The Red-Absorbing Chlorophyll - American Chemical Society

836

J. Phys. Chem. B 2000, 104, 836-847

The Red-Absorbing Chlorophyll a Antenna States of Photosystem I: A Hole-Burning Study of Synechocystis sp. PCC 6803 and Its Mutants M. Ra1 tsep,† T. W. Johnson,‡ P. R. Chitnis,‡ and G. J. Small*,† Ames Laboratory-U.S. Department of Energy and Departments of Chemistry, and Biochemistry, Biophysics, and Molecular Biology, Iowa State UniVersity, Ames, Iowa 50011 ReceiVed: August 19, 1999; In Final Form: NoVember 11, 1999

Low temperature (4.2 K) absorption and hole-burned spectra are presented for the trimeric (wild-type, WT) photosystem I complex of the cyanobacterium Synechocystis sp. PCC 6803, its monomeric form, and mutants deficient in the PsaF, K, L, and M protein subunits. High-pressure- and Stark-hole-burning data for the WT trimer are presented as well as its temperature-dependent Qy-absorption and -fluorescence spectra. Taken as a whole, the data lead to assignment of a new and lowest energy antenna Qy-state located at 714 nm at low temperatures. It is this state that is responsible for the fluorescence in the low-temperature limit and not the previously identified antenna Qy-state near 708 nm. The data indicate that the 714 nm state is associated with strongly coupled chlorophyll a molecules (perhaps a dimer) and possesses significant charge transfer character. The red chlorophylls absorbing at 708 and 714 nm do not appear to be directly bound to any of the above protein subunits. The results are consistent with a location close to the interfacial regions between PsaL and M and the PsaA/B heterodimeric core. It is likely that the red chlorophylls are bound to PsaA and/or PsaB.

1. Introduction An interesting feature of many photosystems is that the longest wavelength Qy-state of at least one antenna or lightharvesting (LH) protein complex [close to the reaction center (RC)] appears to lie at lower energy than that of the primary electron donor (PED) state of the RC at room temperature. Such a conclusion has usually been based on the origin absorption maxima of the antenna and PED chlorophyll (Chl) states. Thus, the optical reorganization energies of the absorption transitions are not taken into account. Their neglect can lead to a significant error in the energy gap. The relevant energies are those of the zero-point vibrational levels of the antenna and PED states since the absorption maximum is blue-shifted relative to the zeropoint level by the optical reorganization energy, ∑i Siωi. Here, ωi is the frequency of the ith phonon coupled to the optical transition and Si is its Huang-Rhys factor. Until spectral holeburning spectroscopies1 were applied to photosynthetic complexes, little was known about the reorganization energies of their excited Chl states (for reviews see refs 2 and 3). It is the zero-phonon hole (ZPH) and its accompanying phonon sideband hole structure that lead to characterization and determination of the linear electron-phonon coupling. Typically, this coupling is weak for antenna Qy-states with ωm ≈ 20-30 cm-1 and Sm ≈ 0.5 where ωm is the peak frequency of the one-phonon profile that carries a width of about 30 cm-1. Thus, the reorganization energy Sωωm is only 10 or so cm-1. An example is the LH1 cyclic BChl a complex of Rhodobacter sphaeroides (a purple bacteria) that encircles the reaction center (RC) complex. The LH1 complex is often referred to as B875 because it exhibits an absorption maximum near 875 nm at room temperature. The PED (the “special pair” P) of the RC is referred to as P870. * Corresponding author. E-mail: [email protected]. † Ames Laboratory. ‡ Department of Biochemistry, Biophysics, and Molecular Biology.

Thus, one might conclude that B875*(* indicating excited state) lies about 70 cm-1 below P870*. However, the electron-phonon coupling of P870 is strong, with an optical reorganization energy of 240 cm-1 (18 nm) due to modes at ≈30 and 120 cm-1.4 Thus, for this system the above reasoning based on absorption maxima is flawed. (The reorganization energies for P960 of the Rhodopseudomonas Viridis RC and P700 of the photosystem I (PS I) RC are also large as reviewed in ref 5.) On the basis of the hole burning and other results given in refs 4, 6, and 7, the zero-point level of the lowest exciton level (B896) of the B875 ring is predicted to lie (on average) ∼100 cm-1 higher in energy than that of P870* at room temperature.8 In the low-temperature limit this gap is reduced to ∼50 cm-1, meaning that energy transfer from LH1 to P870 is still operative.8 An example of a photosystem whose lowest energy antenna state is clearly lower in energy than the PED state at low temperatures is PS II of green plants. The wavelengths of the lowest energy state of the proximal antenna complex, CP47, and the PED state, P680, are 690 and 680 nm, respectively.9-11 Thus, selective excitation of the 690 nm state of CP47 would not be expected to result in charge separation at liquid helium temperatures since the thermal energy available is much smaller than the energy gap of ≈200 cm-1. (The optical reorganization energy of P680 is only ∼50 cm-1.) The hole-burning results of den Hartog12 confirm that this is the case. The situation just described for PS II is similar to that of PS I which is the subject of this paper. Before reviewing the supporting data, it is appropriate to discuss the overall structure of PS I in cyanobacteria. (Unlike PS I of higher plants, PS I of cyanobacteria are devoid of the peripheral LHC I Chl a/Chl b antenna complex and contains only Chl a antenna molecules.) Wild-type cyanobacterial PS I complexes exist in trimeric and monomeric forms13 (referred to hereafter as WT trimer and WT monomer). A monomer of cyanobacterial PS I contains eleven proteins, about 90 Chl a molecules, 10-12 β-carotene mol-

10.1021/jp9929418 CCC: $19.00 © 2000 American Chemical Society Published on Web 01/06/2000

Chlorophyll a Antenna States

Figure 1. Depiction of the protein subunits of cyanobacterial photosystem I. The schematic shows the top view of a monomer and is based on information from X-ray crystallography.

