (fwhm) of 120 ( 10 cm-1. It is remarkable that hole A is absent in spectrum a obtained with a relatively high burn fluence of 48 J/cm2. This is characteristic for all burn fluences higher than 2.2 J/cm2 (not shown); i.e., hole A appears to saturate at relatively low fluence (see curve a of Figure 2). In contrast, hole B corresponding to the upper exciton

level appears almost independent of burn wavelength, even if the burn wavelength is tuned to the spectral region of hole A (see curve c in Figure 2). Based on a Gaussian fit, hole B is located at 14835 ( 5 cm-1 (674.1 nm) and has a width (fwhm) of 120 ( 10 cm-1. A slight change in shape and position of hole B at higher fluence is most probably due to superposition with its own antihole and that of lower-energy hole features at the blue and red sides of hole B, respectively. The position and inhomogeneous broadening of the lower exciton level of Chl a-WSCP has been further investigated by constant fluence hole burning—so-called ZPH action spectroscopy—in the region of hole A. The fractional depths of the ZPHs obtained by action spectroscopy are shown in the lower part of Figure 2 (see black diamonds). This ZPH action spectrum can be fit by a Gaussian with a peak position of 14648 ( 10 cm-1 (682.7 nm) and a width (fwhm) of 150 ( 10 cm-1. This means that the low-energy state(s) represented by the action spectrum possess a broader and slightly blue-shifted absorption band than the terminal state corresponding to hole A. This discrepancy may arise either from the overlap of the nonresonantly burned hole A with its own antihole or from a more complex composition of the low-energy levels of a pigment-protein complex (see Discussion). Chl b-WSCP. Similar NPHB experiments were performed on Chl b-WSCP samples with burn fluences ranging from 0.03 to about 48 J/cm2 at 4.5 K. The 4.5 K absorption spectrum of WSCP reconstituted with Chl b is shown as a black line in Figure 3. The absorption spectrum is composed of a main peak at 655.4 nm, a shoulder at about 663 nm, and a further weak band at about 676 nm. Typical hole spectra of Chl b-WSCP are shown in Figure 3 for four different λB-values of 653.9 (blue line), 656.2 (red line), 664.9 (green line), and 676.1 nm (purple line). The first two λB-values are located close to the main absorption peak at 655.4 nm and thus roughly within the absorption band of the upper exciton level. Analogous to Chl a-WSCP, the corresponding hole spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) coincident with the burn wavelength (see thick arrows in Figure 3), (ii) a number of vibrational ZPHs at lower energy (labeled by small arrows in Figure 3 and listed in Table 1), (iii) a broad hole at ∼665 nm (hole A) most probably due to exciton relaxation to the energetically lower exciton level 4057



Figure 3. 4.5 K absorption (black line) and typical hole-burned spectra of Chl b-WSCP obtained with different burn wavelengths. Hole spectra a-d were generated with a burn fluence of about 48 J/cm2 and a read resolution of 0.4 nm. The burn wavelengths (labeled by thick arrows) were (a) 653.9 nm, (b) 656.2 nm, (c) 664.9 nm, and (d) 676.1 nm, respectively. Letters A and B label the broad satellite holes at ∼665 and ∼658 nm, respectively. The position of the antihole of hole B is marked by an asterisk. Hole spectra are separated by a ΔA of 0.1 for ease of inspection. Thick, broken arrows mark the position of pseudophonon sideband holes located ∼22 cm-1 to the red of the ZPH at 664.9 and 676.1 nm, respectively. Thin black arrows indicate the position of vibrational ZPH. The envelope of the corresponding action spectrum presented in Figure 4 is shown by a smooth red line.

(see below), and (iv) a second broad satellite hole (hole B) at ∼658 nm, i.e., at higher energy than hole A. When the burn wavelength is tuned to 664.9 nm, which is located within the spectral region of hole A and thus within the energetically lower exciton level, the burn efficiency of the ZPH at λB increases drastically. This effect is consistent with a longer excited state lifetime of this exciton level. The broad low-energy hole A disappears, while the ZPH at 664.9 nm is now accompanied by a distinct sideband located about 22 cm-1 toward the red of the ZPH. The latter features are typical for a pseudo phonon sideband (pseudo-PSB). It is remarkable, however, that a broad satellite hole is found at the higher energy side of the ZPH, which is displaced by about 170 cm-1, when burning at lower energy. Thus, this feature seems to be coincident or even identical with hole B produced also upon burning at higher energies (see above). Finally, hole spectra were recorded with λB = 676.1 nm lying within the weak, but distinct, low-energy absorption band at 676 nm. In contrast to the spectra obtained at the other λBvalues, the hole spectrum obtained within this band misses hole B, being only composed of a ZPH and a pseudo-PSB located ∼22 cm-1 to the red of the ZPH. This finding indicates that the 676 nm band is an inhomogeneously broadened absorption band of a widely localized electronic state. Hole burning with burn wavelengths shorter than ∼660 nm always produces the broad hole A as the lowest-energy feature. Broad structures similar to hole A, which have been observed for many photosynthetic antenna complexes,31-34 originate from EET or exciton relaxation to the lowest-energy level(s) of the complex. Figure 4 shows a magnification of two hole spectra from Figure 3, where curve c (λB = 664.9 nm) and curve b (λB = 656.2 nm) represent the cases of selective and nonselective hole burning within the broad hole A, respectively. As shown in more

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Figure 4. Low-energy region of Chl b-WSCP hole spectra obtained with burn wavelengths of 664.9 nm (curve c) and 656.21 nm (curve b), respectively. The burn wavelengths are labeled by thick arrows. The ZPH at 664.9 nm is cut off at ∼50% of its fractional depth. Again, letters A and B mark the broad satellite holes at ∼665 and ∼658 nm, respectively. The gray curve is the same as curve c and represents hole B undisturbed by features due to resonant hole burning. Curve Δ is the difference between curves b and c, respectively, and thus corresponds to spectral features stemming from resonant hole burning at 656.2 nm. Smooth black lines are fits to various hole features discussed in the text. Hole spectra are given arbitrary offsets for ease of inspection. Bottom: ZPH-action spectrum (black diamonds) of Chl b-WSCP obtained with a constant burn fluence of 150 mJ/cm2 and a read resolution of 0.2 nm. The smooth red line is a Gaussian fit of the action spectrum centered at 15040 ( 5 cm-1 (664.9 nm) with a width of 150 ( 5 cm-1, and the smooth blue line is a Gaussian fit centered at 14770 ( 5 cm-1 (677.0 nm) with a width (fwhm) of 190 ( 5 cm-1.