ecules, two phylloquinones, and three [4Fe-4S] clusters. Structural organization of PS I components is known from the X-ray crystallographic analysis at 4 Å resolution of PS I of Synechococcus elongatus.14,15 The function and molecular genetics of photosystem I has recently been reviewed.16 What follows is a brief summary. The PsaA and PsaB proteins of PS I form a heterodimeric core, Figure 1. PsaA and PsaB bind the primary electron donor P700, which is a pair of Chl molecules. One of the monomers of a pair of Chl a molecules (A0) is believed to function as a primary electron acceptor. The pair is bound to PsaA and PsaB which, together, bind about 70 antenna Chl a molecules. Another pair of Chl a molecules (A) is located symmetrically between P700 and the A0 Chl molecules. They are analogous to the accessory BChl molecules of the bacterial reaction center. Because of the basic structural similarity between the electron transfer cofactors of the PS I and bacterial RC, it is likely that at least one of the two A molecules plays a crucial role in the initial phase of charge separation. In addition to the core proteins, the cyanobacterial PS I complex contains three peripheral proteins (PsaC, PsaD, and PsaE) and six integral membrane proteins (PsaF, PsaI, PsaJ, PsaK, PsaL, and PsaM), Figure 1. Organization of these proteins has been deciphered from biochemical studies, electron microscopy and, recently, from crystallographic analyses. We note that the relocation of the K and M subunits shown in Figure 1 is a consequence of further refinement of the X-ray structure (N. Krauss, personal communication). PsaC is a small Fe-S protein that contains the terminal [4Fe-4S] electron accepting clusters FA and FB. The remaining peripheral proteins provide a docking surface for ferredoxin which accepts electrons from the FB cluster in PsaC. PsaL is the integral component of the connecting domain that is responsible for the formation of trimers from three monomers in WT PS I. PsaL contains three transmembrane helices. The innermost transmembrane helix of PsaL binds three antenna Chl molecules. PsaI and PsaM are two short peptides

J. Phys. Chem. B, Vol. 104, No. 4, 2000 837 with one hydrophobic helix in each. They are present on the trimer-forming side of the monomeric PS I complexes. In the absence of PsaM or PsaI, trimers are present at a significantly reduced level. Due to the lack of Chl-coordinating residues, these proteins are not thought to bind Chl molecules directly. PsaF, PsaJ, and PsaK are present on the outer side of PS I monomers and therefore do not face other monomers in a trimer. PsaF probably binds Chl molecules, because it can be isolated as a Chl-protein complex. However, the exact number of chlorophyll molecules bound to PsaF and residues that coordinate these chlorophylls are not known. PsaJ and PsaK contain one and two transmembrane helices, respectively. It is not known if PsaJ and PsaK bind Chl molecules. As mentioned, PS I contains long wavelength absorbing Chl molecules (“red Chls”) whose Qy-state(s) lie lower in energy than P700*. The number of red Chls depends on species. For example, in the cyanobacterium Synechocystis PCC 6803 they show a distinct absorption band at 708 nm at temperatures j77 K and, for that reason, are sometimes referred to as C-708.17,18 It was estimated that the number of Chl a molecules associated with C-708 is two.18 In that work the fluorescence line narrowed spectra indicated that the electron-phonon coupling of C-708 (proposed to be a dimer) is strong, in contrast with the weak coupling observed for previously studied antenna Qy-states. In the case of the cyanobacterium Synechococcus elongatus, the red Chls absorb at 708 and 719 nm at low temperatures.19 It was estimated that five or six Chl a molecules contribute to C-708 and four or five to C-719.19 It is emphasized that there is considerable uncertainty in these numbers as well as in the number of two for C-708 of Synechocystis because of the weak absorption at wavelengths longer than about 700 nm. In ref 19 it was shown that the nonline narrowed fluorescence spectrum of Synechocystis exhibits an origin band near 720 nm at low temperatures. The fluorescence was attributed to the 708 nm absorbing Chls. Thus, the assumed Stokes shift is large, ∼250 cm-1, and was interpreted in terms of strong electronphonon coupling. Very recently, fluorescence origin bands at 77 K of several mutants of Synechocystis were reported.20 The mutants studied were deficient in the L, K, M and F/J/I Psa protein subunits. (In what follows we will refer to these simply as L,... mutants.) Importantly, the fluorescence origin bands of the mutants were very similar to that of WT trimers with maxima near 720 nm. (Again, the deletion of the L subunit results in monomer formation; the F/J/I mutant is also deficient in PsaL and, therefore, also occurs in monomeric form.) Thus, these results suggest that the 708 nm Chls are not bound to the L, K, M, and F/J/I subunits. Related to this is earlier work on the WT monomer (which possesses PsaL) of Synechocystis.17 Monomer formation led to about a 30% decrease in the absorption intensity of the 708 nm Chl. The fluorescence origin band still appeared near 720 nm. We present here the results of low-temperature absorption and persistent nonphotochemical hole burning experiments on WT trimer and WT monomer of Synechocystis as well as its F, K, L, and M mutants. Temperature-dependent fluorescence spectra and high pressure and Stark hole-burning data for WT trimer are also presented. The main objectives were to characterize more completely the Qy-states of the red Chls and to further test the hypothesis that they are not intrinsic to the F, K, L, and M subunits. Taken as a whole, the hole-burning results indicate that, in addition to antenna Chls absorbing near 708 nm, there are red Chls absorbing at 714 nm which were not resolved in previous low-temperature experiments. Although the existence of a new antenna Qy-state at 714 nm is the most

838 J. Phys. Chem. B, Vol. 104, No. 4, 2000

Ra¨tsep et al.

TABLE 1: Cyanobacterial PS I Preparations Used in this Study23 PS I preparation

abbreviation used in the text

cyanobacterial strain

missing proteins

wild type trimer wild type monomer PsaL-less monomer PsaK-less trimer PsaF-less trimer PsaM-less, trimer

WT trimer WT monomer L K F M

wild type wild type ALC7 (PsaL gene inactivated) PsaK1-psaK2-strain AFK6 (psaF deleted) PsaM strain

none none PsaL PsaK PsaF, PsaJ PsaM

obvious interpretation of the data, an alternative explanation is presented that preserves the notion that there is only one type of red Chl in Synechocystis with a peak absorption at 708 nm. It rests on the assumption that higher energy absorbing members of the ensemble of 708 nm Chl are able to transfer energy to P700 as well as assumptions that are not supported by existing data. 2. Experimental Section Cyanobacterial Cultures. Table 1 lists the strains of Synechocystis sp. PCC 6803 that were used in the present studies. The cyanobacterial cells were cultured in BG-11 medium with appropriate antibiotics (30 mg/l chloramphenicol or 40 mg/l kanamycin) at 30 °C and were aerated by bubbling with air. The growth of Synechocystis cells was monitored from absorption of cultures at 730 nm (A730) using an UV-160U spectrophotometer (Shimadzu, Tokyo, Japan). Cells were harvested at the late exponential growth phase, resuspended in 0.4 M sucrose, 10 mM NaCl, 10 mM MOPS-HCl (pH 7.0), and stored at -20 °C for further use. Preparation of Thylakoid Membranes, PS I Complexes, and Donor Proteins. To isolate thylakoid membranes, cells were broken with a bead beater (Biospec Products). The trimeric PS I complexes were purified from membranes by extraction with dodecyl-2-maltoside and ultracentrifugation on sucrose gradient using a published method.21 Isolation of PS I monomers involved incubation of membranes with 25 mM EDTA for 60 min before detergent extraction and ion-exchange chromatography on DEAE cellulose column prior to ultracentrifugation.21 Chlorophyll a concentrations in the thylakoid membranes and PS I complexes were determined in 80% (v/v) acetone. Ultrafiltration was used for concentration or buffer exchange of the PS I samples. The purity of PS I samples was evaluated by polyacrylamide gel electrophoresis. The PS I complexes and photosynthetic membranes were solubilized at 37 °C for 2 h with 1% SDS and 0.1% DTT. Proteins were then resolved by Tricine/urea/ SDS-PAGE and examined by Coomassie blue or silver staining. The relative intensities of different polypeptides indicated the stoichiometry of different PS I proteins. The mutant and WT trimer PS I complexes contained similar levels of expected protein subunits. The monomeric and trimeric PS I complexes had identical protein composition. Therefore, the mutant PS I complexes only lacked the expected subunits (Table 1). Spectroscopic Measurements. The hole-burning apparatus is described in detail elsewhere.22 Briefly, preburn and postburn spectra were obtained using a Bruker IFS 120 HR Fourier transform spectrometer (resolutions used are given in the Figure captions). A Coherent CR 699-21 dye laser pumped by a 6 W Coherent Innova Ar-ion laser was used for hole burning in the 670-710 nm range; line width )0.07 cm-1. A Coherent CR899-31 Ti:sapphire laser pumped by a 15 W Coherent Innova Ar-ion laser was used for hole burning in the 702-722

nm range; line width )0.07 cm-1. Burn intensities and times are given in the figure captions. Samples were stored in the dark at -70 °C prior to use in the spectroscopic experiments. To avoid glass-cracking, sample solutions containing 10 mM MOPS-HCl (pH 7) and 0.05% dodecyl-2-maltoside were diluted with glycerol (2:1 v/v). For absorption and hole burning measurements in the absence of an external electric field or pressure, the samples were contained in polypropylene tubes (i.d. ) 10 mm). The high-pressure apparatus has been described in detail in refs 23 and 24, including the procedure used to measure pressure. To ensure good optical quality, the sample was contained in a gelatin capsule (5 mm outside diameter) purchased from Torpac, Inc., and then housed in a specially designed high pressure cell with four sapphire windows (thickness of 4 mm) providing optical access. The cell was connected to a three-stage hydraulic compressor (Model U11, Unipress Equipment Division, Polish Academy of Sciences) through a flexible thick-walled capillary (o.d./i.d. ) 3.0 mm/0.3 mm). Helium gas was used as the pressure-transmitting medium. A specially designed Janis 11-DT cryostat was used for cooling of the high-pressure cell. High-pressure hole burning was performed at 12 K. At this temperature liquid helium solidifies at ∼75 MPa. Following the procedure given in ref 23, it was confirmed that pressure-induced structural changes are elastic. For studies at higher temperatures and pressures, a Lakeshore Cryotronic temperature controller (model 330) was used to stabilize and measure the temperature. The Stark hole-burning apparatus used was the same as that described in ref 25. Samples were contained in gelatin capsules. Prior to insertion into the Stark cell, the gelatin capsule filled with sample was allowed to soften for about 5 min at room temperature so that it could be mechanically squeezed by the two copper electrodes of the Stark cell. This procedure yielded an optical path length perpendicular to the applied field of ∼6 mm with a distance of ∼2 mm between the electrodes. Teflon spacers were used to set the distance between the electrodes ((0.05 mm). The Stark field could be applied parallel or perpendicular to the burn laser polarization by positioning a polarizer placed in front of the Stark cell. The probing light (unpolarized) was collinear with the burning beam. A Trek, Inc., model 610 C dc high-voltage power supply (0 to ( 10 kV) was used. By changing the polarity of the power supply, a maximum Stark field of ∼ 100 kV/cm was achievable. Holes were initially burned at the highest field with a chosen polarity. All Stark measurements were performed at 1.8 K in a Janis 10 DT liquid helium cryostat. The excitation source for the fluorescence experiments was a Coherent UV Ar-ion laser operating at 351.1 nm. Fluorescence was dispersed by a McPherson 2061 1-m focal length monochromator at a resolution of 0.8 nm and detected by a Princeton Instruments IRY 1024/G/B intensified photodiode array. Samples were contained in 2 mm i.d. quartz tubes. Care was taken to ensure that reabsorption effects were negligible.


J. Phys. Chem. B, Vol. 104, No. 4, 2000 839

Figure 3. Hole burned spectra (4.2 K) obtained with λB ) 670.0 nm of (a) WT trimer, (b) L mutant, and (c) WT monomer. Burn intensity ) 250 mW/cm2; burn time ) 60 min. The read resolution was 2 cm-1. For comparison, the WT trimer absorption spectrum is also shown (d).

Figure 2. Absorption and difference absorption spectra of the PS I complex of Synechocystis PCC 6803 obtained with a resolution of 4 cm-1 at 4.2 K. Part A: the absorption spectrum of trimeric wild-type (WT) complex. The numbers labeling the features are the wavelengths in nanometers. Part B: the PsaL-less monomeric mutant (L) absorption and the WT trimer-L difference absorption (∆A) spectrum. Part C: the PsaF-less trimeric mutant absorption and the WT trimer-F ∆A spectrum. The left and right ordinate scales correspond to the absorption and difference spectra, respectively. The solid and dotted line absorption spectra in parts B and C are the absorption spectrum of WT trimer (as shown in part A) and the mutant, respectively. The scaling procedures used to obtain the difference spectra ensured that ∆A ≈ 0 to the red of the absorption spectrum and that ∆A > 0 or ≈0 throughout.

3. Results Absorption spectra were obtained at room temperature and 4.2 K for WT trimer, WT monomer, and the F, K, L, and M mutants. The 4.2 K spectrum of WT trimer and those of the L and F mutants are shown in Figures 2A, 2B, and 2C, respectively. The structure seen in the spectra is quite remarkable given that the photosystem contains about 90 Chl a molecules per monomer. The wavelengths in nanometers (nm) of the spectral features for WT trimer are indicated in Figure 2A. They are values obtained directly from the spectrum. The dependence of the WT trimer absorption spectrum on temperature between 5 and 295 K was determined. The spectrum obtained at 68 K is very similar to the 77 K spectrum reported by van der Lee et al.17 The focus in this paper is on the longest wavelength bands at 692, 695, 699, and 708 nm and the effects of subunit deletion and monomer formation on them that are discernible by inspection. More extensive experiments (including control) would be required to determine the extent to which the features at wavelengths shorter than 692 nm in the difference absorption spectra are reliable and whether such features are excitonically correlated with the longest wavelength bands. One observes that