detail for curve b in Figure 4, a Gaussian fit of hole A yields a peak position of 15020 ( 5 cm-1 (665.8 nm) and a width (fwhm) of 110 ( 10 cm-1. Interestingly, this means that hole A is located almost 3 nm lower in energy than the absorption shoulder at 663 nm. Resonant hole burning in the spectral region of hole A produces a narrow ZPH accompanied by pseudo-PSB (see curve c in Figure 4). This finding indicates that the width of hole A is mainly due to inhomogeneous broadening and that the electron-phonon coupling of the corresponding excitonic state is moderate (see further discussion below). A comparison of curves a and b of Figure 3 reveals that the position of hole A is widely independent of burn wavelength except for some distortion due to the superposition of hole A with several vibrational ZPHs. Furthermore, the position of hole A does not exhibit a notable dependence on burn fluence (see Figure 5 and discussion below). These results further corroborate the idea that hole A can be identified as the absorption band of the lower exciton component of Chl b-WSCP. As similarly observed for Chl a-WSCP, the hole-burned spectra of Chl b-WSCP displayed in Figure 3 reveal a multitude of vibrational ZPHs corresponding to S0fS1 vibrational frequencies in the wavenumber range between 180 and 1665 cm-1. In the case of curve c these holes are produced resonantly by origin hole burning within 0-0 transitions at 664.9 nm, while they appear due to nonresonant vibrational hole burning in curves a and b of Figure 3. The full set of S0fS1 vibrational frequencies determined for Chl b-WSCP is listed in Table 1 and 4058



Figure 5. Frame I: hole-burned spectra of Chl b-WSCP obtained with a burn wavelength of 656.2 nm and different burn fluencies ranging from 0.06 to 40.92 J/cm2. Smooth black lines are fits to various hole features discussed in the text. The broad satellite holes A and B are labeled as in Figures 1 and 2. Hole spectra are given arbitrary offsets for ease of inspection. Smooth black lines are Gaussian fits to the narrow ZPH, hole A, and hole B (see text for details). Frame II: peak hole depth as a function of burn fluence of the narrow ZPH at 656.2 nm (triangles), hole B (squares), and hole A (diamonds) according to the Gaussian fits shown in Frame I. Symbols are connected by black lines as a guide to the eye. The red line shown along with the hole B data is the same as that shown along with the hole A data but normalized to fit the hole B depth.

compared to the corresponding S1fS0 frequencies reported based on delta-FLN experiments in part I (DOI 10.1021/ jp111455g). Constant fluence hole burning (ZPH-action-) spectroscopy was employed to further investigate the spectral position and inhomogeneous width of the lowest state’s absorption band. The peak absorption changes of the ZPH burned across the lowenergy region of the absorption spectrum are indicated by black diamonds in the lower part of Figure 4. The fractional depths of the ZPH were smaller than 10% so that the action spectrum should not be significantly affected by saturation effects. Furthermore, there is no marked contribution from the ZPH of the higher excitonic state on the blue side of the action spectrum because of the much shorter lifetime due to fast exciton relaxation into the lower excitonic state. The widths of the ZPH are resolution-broadened with a width (fwhm) of ∼2 cm-1, while their relative intensities are preserved, so that the shape of the action spectrum is not distorted by this type of measurement. Higher-resolution experiments are beyond the scope of this study because the excited state lifetime of the lower excitonic level has already been studied in detail by Schmitt et al.6 The action spectrum shown in the lower part of Figure 4 is composed of two main distributions of ZPHs, one in the spectral region of hole A and a second at much lower energy, which is widely coincident with the faint absorption peak at ∼676 nm (see Figure 3). The shape can be well approximated by two Gaussian lineshapes. The Gaussian distribution in the vicinity of hole A (smooth red line in Figure 4) has a peak position of 15040 ( 10 cm-1 (664.9 nm) and a width (fwhm) of 150 ( 10 cm-1. This means that the distribution represented by the action spectrum is not identical to hole A but slightly blue-shifted by about 1 nm and somewhat broader. In this regard it is important to note that the shape of hole A does not exhibit strong distortions due to an antihole, which may easily explain such a seeming contradiction. These findings indicate that the first Gaussian line shape cannot

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be immediately identified with the inhomogeneously broadened distribution of 0-0 transitions of the lower exciton level of Chl bWSCP (see discussion section). The second Gaussian distribution at lower energy (smooth blue line in Figure 4) has a peak position of 14770 ( 10 cm-1 (677 nm) and a width (fwhm) of 190 ( 10 cm-1, which is close to the 676 nm absorption band and broader than that of the first Gaussian distribution. It is interesting to note that there is no discernible low-energy hole at ∼677 nm in spectra a and b of Figure 3, which would indicate efficient EET to a Qy-state represented by the 677 nm ZPH distribution. Finally, a small deviation from the Gaussian shape is visible at the blue side of the first distribution at about 660 nm. In summary, these findings indicate a very complex composition of the low-energy level structure of bulk Chl b-WSCP, which will be analyzed in more detail in the Discussion section. The hole spectra shown in Figures 3 and 4 further reveal that hole burning in the spectral region of hole A generally produces a broad higher-energy satellite hole at ∼658 nm (hole B) which is shifted by ∼170 cm-1 toward the blue of hole A. As demonstrated by curve c in Figure 4, hole B is even present when burning selectively at lower energy, i.e., here at 664.9 nm. In the latter case, hole B is clearly too broad and too intense to be attributed to vibrational hole burning. Thus, hole B can be identified with the excitonic satellite hole of the upper exciton state that appears as a response upon hole burning within the corresponding lower excitonic level. Because of the origin of hole B as a nonselective feature, its width is naturally contributed to by inhomogeneous broadening so that no conclusions about the lifetime of the upper excitonic level are possible at this point. The composition of the hole spectrum burned selectively within the spectral region of hole B (see curve b in Figure 4) is even more complex because it may generally consist of features due to selective and nonselective hole burning, where the latter builds on hole A. The nonselectively burned contribution can be approximated by the shape of hole B in curve c of Figure 4. This contribution is shown as a gray curve in Figure 4 and normalized to the low-energy wing of hole B in curve b of Figure 3. The use of this line shape as the nonselective contribution can also be motivated by the data shown in Figure 3, where the shape of hole B in spectra a and c is quite similar although the burn wavelengths are located at the high- and low-energy side of hole B, while spectrum b shows a clearly different shape of hole B due to a superposition of selective and nonselective contributions. This observation clearly indicates that hole B in spectra a and c builds on hole A as an excitonic satellite hole; i.e., it is a nonselectively burned feature. Returning to the discussion of Figure 4, it follows from the argument above that the difference between curves b and c represents the shape of the selectively burned hole at 656.2 nm (curve Δ). The difference spectrum reveals more structure than the narrow ZPH and can be fit by two components: a narrow Gaussian band with a width (fwhm) of 9 cm-1 and a broader Lorentzian with a width (fwhm) of 65 cm-1. The steeper, nonLorentzian tailing toward the high-energy side is due to the overlap with the antihole produced when burning hole B. The width of the narrow ZPH is close to the experimental resolution of 0.4 nm, thus precluding the determination of a corresponding lifetime (T1). This could be the equivalent of the slower ∼7-8 ps EET component observed by femtosecond absorption spectroscopy.2 The width of the broader Lorentzian contribution can be attributed to the lifetime (T1) of the Qy-state excited directly at λB. Using T1 = (πcΓhole)-1, where c is the speed of 4059