deletion of the L subunit leads to a significant reduction in 708 nm absorption, about 30% at 708 nm. The WT trimer-L difference spectrum leads to a width of 400 cm-1 for the 708 nm difference band that tails out to 720 nm, as does the absorption spectrum. Importantly, the WT trimer-M and WT trimer-WT monomer difference spectra (not shown) are essentially identical to that of the WT trimer-L spectrum for λ > 690 nm. For all three the integrated intensity of the 708 nm difference band relative to that of the WT trimer absorption spectrum (650 to 730 nm) led to a percentage absorption loss of ∼2. That the WT monomer exhibits a decrease in intensity of the 708 nm band of WT trimer was previously observed at 77 K.17 In frame B of Figure 2 one sees that deletion of the L subunit has little effect on the 692 and 695 nm bands (shoulders). This was also the case for the M mutant and WT monomer. The WT trimer-L, WT trimer-M, and WT trimer-WT monomer difference spectra obtained at room temperature (not shown) are very similar to those of Soukalis et al.20 All three showed a prominent and lowest energy difference band near 702 nm. It is likely that this profile corresponds to the 708 nm profile at 4.2 K, vide infra. Concerning the F mutant, the spectra in frame C of Figure 2 reveal that deletion of the F subunit has no effect on the 708 nm band and, probably also, the 699 nm band. (Deletion of the K subunit also had no effect on the 708 nm band, results not shown.) However, there is a significant loss of intensity centered near 689 nm. At room temperature the rather broad, flat-topped feature at 689 nm is smoothed and quite symmetric but is still located at 689 nm (results not shown). It is reasonable to conclude that some of the Chls of the F subunit contribute to absorption between about 686 and 695 nm at 4.2 K. Figure 3 shows persistent nonphotochemical hole-burned (NPHB) spectra for WT trimer, the L mutant, and WT monomer obtained at 4.2 K with a burn wavelength (λB) equal to 670.0 nm that coincides with the relatively sharp zero-phonon hole (ZPH). For ease of discussion, the WT trimer absorption spectrum is also shown. A number of conclusions can be drawn. First, the three NPHB spectra are very similar. (The NPHB spectrum of the M mutant, not shown, is also very similar.) Second, and importantly, the relatively broad hole (fwhm ∼ 230 cm-1) at 714 nm does not coincide with the maximum of the 708 nm absorption band. Third, the low energy satellite holes produced by burning at 670 nm correlate quite well with features of the absorption spectrum, the only exception being the just-


Figure 4. Hole burned spectra (4.2 K) of WT trimer obtained with burn wavelengths (λB) given in the figure. The solid vertical lines indicate the real- and pseudo-phonon sideband holes with a peak frequency of 16 cm-1. The dashed arrows in the λB ) 714 and 718 nm spectra locate a higher frequency phonon (≈ 100 cm-1) that couples to the electronic transition. The solid arrows in the λB ) 702 and 706 nm spectra mark the broad 714 nm hole while those in the λB ) 710 and 714 nm mark the high energy satellite holes at 692, 695, and 699 nm. See text for burn fluences. The read resolution was 1 cm-1.

mentioned 714 nm hole. Fourth, the interactions between the monomers of the PSI trimer have, at most, a weak effect on its Qy-electronic spectrum (structure). Fifth, except for the 714 nm hole the satellite holes are quite narrow, which suggests that the contribution from inhomogeneous broadening to the absorption bands cannot be larger than about 70 cm-1 (see, for example, the 692 and 699 nm holes). To investigate further the nature of the red-most states of PSI, NPHB spectra of WT trimer were obtained with λB-values between 690 and 722 nm. At each burn wavelength spectra were obtained with burn fluences of 6, 30, 150, and 450 J/cm2. Figure 4 shows the spectra for λB ) 702, 706, 710, 714, 718, and 722 nm obtained with a burn fluence of 150 J/cm2 except for the λB ) 722 nm spectrum that was obtained with a burn fluence of 450 J/cm2. In each spectrum the ZPH coincident with the burn wavelength is labeled by λB. The features (indicated by solid vertical lines) just to the left and right of the ZPH are the realand pseudo-phonon sideband holes (PSBH).2,26 In each spectrum they are displaced from the ZPH by 16 cm-1 which is the peak wavenumber value of the phonons that couple to the electronic transitions. The solid arrow in the λB ) 702 and 706 nm spectra indicates the location of the broad 714 nm hole, vide supra. It is more apparent in the spectra obtained with a burn fluence of 450 J/cm2 (not shown). The solid arrows in the λB ) 710 and 714 nm spectra at 699, 695, and 692 nm locate high energy satellite holes that coincide with features in the absorption spectrum, see Figure 2. The 699 and 692 nm holes can also be seen in the λB ) 706 and 718 nm spectra. It is not clear if the states associated with the satellite holes are excitonically correlated with the states directly excited or are responses to the structural changes that accompany nonphotochemical hole burning of the states directly excited. We note that the 699 and 695 nm holes are superimposed on the broad antihole (positive absorption) that is associated with and blue-shifted relative to

Ra¨tsep et al. the hole formed by burning at λB; see, for example, the λB ) 706, 710, and 714 nm spectra. The presence of such an antihole is the signature for NPHB of S1 (ππ*) states.27,28 In comparing the NPHB spectra in Figure 4 we conclude, based on the theory of spectral hole profiles29 and earlier studies5 of photosynthetic systems that the linear electron-phonon coupling increases from weak (S < 1) at 702 nm to strong (S > 1) at λB’s J710 nm (see section 4). Here S, is the total HuangRhys factor for the low-frequency phonons. This variation in coupling strength, together with the observation of the 714 nm hole under nonline narrowing conditions, suggest that the 708 nm absorption band is contributed to by at least two Qy-states, one being the 714 nm state characterized by strong electronphonon coupling and large inhomogeneous broadening, cf. section 4. The absorption maximum of the other state(s) would need to be near 708 nm in order to account for the profile of the 708 nm absorption. As reviewed in ref 5, strong electron-phonon coupling of Qy r S0 transitions in photosynthetic complexes can be associated with a large change in permanent dipole moment. An important example is the BChl a special pair (P870) state of the Rb. sphaeroides reaction center for which f • ∆µ ) 5.2 D,30 where f is the local field correction and ∆µ is the magnitude of the permanent dipole moment change. A value for f of ∼1.5 based on the Lorentz local field correction has sometimes been used, but since the dielectric constant is not known, we prefer to report f • ∆µ values. The optical reorganization energy of the P870* r S0 transition is large, 235 cm-1, and due to 30 and 120 cm-1 modes with S-factors of 1.8 and 1.5, respectively. In sharp contrast, the electron-phonon coupling of the Qy r S0 transitions of antenna complexes studied to date is weak with an optical reorganization of only 10-20 cm-1.2,3,5 An example is the Fenna-Matthews-Olson BChl a antenna complex of Cb. tepidum for which f • ∆µ ) 0.6 D25 and the reorganization energy equals ∼10 cm-1.31 As reviewed in ref 5 and discussed more recently in refs 32 and 33, large dipole moment changes in photosynthetic complexes appear to arise from electronexchange coupling between closely spaced Chls. The coupling endows the Qy-state with significant charge-transfer character. With this in mind we performed Stark-hole-burning experiments on WT trimer in order to gain further insight on the nature of the 714 nm and other red absorbing states. Results were obtained for λB ) 690.0, 692.0, 695.0, 698.5, 702.0, 706.5, 708.0, 712.0, 714.0, and 716.0 nm. Stark splitting of the ZPH was not observed, only Stark broadening for laser polarization parallel and perpendicular to the applied electric field. Within experimental uncertainty, the f • ∆µ values were identical for both polarizations. Stark broadening data for λB ) 692.0, 706.5, and 714.0 nm are shown in Figure 5. The solid curves are fits obtained using the following expression for the Stark broadening, Γ(F):

Γ(F) ) 2γ(1 + F2)1/2

(1)

where

F)

2f‚∆µEs pγ

(2)

and γ is the width of the ZPH at zero applied field (ES). Equation 1 follows from the theory of Kador et al.34 It is valid when the quadratic dependence of the zero-phonon line frequency on applied field is negligible and F j3.5. Given that our maximum field strength is only 100 kV/cm, the first condition is met.25 We confirmed that the F j3.5 condition is also satisfied. The



TABLE 2: Stark-hole Burning Results for WT Photosystem I Trimer of Synechocystis and Other Systems Containing Chl a λB, nm

f • ∆µ (D)

WT trimer PS I of Synechocystis

690.0 692.0 695.0 698.5 702.0 706.5 708.0 710.0 712.0 714.0 716.0

0.50 ( 0.20 0.56 ( 0.10 0.48 ( 0.10 0.67 ( 0.10 0.64 ( 0.10 0.78 ( 0.15 1.0 ( 0.40 2.0 ( 0.40 1.8 ( 0.20 2.3 ( 0.20 2.4 ( 0.20

this work this work this work this work this work this work this work this work this work this work this work

Chl a:PVB polymer films

666.0 674.0 670.0 672.0 680.0

0.52 ( 0.05 0.52 ( 0.05 0.50 ( 0.05 0.49 ( 0.05 0.63 ( 0.10

Small and Ra¨tsep, unpublished results

system

Chl a:PS II reaction center + Triton X-100 Chl a:LHC II

Figure 5. Dependence of the 1.8 K widths of the zero-phonon holes of WT trimer burned at λB ) 692.0, 706.5, and 714.0 nm on electric (Stark) field. Diamonds are the experimental points. The solid curves are theoretical fits, cf. text. From top to bottom the f • ∆µ values are 0.56, 0.78, and 2.3 D, respectively. The laser polarization was perpendicular to the applied Stark field.

fits for λB ) 692.0, 706.5, and 714.0 nm result in f • ∆µ values of 0.56 ( 0.10, 0.78 ( 0.15, and 2.3 ( 0.2 D, respectively. The f • ∆µ values for all λB-values are given in Table 2. For comparison, f • ∆µ values from Stark-hole burning are given for Chl a monomer in a poly(vinylbutyral) film, the lowest energy Chl a Qy-state of the LHC II antenna complex of photosystem II of green plants, and Chl a disrupted by Triton X-100 in the photosystem II reaction center. In section 4 we argue that the value of f • ∆µ for a weakly coupled (negligible electron-exchange coupling) Chl a molecule in proteins is ≈0.6 D. Accepting this, the results in Table 2 for PSI indicate that the Chl a molecules contributing to absorption at λ-values j706 nm are weakly coupled. In contrast, for example, the Chl a molecules responsible for absorption near 714 nm are strongly coupled, consistent with the strong electron-phonon coupling in this region, see section IV. That the f • ∆µ values of 0.78 and 1.0 D PS I of Synechocystis at 706.5 and 708.0 nm are a factor of 2-3 times smaller than the 2.3 D value at 714 nm is

ref

Small and Ra¨tsep, unpublished results Small and Ra¨tsep, unpublished results

consistent with the 708 nm band being contributed to by the 714 nm state and a state near 708 nm. It has recently been shown that the pressure dependence of hole-burned and low-temperature absorption spectra are useful for identifying Qy-states of photosynthetic complexes that are associated with strongly coupled Chl molecules.7,35,36 Specifically, states can be categorized as weakly or strongly coupled based on the linear shifting (to the red) of their absorption bands and/or ZPH burned into the bands with increasing pressure. Strong and weak coupling is defined by shift rates j0.15 cm-1/ MPa and J0.2 cm-1/MPa, respectively. Examples of shift rates are given in Table 3. We note that shift rates of -0.05 to -0.15 cm-1/MPa are typical for ππ* states of isolated chromophores in polymers and glasses. The dependence of the WT trimer absorption spectrum on pressure between 16 and 803 MPa was determined at 62 K. Only the 16 and 803 MPa spectra are shown in Figure 6. The shift rate for the 708 nm band is by far the largest (∼ -0.3 to -0.4 cm-1/MPa) consistent with contributing Chls that are strongly coupled. The estimated shift rates of -0.10 cm-1/MPa for the 672 and 680 nm bands and -0.15 cm-1/ MPa for the ∼695 nm shoulder indicate weak coupling. More selective and accurate pressure shift rates are provided by hole burning. Table 3 summarizes the shift rates for eight λB-values between 688.0 and 718.0 nm. The data for λB ) 690.0, 710.0, and 718.0 nm are shown in Figure 7. The shift rates for λB between 688.0 and 697.0 nm (-0.17 to -0.20 cm-1/MPa) are somewhat larger than -0.15 cm-1/MPa which is our rough demarcation for the onset of strong coupling, vide supra. However, these shift rates are a factor of 2 lower than the values (-0.42 to -0.49 cm-1/MPa) obtained with λB ) 710.0, 713.0, and 718.0 nm, values that definitely indicate strong electronexchange coupling and are comparable to those observed for the strongly coupled BChl a molecules of the B850 and B875 rings of the LH2 and LH1 antenna complexes of purple bacteria, Table 3. Experiments are planned to determine the linear pressure shift rates for λB-values between 696 and 710 nm. (At the time we performed the experiments our main objective was to determine whether the linear pressure shift rates for λB-values in the vicinity of 714 nm are consistent with strong coupling.) During the initial stage of our studies it was observed that the NPHB efficiency at the blue side of the Qy-absorption spectrum is significantly lower than at the red side. Since the efficiency depends sensitively on the homogeneous width of the zero-phonon line at λB and the lifetime of the state excited, more detailed experiments were performed. The results for WT trimer are shown in Figure 8 where the diamonds are the


Ra¨tsep et al.