The Journal of Physical Chemistry B light and Γhole (cm-1) the Lorentzian holewidth, one obtains a lifetime of 165 ( 10 fs for λB = 656.2 nm. The latter lifetime T1 most probably represents fast exciton relaxation to the lower energetic level of Chl b-WSCP, which is somewhat larger than, but in a similar order of magnitude as, the value of 60 fs calculated by Renger et al. for the case of Chl b-containing WSCP.7 Frame I of Figure 5 shows hole spectra obtained with a burn wavelength of 656.2 nm and different burn fluences ranging from 0.06 to ∼41 J/cm2, where the red curve (14.46 J/cm2) is identical to curve b of Figure 3. All spectra consist of the three main features discussed above: a narrow ZPH at 656.2 nm as well as the broad satellite holes A and B. In addition, the spectra obtained at higher fluence clearly show a set of shallow vibrational ZPHs in the vicinity of hole A. As already mentioned above, the position and width of hole A are almost independent of burn fluence. The situation is more complex for hole B, which is the superposition of two selectively burned and one nonselectively burned hole features (see above). Nevertheless, the shape of hole B also appears to be similar at different values of burn fluence except for a steeper high-energy wing at higher burn fluence, which is typical for hole features with closely spaced hole-/antihole-contributions. The nonselectively burned contribution to hole B (excitonic satellite hole) exhibits virtually no burn fluence dependence for a burn wavelength of 653.9 nm (not shown), where this nonselectively burned satellite hole is visible as a distinct feature (see also Figure 3). To evaluate the growth kinetics, the hole features discussed above were fit by three Gaussian profiles (see lowest curve in Frame I of Figure 5): one for the narrow, resolution-broadened ZPH (fwhm of 9 cm-1), one for the inhomogeneously broadened hole A (fwhm of 110 cm-1), and one encompassing the excitonic satellite hole B plus the ∼165 fs-component. The hole depths of these profiles are plotted in Frame II of Figure 5 as a function of burn fluence. It is remarkable that the growth kinetics of hole A can be easily normalized on that of hole B by simultaneously applying a factor of 3 (see red line in Frame II of Figure 5). This means that the growth of the three hole features is correlated. This finding provides further support for the assumption that the 165 fscomponent is due to a fast relaxation into the lower exciton level represented by hole A and that hole B is a satellite hole of the upper exciton state. On the other hand, the growth of the narrow, resolution-broadened ZPH (fwhm of 9 cm-1) appears to be uncorrelated with that of the broader hole features (see dashed line in Frame II of Figure 5). As a result, there are essentially two possible interpretations for the presence of the narrow ZPH: (a) vibrational relaxation upon direct excitation of the lower exciton level at ∼666 nm or (b) a slower (incoherent) EET pathway to the lowest-energy state represented by hole A.

4. DISCUSSION The hole-burning data presented above provide detailed information on spectral positions and homogeneous and inhomogeneous broadening of the excitonic energy states of Chl homodimers bound by recombinant type-IIa WSCP. In addition, the line-narrowed fluorescence spectra of WSCP reported in the accompanying paper (part I, DOI 10.1021/jp111455g) characterize the exciton-phonon and vibrational coupling of the lower exciton level of WSCP. Most prominent is the satellite hole structure consisting of two intense holes split by about 170 and 200 cm-1 for Chl b- and Chl a-WSCP, respectively, which is in agreement with strong excitonic coupling within a Chl dimer in