TABLE 3: Linear Pressure Shifts for PS I of Synechocystis (WT trimer) and Other Photosynthetic Complexes at 12 K complex

banda/stateb/ λB,c nm

pressure shift, cm-1/MPa

688.0c 690.0c 692.0c 694.0c 696.0c 710.0c 713.0c 718.0c B800a B850a B870b B875a B896b B800a B850a B870 B824a B824b

-0.17 ( 0.01 -0.19 ( 0.01 -0.19 ( 0.01 -0.20 ( 0.01 -0.20 ( 0.01 -0.42 ( 0.02 -0.45 ( 0.02 -0.49 ( 0.02 -0.15 -0.38 -0.54 -0.63 -0.67 -0.09 -0.39 -0.51 -0.10 -0.15

WT trimer PS I of Synechocystis

LH2 antenna complex of Rb. sphaeroides LH1 antenna complex of Rb. sphaeroides LH2 antenna complex of Rps. acidophila FMO antenna complex of Cb. tepidum

ref this work this work this work this work this work this work this work this work 46 7 46 54

a Shift rate for absorption band. b Shift rate of zero-phonon holes burned into the absorption band of a single Qy-state. c The pressure shifts of zero-phonon holes burned at the indicated wavelengths. d B870 and B896 are, respectively, the lowest exciton levels of the LH2 and LH1 complexes. Their pressure shift rates were determined by monitoring zero-phonon holes.

Figure 6. The absorption spectra of WT trimer at pressures of 16 and 803 MPa obtained at 63 K. Read resolution was 4 cm-1.

Figure 7. Linear pressure shifting of zero-phonon holes of WT trimer burned at 12 K and a pressure of 16 MPa. The shift rates for λB ) 690, 710 and 718 nm are -0.19, -0.42, and -0.49 cm-1/MPa.

∆-absorbance (∆A) values for the ZPH burned with a constant fluence of 60 J/cm2 and read with a resolution of 2 cm-1. The resulting spectrum is referred to as a ZPH action spectrum. Such spectroscopy has been used to identify the lowest exciton level of the B850 and B875 BChl a rings of the LH2 and LH1 antenna complexes of purple bacteria,7,37 the lowest Qy-state of the LHC II trimer antenna complex of PS II of green plants and the lowest energy Qy-state of the isolated PS II reaction center.38,39 The action spectrum in Figure 8 shows a broad feature with a

Figure 8. The zero-phonon hole action spectrum (diamonds) of WT trimer obtained at 4.2 K with a constant burn fluence of 60 J/cm2 and a read resolution of 2 cm-1. For comparison, the WT trimer absorption spectrum is also shown.

maximum near 712 nm. An approximate gauge of relative holeburning efficiencies is given by the ∆A/A values. It is apparent that the efficiency decreases rapidly for λB shorter than about 688 nm. Between 670 and 684 nm the ∆A/A values are essentially constant and a factor of about 20 times smaller than those between 690 and 698 nm. They are a factor of about 10 times smaller than the values between 710 and 718 nm. A qualitative understanding of the differences in hole-burning efficiency emerges from the theoretical expression for hole growth kinetics41 that contains the term exp(-Pσφt) where P is the burn photon flux, σ the peak absorption cross-section of the zero-phonon line, φ the hole burning quantum yield, and t the burn time (Pt was held constant in the experiments). Generally (and as is the case here) φ , 1 even for nanosecond excited-state lifetimes (τ).41 Thus, φ ≈ kNPHB τ, where kNPHB is the rate constant for hole-burning. σ is proportional to exp(S), the Franck-Condon factor for the zero-phonon line, and inversely proportional to its homogeneous width. For τ j10 ps and T j4 K, the contribution to the homogeneous width from pure dephasing should be negligible.42,43 Thus, this width is inversely proportional to τ. One has, therefore, that the hole growth kinetics depend on an exponential with an argument containing τ2 exp(-S). The excited lifetime is given by τ ) (πcΓ)-1 where Γ is the width of the zero-phonon hole in cm-1


Figure 9. Temperature dependence of the origin band region of the fluorescence spectrum of WT trimer obtained with laser excitation at 351.1 nm. The intensities of the fluorescence at different temperatures can be directly compared.

and c ) 3 × 1010cm s-1. Hole widths were measured with a resolution of 1 cm-1 at 4.2 K for λB in the range 670-684 nm. The widths were reasonably constant, 4-5 cm-1, corresponding to τ-values of about 3 ps. Hole widths at λB ) 692, 695, and 698 nm led to lifetimes of close to 10 ps. Because S < 1 at these and shorter wavelengths, the higher hole-burning efficiency for the 690-698 nm region relative to the 670-684 nm region can be qualitatively understood in terms of the τ2 term, assuming that the kNPHB and exp(-S) is reasonably constant. The resolution (1 cm-1) used to read the holes for λB-values >702 nm was too low to permit resolution. Higher resolution (20 MHz) fluorescence excitation experiments are planned. It is likely that the hole widths at 4.2 K of the lowest energy fluorescent state will be dominated by pure dephasing induced by the two-level systems of the protein as recently observed in other photosynthetic complexes.12,38,44 Unfortunately, the above 3 and 10 ps energy transfer lifetimes cannot be compared with those from ultrafast time domain experiments since they have only been performed at room temperature.45,46 Van der Lee et al.17 reported that the fluorescence origin band of WT trimer of Synechocystis sp. PCC 6803 at 77 K lies at 724 nm. (The origin band of the WT monomer at 77 K was reported to be at 720 nm.18) They assigned the fluorescence to the state responsible for the 708 nm absorption band and interpreted the large Stokes shift (≈300 cm-1) in terms of strong electron-phonon coupling. Recently, Soukalis et al.20 reported that the fluorescence origin bands of WT trimer and the L, M and F/J/I (also missing L) mutants are very similar at 77 K and centered at 722 nm. Figure 9 shows the temperature dependence of the WT trimer fluorescence origin region obtained with excitation at 351.1 nm. The same detection sensitivity was used for all temperatures. The peak position at 4.2 K is 722.5 nm. Within experimental uncertainty this position is unchanged up to and including T ) 120 K. At higher temperatures the band blue-shifts and at 220 K lies at 712.4 nm. The conclusion of Gobets et al.18 that the marked decrease in the intensity of the fluorescence band at ≈ 722 nm with increasing temperature is due to thermal population of P700* from the red absorbing state


Figure 10. Temperature dependence of the absorption spectrum of WT trimer.

responsible for the 722 nm fluorescence seems reasonable, cf. section 4. The integrated intensity of the origin band at 4.2 K in Figure 9 is a factor of 30 greater than that at 220 K. Given that the position of the fluorescence origin band is independent of temperature up to 120 K, we determined the temperature dependence of the WT absorption spectrum, Figure 10. The 708 nm absorption band is discernible up to 170 K and can be seen to blue shift with increasing temperature. The second derivative spectra led to a linear shift rate of 0.015 cm-1/K which, if extrapolated, leads to a wavelength of 704 nm for the 708 nm band at room temperature which is close to 702 nm of the lowest energy band in the WT trimer-L, WT trimer-M, and WT trimer-WT monomer difference spectra discussed earlier. This provides support for the 702 nm band at room temperature corresponding to the 708 nm band in the lowtemperature limit. (The shift rate above 170 K could be higher than the value given above.) 4. Discussion The 708 nm Absorption Band. Comparison of the 4.2 K absorption spectra of the F and K mutants with the WT trimer spectrum (see Figure 2 for the spectrum of the F mutant) establishes that the 708 nm band is not due to Chls of the F and K subunits nor other Chls in the near vicinity of their boundaries whose excitation energies would be altered by deletion of the F and K subunits. The WT trimer-F difference spectrum, Figure 2C, indicates that the F subunit contributes to the 692 and 695 nm bands (shoulders) in the WT spectrum. The absorption spectrum of the K mutant (not shown) was found to be essentially identical to that of WT trimer. That is, the WT trimer-K difference spectrum is consistent with the K subunit being devoid of Chls, cf., Introduction. In contrast with the F and K mutants, the WT trimer-L mutant difference spectrum (Figure 2B) shows that deletion of the L subunit leads to a significant (≈ 30%) decrease in the intensity of the 708 nm band. The 708 nm WT trimer-M difference profile and

844 J. Phys. Chem. B, Vol. 104, No. 4, 2000 intensity (not shown) were found to be very similar to that of WT trimer-L. This was also the case for the WT trimer-WT monomer difference spectrum. We remind the reader that the L mutant is devoid of the L subunit but that it is present in the WT monomer. These results suggest that the 708 nm band is not due to Chls belonging to either the L or M subunits but, rather, to other Chls located close to the boundaries between these subunits and others. We return to this possibility following discussion of the pressure dependent, Stark, and electronphonon coupling data. As discussed in the preceding section, the temperature dependence of the 708 nm absorption band between 4.2 and 170 K (Figure 10) leads to a predicted wavelength of 704 nm at room temperature. Thus, it is likely that the 702 nm band observed in the room-temperature WT trimer-L, WT trimerM, and WT trimer-WT monomer difference spectra corresponds to the 708 nm band. The 6 nm (120 cm-1) red shift that occurs as the temperature is reduced to 4.2 K, although not as large as the ∼ 300 cm-1 values for the P870 and P960 special pair bands of the Rb. sphaeroides and Rps. Viridis reaction centers,6 is consistent with the Chls that contribute to the 708 nm band being quite strongly coupled. By way of contrast, Figure 10 shows that the position of the main band at 680.2 nm is temperature independent. Accepting that the fluorescence origin band at 722 nm is due to the lowest energy state of the 708 nm absorption band at lower temperatures,18 one might have expected to observe it undergo a slight blue shifting as the temperature is increased from 4.2 to 120 K, as is the case for the 708 nm band. A plausible explanation for the absence of a shift is linked to the decrease in fluorescence intensity as the temperature is increased. It is that18 the expected blue shift is canceled by a red shift that arises because the higher energy sites of the distribution of absorbing complexes that fluoresce are preferentially thermally activated to populate P700*. Evidence for a 714 nm Antenna State. In what follows we discuss data which indicate that the 708 nm absorption band is not due to a single Qy-state but rather to more than one state with the lowest energy state lying at 714 nm. The existence of a 714 nm state is suggested by the NPHB spectra in Figure 2 obtained with λB ) 670.0 nm. They show a broad (fwhm ≈ 230 cm-1) hole at 714 nm. (This hole appears for other λBvalues located within the main part of the absorption spectrum, results not shown.) If the 708 nm band was due to only one state, one would expect a hole centered near 708 nm upon excitation of higher energy states. Such is the case for the special pair band (P870) of the Rb. sphaeroides reaction center (see ref 5 and refs. cited therein). The existence of a 714 nm state is supported by the Stark-hole-burning results, Table 2. The value 0.63 D for f • ∆µ of the isolated LHC II trimer antenna complex of PS II is that of the lowest energy Qy-state at 680 nm. This state is due to the lowest energy Chl a molecule of the subunit. The results of recent hole-burning experiments (including pressure dependent studies) indicate that this Chl a molecule is weakly excitonically coupled to the other 11 Chl a and Chl b molecules of the subunit. That the 0.63 D value for f • ∆µ is close to the average of the f • ∆µ|| and f • ∆µ⊥ values for Chl a monomer in poly(vinylbutyral) is consistent with weak coupling. The average value for the isolated PS II reaction center in the presence of Triton X-100 detergent is similar for λB in the 670-672 nm range. This detergent produces disrupted (disconnected) Chl a molecules that dominate the hole-burning near 670 nm.47 Thus, the f • ∆µ values of 0.60 and 0.49 D can be viewed as those of an isolated Chl a molecule in a Triton