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open sandwich geometry as reported in a number of preceding studies on recombinant type-IIa WSCP.2,5-7 In this regard it is important to note that Theiss et al.2 concluded that recombinant type-IIa WSCP from cauliflower as used here binds up to two Chl molecules per WSCP tetramer. X-ray diffraction data are available only for type-IIb WSCP, which reveal a total of four Chl molecules bound by a WSCP tetramer.4 In the following, we discuss the hole-burning data obtained here and compare the results with those reported in preceding studies.2,5-7 Assignment of Low-Energy Excitonic States. The position and width of the lowest excitonic energy level of Chl a- and Chl bWSCP have been investigated using two different approaches: (a) via nonselective hole burning producing hole A as the lowestenergy feature, which appears most probably as a result of EET or exciton relaxation (see below), and (b) by constant fluence (ZPH action-) spectroscopy yielding the shape of the lowestenergy level through selective hole burning. As to the latter, ZPH action spectroscopy is based on the fact that the hole-burning quantum yield is directly proportional to the excited state lifetime (see, e.g., Kenney et al.35). Therefore, hole-burning resonant to λB is especially efficient in long-living excited Qy-states, and a scan of the low-energy absorption region with constant fluence hole burning is expected to yield the shape of the inhomogeneously broadened lowest-energy Qy-state or exciton state, respectively.31 In the case of Chl a-WSCP, ZPH action spectroscopy (see Figure 2) revealed an almost Gaussian distribution of narrow ZPH peaking at ∼682.7 (14648 ( 5 cm-1), which is roughly 17 nm to the red of the action spectrum found for Chl b-WSCP (see Figure 4). This value is approximately the same as the shift between the Qy-absorption spectra of WSCP reconstituted with Chl a and Chl b, respectively. The profile has a Gaussian width (fwhm) of ∼150 cm-1 and is located within the low-energy shoulder of the absorption spectrum at ∼681 nm (see Figure 1). The corresponding nonresonantly burned feature is hole A, located at lower energy than the ZPH action spectrum peaking at 683.3 nm and having a Gaussian width (fwhm) as narrow as 110 ( 10 cm-1. The fluorescence spectrum of Chl a-WSCP is located at ∼683.8 nm (14624 cm-1) having a width (fwhm) of ∼150 cm-1 (see Figure 6). The calculated spectrum shown in Figure 6 was based on the assumption that hole A reflects the fluorescing energy level of Chl a-WSCP and on the parameters of electron-phonon coupling determined in the accompanying paper (see part I, DOI 10.1021/jp111455g). The agreement with the experimentally obtained fluorescence origin band is very good except for deviations due to neglect of higher-frequency vibrational modes (see Figure 6) at wavelengths longer than ∼687 nm. On the other hand, a fit of the fluorescence spectrum of Chl a-WSCP using the parameters of the ZPH action spectrum turns out to be too broad and slightly blue-shifted (not shown). These findings suggest that the lowest-energy level of Chl a-WSCP is represented by hole A, while the action spectrum appears to have a more complex composition (see below). The hole features of Chl b-WSCP closely resemble those discussed above. In the case of Chl b, action spectroscopy led to the identification of two distributions of narrow ZPHs peaking at ∼664.9 (15040 ( 5 cm-1) and 677.0 nm (14770 ( 10 cm-1), respectively (see Figure 3). The former profile has a Gaussian width (fwhm) of ∼150 cm-1 and is located within the lowenergy shoulder of the absorption spectrum of Chl b-WSCP at ∼663 nm (see Figure 3). The latter has a much broader Gaussian 4060



Figure 6. Simulations (black lines) of the experimental 4.5 K absorption (blue diamonds) and fluorescence spectra (red diamonds) of Chl aWSCP based on the parameters determined in this study (see text for details). The red lines give the positions of the lower exciton level in two different protein conformations, where the energetically lower one is the fluorescent state at 4.5 K. Parameters are summarized in Table 2.

width (fwhm) of ∼190 cm-1 and is almost coincident with the weak absorption band of Chl b-WSCP at ∼677 nm (see Figure 3). The fluorescence spectrum of Chl b WSCP is found at ∼666.1 nm (15013 cm-1) having a width (fwhm) of ∼150 cm-1 (see Figure 7); i.e., it is located ∼27 cm-1 to the red compared to the first Gaussian ZPH distribution peaking at 664.9 nm. With an average Huang-Rhys factor S of ∼0.8 and a mean phonon frequency of 23 cm-1, however, the 0-0 transition and the emission spectrum should be displaced by a Stokes shift Sωm of only ∼18 cm-1, yielding a fluorescence maximum at ∼665.7 nm. This is a shortfall of about 33% compared with the experimentally obtained emission maximum. As the formula Sωm gives only a rough estimate of the Stokes shift in the case of a strongly asymmetric and highly structured one-phonon profile as determined above for WSCP, a fluorescence spectrum was calculated according to eq 2. For this simulation it was assumed that the ZPH profile obtained by action spectroscopy represents the inhomogeneously broadened lowest-energy level of Chl b-WSCP and is coupled to the one-phonon profile determined in the accompanying paper (see part I, DOI 10.1021/jp111455g). Nevertheless, the calculated fluorescence spectrum was peaking too far to the blue of the experimentally obtained fluorescence band and turned out to be too broad (not shown) indicating that the action spectrum does not directly reflect the lowest (fluorescent) energy state of Chl b-WSCP. In this regard, it is interesting to note that the nonselectively burned hole A was found at 15020 ( 5 cm-1 (665.8 nm) having a Gaussian width (fwhm) of only 110 ( 10 cm-1; i.e., it is found at lower energy compared with the ZPH action spectrum and much narrower. The fluorescence spectrum shown in Figure 7 was calculated according to eq 2, assuming that hole A reflects position and inhomogeneous width of the fluorescing energy level of Chl b-WSCP and using the parameters of electronphonon coupling determined in the accompanying paper (see part I, DOI 10.1021/jp111455g). The agreement with the experimentally obtained fluorescence origin band, but especially with its spectral position, is almost perfect except for deviations at wavelengths higher than ∼670 nm. These deviations are most

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Figure 7. Simulations (black lines) of the experimental 4.5 K absorption (blue diamonds) and fluorescence spectra (red diamonds) of Chl bWSCP based on the parameters determined in this study (see text for details). The red lines give the positions of the lower exciton level in two different protein conformations, where the energetically lower one is the fluorescent state at 4.5 K. The inset shows a potential model of conformational substates in the vicinity of the pigment molecules to describe the observed wavelength dependence of S, where the potential curves of the ground and excited electronic states are labeled by S0 and S1, respectively. Furthermore, letters a-d label the following processes: (a) absorption, (b) “blue” fluorescence, (c) protein relaxation, and (d) “red” fluorescence after protein relaxation and fast vibrational relaxation (see text for details). Parameters are summarized in Table 3.

probably due to the higher-frequency modes which are not taken into account in the calculation. In addition, fluorescence from the weak absorption band at ∼677 nm will certainly contribute to the pronounced shoulder in fluorescence at ∼680 nm. In summary, this finding reveals that the fluorescence of Chl b WSCP originates from a lowest-energy level at 665.8 nm represented by hole A, which appears as a result of EET and/or exciton relaxation from higher energy levels. In turn, this raises the question for the blue-shift and broadening of the ZPH action spectrum compared to hole A. The apparent mismatch between positions and shapes of hole A and the ZPH action spectrum for both Chl a- and Chl b-WSCP could be most easily explained by a superposition of hole A with its own antihole, as outlined, e.g., for the case of an excitonically coupled Chl dimer by Reppert et al.36 However, this effect would not account for the narrower and red-shifted fluorescence spectra obtained in this study. This feature is quite difficult to explain within the model of only two strongly coupled pigment molecules. Therefore, three alternative scenarios have to be considered: (a) the presence of more than two pigment molecules in a WSCP tetramer as found for type IIb-WSCP,4 (b) the presence of weakly and strongly coupled Chl dimers in individual WSCP complexes,2 and (c) a slow protein relaxation within the lowest excitonic energy level. The first explanation would assign hole A to the lowest exciton level of one of two excitonically coupled dimers, while the action spectrum encompasses the lower levels of both energetically inequivalent Chl dimers linked by slow incoherent EET. Note in this regard that Theiss et al.2 have unambiguously shown that the WSCP type used here binds two Chl molecules per protein tetramer so that a larger number of excited states can be ruled out. As to the second, the origin of the 4061


The Journal of Physical Chemistry B complex composition of the action spectrum may also lie in the presence of geometrically inequivalent and thus weakly and strongly coupled Chl dimers as inferred by Theiss et al.2 based on fast and slow EET components in WSCP. Within this model, hole A would correspond to a strongly coupled Chl dimer exhibiting a stronger excitonic splitting and thus a larger redshift of the lower exciton level. This idea would be in agreement with the appearance of a higher-energy satellite hole upon selective hole burning within the spectral region of hole A. Weakly coupled Chl dimers, however, would exhibit a smaller excitonic splitting and could therefore constitute the blue-shifted part of the ZPH action spectrum. Since both types of low-energy states would be the terminal states of EET and/or exciton relaxation in their respective WSCP molecule, hole burning in either state’s position would result in (almost) equally narrow ZPH. The superposition of the two ZPH distributions would eventually form the complete ZPH action spectrum. Although this argument appears consistent at first glance, this idea does not explain that the fluorescence appears from only the red-shifted form of the two types of Chl dimers. Rather, both types of individual low-energy states should exhibit fluorescence in this case, which disagrees with the above assignment of hole A as the fluorescent state. Note in this regard that the situation of recombinant type IIa-WSCP binding only two Chls cannot be directly compared to complex systems like CP43 or LH2, where bulk spectra may exhibit multiple fluorescence origins because in certain individual complexes in the bulk sample the on average second lowest state may lie lower in energy than the on average lowest-energy state taking into account the typically broad inhomogeneous widths of pigment-protein complexes.37,38 Therefore, another interpretation is required. Alternatively, it may be possible that there is a slow protein relaxation within the lowest excited state from sites at the blue of the ZPH action spectrum to those at the red side; i.e., the action spectrum also encompasses a number of protein conformations of one and the same excitonic level that do not contribute to fluorescence at 4.5 K. As the widths of the ZPH within the action spectrum are limited by read resolution (see above), both scenarios cannot be directly compared based on the data of the present study. Fluorescence lifetime measurements of Schmitt et al.6 on Chl b-WSCP revealed a slow—roughly 100 ps—rise term at the red side of the fluorescent state at 10 K, which appeared in addition to the overall nanosecond fluorescence decay. As discussed by Schmitt et al.,6 the 100 ps component is too slow to be attributed to incoherent EET but lies in the time range expected for pure dephasing/spectral diffusion within asymmetric two-level systems (TLS) of the excited state.12-14 Each potential minimum then corresponds to a particular protein conformation in the vicinity of the chromophore. An adaption of this model is shown in the inset of Figure 7. In brief, light absorption in (or EET to) the lowest-energy level causes an excitation of the pigmentprotein system from its ground state (S0) into an excited state (S1) potential surface (full red arrow). As the electronic/excitonic excitation appears very fast, the heavy system of protein nuclei follows slowly, and the system subsequently relaxes into (at least) another potential surface within the excited state (S1) (see process labeled with c). Fluorescence (process d) then appears red-shifted from the energetically lowest potential surface. “Blue” fluorescence (process b) occurs only at temperatures which permit a thermal population of the higher-energy potential surface in the excited state (S1), which should result in a very complex temperature dependence of both fluorescence lifetime

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and fluorescence spectrum. A similar scenario has been proposed earlier for the trimeric FMO complex by Freiberg et al.39 This complex landscape of the potential surface may thus be a general phenomenon in pigment-protein complexes, which is otherwise hidden by the complexity of native antenna proteins. The observation of this striking effect underlines the importance of studies of relatively simple model systems like WSCP. It is also interesting to note that although the ZPH action spectra of WSCP reconstituted with either Chl a or Chl b are shifted by about 17 nm relative to each other they have two important properties in common: (a) they exhibit a nearly Gaussian shape with a width (fwhm) of about 150 cm-1 and (b) at 4.5 K fluorescence originates only from the lowest-energy levels red-shifted in comparison to the total action spectrum; i.e., only some of the protein conformations in the vicinity of the pigment are thermally populated. The 17 nm shift between the two action spectra is readily explained by the different absorption properties of the two Chl types used for reconstitution of WSCP. A small component of the overall shift may also be due to different pigment-pigment interactions within the Chl a- and Chl b-homodimers, respectively. The similar shape and widths of the total action spectrum as well as the selection of a narrow, redshifted state as the fluorescent energy level, however, point to a highly similar manifold of protein conformations around the fluorescing chromophore and to concomitantly similar potential surfaces characterizing the pigment-protein system in its excited states. One notable exception to this overall similarity is the difference of the burn efficiencies observed for hole A in WSCP reconstituted with Chl a and Chl b, respectively; i.e., hole A generally saturates at low fluence but at even lower fluence in the case of Chl a compared with Chl b. This pronounced difference in hole-burning efficiency observed for very similar pigment molecules bound by the same protein may permit a structural assignment of the underlying burn process. The only structural difference between Chl a and Chl b lies in the replacement of the methyl group at the C-7 position in the case of Chl a by a formyl group in the case of Chl b. The formyl group is reported to form a H-bond with the Leu 91-residue of the class IIb-WSCP protein backbone,4 which is schematically shown in the inset of Figure 1 of part I (DOI 10.1021/jp111455g). This H-bond could lead to a more rigid protein environment in the case of Chl b, causing higher potential barriers between conformational substates and concomitantly lower burn efficiency in the case of Chl b. The generally low burn efficiency for the nonresonant hole A can also be explained in terms of the WSCP structure. Hole growth in photosynthetic pigment-protein complexes is typically modeled using at least two sets of twolevel systems (TLSs), internal and external, where the external TLSs are attributed to the solvent surrounding the protein.35,38 In the case of WSCP, however, the Chl binding pocket is well shielded from the solvent,4 so that the number of TLSs effective in WSCP may be much smaller than in other pigment-protein complexes. As a consequence, the probability that a protein conformation in the vicinity of a Chl molecule can be converted into another one in a bulk sample is lower than typically observed for other pigment-protein complexes. Excited State Dynamics. Femtosecond transient absorption spectroscopy on Chl a/Chl b heterodimers2 revealed two exponential kinetics with time constants of 400 fs and 7 ps at room temperature. These two components were assigned to fast excited state relaxation between the upper and lower exciton level 4062


The Journal of Physical Chemistry B of the Chl a/Chl b heterodimer and to slower EET within weakly coupled Chl a/Chl b dimers, respectively. Given the experimental resolution of ∼200 fs, the exciton relaxation in Chl homodimers could not be directly resolved by Theiss et al.;2 however, a time constant of less than 100 fs was predicted based on exciton calculations by Renger et al.7 In the case of Chl b-WSCP, hole spectra burned at 656.2 nm indeed revealed two selectively burned hole contributions within the upper exciton state (see Figure 4). Taking into account a blue-shifted antihole, the broad component has a Lorentzian shape with a width (fwhm) of 65 cm-1 corresponding to an excited state lifetime T1 of about 160 fs at 4.5 K. Although this value is not directly comparable to those of Theiss et al.2 and Renger et al.7 obtained at room temperature, it may be the equivalent of the fast relaxation component at 4.5 K. Thus, we conclude that the ∼160 fs component can most likely be attributed to excited state relaxation between the upper and lower exciton level of the Chl b homodimer at 4.5 K. This finding demonstrates the ability of hole-burning spectroscopy to determine excited state lifetimes directly from widths of selectively burned holes. The narrow hole burned at 656.2 nm is limited by the read resolution of 0.4 nm and would thus correspond to a lifetime T1 longer than 1.2 ps. Thus, the narrow ZPH could be the equivalent of the 7 ps component determined by Theiss et al.,2 which was attributed to EET between weakly coupled Chl dimers. However, the presence of a significant number of weakly coupled Chl dimers was ruled out above because fluorescence appears to originate solely from a low-energy exciton level represented by hole A. In contrast, the narrow ZPH at 656.2 nm appears along with a number of vibrational ZPHs in the vicinity of hole A. This finding suggests that the narrow ZPH represents vibrational relaxation within the lower exciton level rather than incoherent EET. In summary, the population of the lower exciton level of Chl b-WSCP seems to proceed via two major channels: (a) ultrafast relaxation from the higher into the lower excitonic level within 160 fs at 4.5 K and (b) slower vibrational relaxation within the lower exciton level. Pigment-Pigment Coupling. The most prominent feature of the WSCP hole spectra shown in Figures 2 and 4 is the nonselectively burned doublet of holes A and B. In the case of Chl b-WSCP, holes A and B are found at ∼666 and ∼658 nm, respectively, while they are shifted to ∼683.3 and ∼673.5 nm, respectively, for Chl a-WSCP. An important observation is that hole B also appears upon selective hole burning within the spectral region of the lower-energy hole A; i.e., it cannot be produced by downhill-EET. Furthermore, it is too intense to reflect a vibrational feature and located at much higher energies than are typical for protein phonons. This underlines that hole B is a satellite hole of hole A, which appears as a result of excitonic coupling. Excitonic satellite holes occur in systems with strong pigment-pigment interaction because hole burning in any excitonic level evokes responses from other exciton levels due to the delocalization of the excited state wave functions over the interacting chromophores. Satellite hole structures have been observed, for example, for the special pair of Rhodopseudomonas viridis40 and for the B800-less mutant of the bacterial LH2 antenna.34 As such, the doublet of holes A and B in WSCP is one of the clearest examples of an excitonic satellite hole structure available in the literature and, thus, an indication for an excitonically coupled Chl dimer. Assuming identical site energies of the excitonically coupled Chl molecules, the

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difference between the satellite hole positions is equal to the excitonic splitting 2J. Then, the dipole-dipole interaction energy J can be directly determined to be 85 and 100 cm-1 for Chl b- and Chl a- WSCP, respectively. Interestingly, excitonic model calculations of nonline-narrowed hole spectra for a strongly coupled open-sandwich Chl dimer with roughly comparable excitonic coupling constant and angle between the transition dipole moments36 reveal similarities to the experimentally obtained spectra presented here for both types of WSCPs. The results are also in agreement with a number of previous studies on type-IIa WSCP2,5,7 suggesting a strongly coupled Chl dimer in open sandwich geometry in WSCP. In contrast, hole spectra of systems with multiple chromophores exhibit a complex interplay of hole, satellite hole, and antihole structures.26 On the basis of the findings presented above, the Qy-absorption spectra of WSCP reconstituted with Chl homodimers should be entirely described by the absorption bands of the two excitonic energy levels of the strongly coupled pigment dimer. Most of the parameters determining shape and position of these absorption bands are directly obtained in this study by evaluating hole-burned spectra. These fixed parameters comprise: (i) position and inhomogeneous width of the fluorescent excitonic energy level, (ii) the excitonic splitting between the two excitonic levels (see below), (iii) the homogeneous or lifetime broadening of the higher exciton level, and (iv) the parameters of electron phonon coupling, which are determined directly for the lower exciton state and are assumed to be similar for both exciton states because of the similar protein environment. It appears to be justified to use the same parameters for inhomogeneous width and electron phonon coupling for both excitonic states because the protein environment of both Chl molecules should be widely similar. In addition, it can be concluded from the resolutionlimited narrow ZPH burned within the lower exciton level that the homogeneous contribution to the width of the entire absorption band is much smaller than its inhomogeneous broadening and can thus be neglected at 4.5 K. The fits of the Qy-absorption spectra of Chl a- and Chl bWSCP shown in Figures 6 and 7, respectively, have been constructed in a stepwise procedure using the parameters determined above for the same systems. First, the absorption band of the fluorescent part of the lower excitonic level has been calculated as the mirror image of the fluorescence line shape accounting for position and width of hole A and the corresponding parameters of electron-phonon coupling (see above). This line shape has been tested to fit hole A (not shown) and then fit to the low-energy wing of the Qy-absorption spectra (see right full red line in Figures 6 and 7). It is apparent in both figures that this absorption band is not sufficient to account for the entire low-energy shoulder located at 663 and 681 nm for Chl b- and Chl a-WSCP, respectively. This is another hint that the ZPH action spectrum represents (at least) two but most probably a distribution of conformational substates in the vicinity of the pigments, which are related by slow protein relaxation. Thus, only the energetically lowest substates contribute to fluorescence at 4.5 K. Again, at first glance the same discrepancy could also be accounted for involving additional states originating from weakly coupled Chl dimers, which would be found to the blue of hole A. However, the energetically lower state should appear as an additional broad hole following incoherent EET to this state and contribute to the fluorescence emitted by the bulk sample. As this emission is not detected as a separate peak or shoulder on the blue wing of the WSCP fluorescence spectra, this interpretation 4063



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Table 2. Fit Parameters of the 4.2 K Absorption and Fluorescence Spectra of Chl a-WSCP Shown in Figure 6 a upper exciton state Chl a position inhomogeneous width

B

A

B

A

λC [nm]

670.2

674.0

679.3

683.2

ωC [cm-1]

14926

14842

14720

14636

Γinh [cm-1]

110

110

110

110

4.07

4.28

0.95

1.00

relative intensity a

lower exciton state

Letters A and B refer to the energetically lower and higher conformational substate, respectively.

Table 3. Fit Parameters of the 4.2 K Absorption and Fluorescence Spectra of Chl b-WSCP Shown in Figure 7 a upper exciton state Chl b position inhomogeneous width relative intensity a

lower exciton state

B

A

B

A

λC [nm]

655.9

658.3

663.3

665.8

ωC [cm-1]

15249

15194

15075

15020

Γinh [cm-1]

120

120

120

120

6.75

3.31

2.04

1.00

Letters A and B refer to the energetically lower and higher conformational substate, respectively.

would require widely larger S-factors than determined above for both WSCP types. In a second step, the position and shape of the second conformational substate of the lower exciton level have to be determined. Thus, the ZPH action spectrum has been fitted with two Gaussians with a width of 110 cm-1 representing the inhomogeneously broadened 0-0 transitions of the fluorescent and nonfluorescent coupled to phonons with the lowest S-factor obtained by delta-FLN spectroscopy at the blue edge of the fluorescence spectrum. The latter line shape was scaled with an arbitrary parameter to fit the low-energy shoulder of the Qy-absorption spectra shown in Figures 6 and 7. The use of only one nonfluorescent protein conformation is somewhat arbitrary because it may represent a distribution of protein conformations provided that this model applies. However, it represents the equivalent of the 100 ps component determined in fluorescence lifetime measurements6 and is thus the minimal consistent model available. Finally, to model the upper exciton level the envelope of the absorption bands of the two conformational substates has been shifted by the excitonic splitting determined above (within experimental uncertainty) and scaled to fit the main peak of the Qy-absorption spectra shown in Figures 6 and 7. The parameters of the fits are compiled in Tables 2 and 3 for Chl a- and Chl b-WSCP, respectively. This approach appears to be justified since the environment of the two coupled Chl molecules —each bound to a WSCP monomer—can be assumed to be quite similar and should thus be characterized by similar conformational substates. The fits of the entire absorption spectra of Chl a- and Chl b-WSCP show a good agreement with the experimental data except for deviations in the region of vibrational transitions and electronic Qx-transitions, thus providing further proof for the quality of the parameters obtained by spectral hole burning in this study.

interactions of excitonically coupled Chl homodimers in recombinant class-IIa WSCP from cauliflower. As such, the results reveal detailed knowledge about pigment-pigment and pigmentprotein interactions in a relatively simple, but excitonically coupled, model system, which are of general relevance for more complex photosynthetic antenna and reaction center proteins. As the most prominent feature, the hole-burned spectra exhibit a doublet of broad satellite holes which are identified as the signature of the excitonically coupled chlorophyll dimer. Taking into account the parameters of electron-phonon coupling determined in the accompanying paper (DOI 10.1021/ jp111455g), the lower exciton state can be assigned as the fluorescence origin. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm-1 for chlorophyll b- and chlorophyll a-WSCP, respectively. These parameters can serve as benchmarks for theoretical simulations. Furthermore, the hole spectra reveal a set of S0fS1 vibrational frequencies in the wavenumber range between 180 and 1665 cm-1, which are complementary to the S1fS0 frequencies reported in the accompanying paper (DOI 10.1021/jp111455g). Constant fluence hole-burning data suggest that the lower exciton state cannot be entirely described by an inhomogeneously broadened Gaussian band. The broader action spectrum may be explained by spectral heterogeneity or by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (2008). The rich information presented above illustrates the capability of hole-burning spectroscopy to reveal spectral substructures, which are otherwise hidden by significant inhomogeneous broadening in pigment-protein complexes.

’ AUTHOR INFORMATION

5. CONCLUDING REMARKS The 4.5 K hole-burning experiments presented above provide a detailed characterization of the excitonic energy level structure including excited state dynamics and electron-vibrational

Corresponding Author

*Berlin Institute of Technology, PC14, Strasse des 17. Juni 135, 10623 Berlin, Germany. Phone: þ49-30-31427782. Fax: þ49-30-31421122. E-mail: [email protected]. 4064


The Journal of Physical Chemistry B Present Addresses #

Institute of Physics, University of Tartu, Riia 142, 51014 Tartu, Estonia. Phone.: þ(372) 737 4627. Fax: þ(372) 738 3033. E-mail: [email protected].

’ ACKNOWLEDGMENT Estonian Science Foundation (Grant No. 8674) and Estonian Ministry of Education and Science (Grant No. SF0180055s07) have supported this work. J.P., F.-J.S., C.T., H.J.E., and G.R. gratefully acknowledge support from Deutsche Forschungsgemeinschaft (SFB 429, TP A1). We are also grateful to S. Kussin and M. Wess (TU Berlin) for their help in sample preparation. ’ REFERENCES (1) Schmidt, K.; Fufezan, C.; Krieger-Liszkay, A.; Satoh, H.; Paulsen, H. Biochemistry 2003, 42, 7427. (2) Satoh, H.; Nakayama, K.; Okada, M. J. Biol. Chem. 1998, 46, 30568. (3) Theiss, C.; Trostmann, I.; Andree, S.; Schmitt, F. J.; Renger, T.; Eichler, H. J.; Paulsen, H.; Renger, G. J. Phys. Chem. B 2007, 111 (46), 13325. (4) Horigome, D.; Satoh, H.; Itoh, N.; Mitsunaga, K.; Oonishi, I.; Nakagawa, A.; Uchida, A. J. Biol. Chem. 2007, 282 (9), 6525. (5) Hughes, J. L.; Razeghifard, R.; Logue, M.; A. Oakley Wydrzynski, T.; Krausz, E. J. Am. Chem. Soc. 2006, 128, 3649. (6) Schmitt, F.-J.; Trostmann, I.; Theiss, C.; Pieper, J.; Renger, T.; Fuesers, J.; Hubrich, H.; Paulsen, H.; Eichler, H. J.; Renger, G. J. Phys. Chem. B 2008, 112, 13951. (7) Renger, T.; Trostmann, I.; Theiss, C.; Madjet, M. E.; Richter, M.; Paulsen, H.; Eichler, H. J.; Knorr, A.; Renger, G. J. Phys. Chem. B 2007, 111 (35), 10487. (8) Reddy, N. R. S.; Lyle, P. A.; Small, G. J. Photosynth. Res. 1992, 31, 167. (9) Jankowiak, R.; Hayes, J. M.; Small, G. J. Chem. Rev. 1993, 93, 1471. (10) Purchase, R.; V€olker, S. Photosynth. Res. 2009, 101, 245. (11) R€atsep, M.; Blankenship, R. E.; Small, G. J. J. Phys. Chem. B 1999, 103, 5736. (12) Creemers, T. M. H.; De Caro, C. A.; Visschers, R. W.; van Grondelle, R.; V€olker, S. J. Phys. Chem. B 1999, 103, 9770. (13) Pieper, J.; Irrgang, K.-D.; R€atsep, M.; Voigt, J.; Renger, G.; Small, G. J. Photochem. Photobiol. 2000, 71, 574. (14) Pieper, J.; R€atsep, M.; Jankowiak, R.; Irrgang, K.-D.; Voigt, J.; Renger, G.; Small, G. J. J. Phys. Chem. A 1999, 103, 2412. (15) Reddy, N. R. S.; Picorel, R.; Small, G. J. J. Phys. Chem. 1992, 96, 6458. (16) Wu, H.-M.; R€atsep, M.; Jankowiak, R.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1997, 101, 7641. (17) Zazubovich, V.; Jankowiak, R. J. Lumin. 2007, 127, 245. (18) Neupane, B.; Dang, N. C.; Acharya, K.; Reppert, M.; Zazubovich, V.; Picorel, R.; Seibert, M.; Jankowiak, R. J. Am. Chem. Soc. 2010, 132, 4214. (19) Chang, H.-C.; Small, G. J.; Jankowiak, R. Chem. Phys. 1995, 194, 323. (20) den Hartog, F. T. H.; Dekker, J. P.; van Grondelle, R.; V€olker, S. J. Phys. Chem. B 1998, 102, 11007. (21) Pieper, J.; Voigt, J.; Renger, G.; Small, G. J. Chem. Phys. Lett. 1999, 310, 296. (22) Hayes, J. M.; Matsuzaki, S.; R€atsep, M.; Small, G. J. J. Phys. Chem. B 2000, 104, 5625. (23) Hughes, J. L.; Picorel, R.; Seibert, M.; Krausz, E. Biochemistry 2006, 45, 12345. (24) Gillie, J. K.; Small, G. J.; Golbeck, J. H. J. Phys. Chem. 1989, 93, 1620.

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(25) Zazubovich, V.; Tibe, I.; Small, G. J. J. Phys. Chem. B 2001, 105, 12410. (26) Reppert, M.; Zazubovich, V.; Dang, N. C.; Seibert, M.; Jankowiak, R. J. Phys. Chem. B 2008, 112, 9934. (27) Hobe, S.; Fey, H.; Rogl, H.; Paulsen, H. J. Biol. Chem. 2003, 278 (8), 5912–5919. (28) Hayes, J. M.; Lyle, P. A.; Small, G. J. J. Phys. Chem. 1994, 98, 7337. (29) Avarmaa, R. A.; Rebane, K. K. Spectrochim. Acta 1985, 41, 1365. (30) R€atsep, M.; Linnanto, J.; Freiberg, A. J. Chem. Phys. 2009, 130, 194501. (31) Reddy, N. R. S.; Lyle, P. A.; Small, G. J. Photosynth. Res. 1992, 31, 167. (32) Pieper, J.; R€atsep, M.; Jankowiak, R.; Irrgang, K.-D.; Voigt, J.; Renger, G.; Small, G. J. J. Phys. Chem. A 1999, 103, 2412. (33) Dang, N. C.; Zazubovich, V.; Reppert, M.; Neupane, B.; Picorel, R.; Seibert, M.; Jankowiak, R. J. Phys. Chem. B 2008, 112, 9921–9933. (34) R€atsep, M.; Hunter, C. N.; Olsen, J. D.; Freiberg, A. Photosynth. Res. 2005, 86 (1-2), 37. (35) Kenney, M. J.; Jankowiak, R.; Small, G. J. Chem. Phys. 1990, 146, 47. (36) Reppert, M.; Naibo, V.; Jankowiak, R. J. Chem. Phys. 2008, 131, 11. (37) Zazubovich, V.; Jankowiak, R. J. Lumin. 2007, 127, 245. (38) Grozdanov, D.; Herascu, N.; Reinot, T.; Jankowiak, R.; Zazubovich, V. J. Phys. Chem. B 2010, 114, 3426. (39) Freiberg, A.; Lin, S.; Timpmann, K.; Blankenship, R. E. J. Phys. Chem. B 1997, 101, 7211. (40) Reddy, N. R. S.; Kolaczkowski, S. V.; Small, G. J. J. Phys. Chem. 1993, 97, 6934.

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