Ra¨tsep et al. X-100 environment. We conclude that f • ∆µ for a weakly coupled Chl a molecule in a protein is about 0.6 D. Weak coupling does not necessarily mean that its electrostatic excitonic interactions with other Chl molecules are insignificant. Rather, what is meant is that electron-exchange interactions, which result in charge-transfer character for the excited state, are weak. Accepting that f • ∆µ ≈ 0.6 D corresponds to a weakly coupled Chl a molecule (or Qy-state) in a protein, the f • ∆µ results for PSI in Table 2 for λB in the 690.0-702.0 nm range can be said to be monomer-like. Even for λB ) 706.5 and 708.0 nm, which are close to the maximum of the 708 nm absorption band, the f • ∆µ values are not much larger than 0.6 D. What is striking is that the f • ∆µ values for λB in the vicinity of 714 nm are large, 1.8-2.4 D. If the 708 nm absorption band were due to only one antenna state, we would expect that the variation in f • ∆µ values would be small as one tunes λB across the band. Stark-hole-burning spectroscopy has been performed on the B800 absorption band and absorption band of the lowest exciton level (B870) of the LH2 antenna complex of purple bacteria,48 the absorption band of the lowest exciton level (B896) of the LH1 antenna complex of purple bacteria and the lowest energy absorption band of the Fenna-Matthews-Olson BChl a complex of Cb. tepidum.25 The variations in f • ∆µ values as λB was tuned across the inhomogeneously broadened bands were no greater than 20%, much smaller than the difference between the values of f • ∆µ for PS I at λB ) 706.5 and 714.0 nm. Thus, to assert that the strong variation in f • ∆µ values is consistent with the 708 nm band being due to a single Qy-state is not supported by existing data. As mentioned in the preceding section, there is a positive correlation between the magnitude of f • ∆µ and the linear electron-phonon coupling strength (S). The hole profiles shown in Figure 4 provide another example of this correlation. According to theory,6 the integrated intensity of the ZPH divided by the intensity of the entire profile is equal to exp(-2S) in the shallow burn limit. Since the hole profiles in Figure 4 do not satisfy this limit, the S-values for the indicated λB-values were determined using spectra obtained with lower burn fluences. The values obtained for λB ) 702, 706, 710, 714, 718, and 722 nm are 0.6, 1.2, 1.7, 1.8, 1.6, and 1.9, respectively, with an estimated uncertainty of (0.1. A value of 0.3 for λB ) 692 nm was also determined. In all cases the peak frequency ωm of the coupling phonons was near 16 cm-1. There is clearly a correlation between the strong electron-phonon coupling for λB J710 nm and the large f • ∆µ values observed for λB near 714 nm. The large linear pressures shift rates for λB near 714 nm (Table 3), which were discussed in the preceding section, also correlate with the large f • ∆µ values and strong electronphonon coupling near 714 nm. As discussed in ref 7, it is difficult to explain shift rates as large or larger than -0.4 cm-1/ MPa in terms of electrostatic coupling between neighboring Chl molecules; i.e., it is necessary to invoke electron-exchange coupling that brings charge-transfer character to the excited state. Such character would explain the large f • ∆µ values observed for λB J710 nm. An Interpretation Other than a 714 nm State. In the preceding subsection the broad hole at 714 nm seen in the spectra of Figure 3 was taken as one piece of evidence for a 714 nm antenna state. Its existence would require that there is another state absorbing near 708 nm, the maximum of the 708 nm absorption band. As discussed in the Introduction, it had been previously thought that the 708 nm band is due to one state. Assuming that this is the case, an explanation for the 714

Chlorophyll a Antenna States nm hole is required. The only interpretation we can think of is that the 714 nm hole is a consequence of nonuniform holeburning efficiency. That is, in the ensemble of PS I particles the hole-burning efficiencies for particles contributing to the 708 nm band at higher energies are considerably lower than those that contribute at lower energies. This, in turn, requires an explanation. The most plausible one is that P700, the special pair of the reaction center, serves as a trap for those complexes that absorb at higher energies in the 708 nm band. This suggests that the 708 nm Chls are in close proximity to P700. (Our 4.2 K difference spectra, such as those shown in Figure 2, argue strongly against the 708 nm absorbing Chls belonging to the reaction center.) The question then arises as to the location and absorption profile of the P700 absorption band at liquid helium temperatures. The most detailed information on this is from the 4.2 K hole burning study of Gillie et al.49 of 45:1 enriched PS I particles from spinach. The mechanism for persistent hole burning of P700 in that work was photochemical due to irreversible formation of P700+ FA/B. It was concluded that the absorption band of P700 is centered at 702 nm and that its fwhm is ∼350 cm-1 with ∼100 cm-1 due to inhomogeneous broadening and the remainder to homogeneous broadening from strong electron-phonon coupling. Theoretical analysis led to an average wavelength of 710 nm for the zero-point level of P700*.49 Thus, it is possible that in Synechocystis P700 could serve as a trap at liquid helium temperatures for the 708 nm antenna state in complexes whose absorptions contribute to the 708 nm band at higher energies. In this regard, we note that the results of Pålsson et al.19 for PS I of Synechoccus elongatus, which exhibits antenna states at 708 and 719 nm, show that at room-temperature excitation at wavelengths as long as 750 nm results in primary charge separation as efficient as that observed with high energy excitation. The P700 hole spectra reported by Gillie et al.49 were obtained with a double beam spectrometer which ensured that P700 was not oxidized during recording of absorption spectra. This is not the case in the present study since a FT spectrometer was used. On the basis of the burn intensities used by Gillie et al., we conclude that P700 of those PS I particles that undergo irreversible formation of P700+ FA/B were bleached by the white light of the FT spectrometer during the recording of pre-burn absorption spectra in the present work. The results of experiments on WT samples to which sodium ascorbate was added at room temperature to ensure that all P700 was in its reduced state were consistent with white light bleaching at liquid helium temperatures. As reviewed by Brettel,50 the fraction of PS I reaction centers that undergo irreversible charge separation at temperatures below about 100 K is dependent on the species studied and even cool down procedure. Of relevance to our work is that ∼45% of PS I particles of Synechococcus elongatus undergo irreversible charge separation at low temperatures. It seems reasonable to assume a similar percentage for Synechocystis PCC 6803 (the results of Gillie et al.49 for 45:1 enriched particles from spinach are consistent with a percentage close to 45%), meaning that in our absorption spectra (Figure 2) only about half of the PS I particles contribute to the absorption of P700. On the basis of the results of Gillie et al., this absorption should tail out to about 710 nm. We note that their results showed no evidence for red absorbing antenna states in the 45:1 enriched particles even though PS I-200 particles from spinach exhibit an antenna absorption at 708 nm.17 It appears, therefore, that the preparation and isolation of the 45:1 particles either removed the redabsorbing Chl a molecules or disrupted their structure in a way that shifts their absorption to higher energies.

J. Phys. Chem. B, Vol. 104, No. 4, 2000 845 The ZPH action spectrum shown in Figure 8 does not provide support for the alternative interpretation of the broad 714 nm hole because between 700 and 722 nm it is smoothly varying. There is no indication of a large decrease in hole-burning efficiency between about 708 and 702 nm which is required by the alternative interpretation. However, it is possible that the holes observed in this region are due to Chls associated with other Qy-states. P700 is one possibility. However, in 45:1 enriched particles from spinach, sharp ZPH from P700 were not observed because of strong electron-phonon coupling.49 To further test the alternative interpretation, the intensity of the 722 nm fluorescence origin band at 4.2 K was monitored at 14 excitation wavelengths between 694 and 714 nm under constant excitation intensity conditions. The results (not shown) yielded an intensity profile that quite closely mirrors the absorption spectrum. That is, the data show no indication of a significant decrease in fluorescence intensity for excitation wavelengths between about 708 and 702 nm as would be expected if the alternative interpretation were correct. These fluorescence results and the ZPH spectrum argue strongly against this interpretation. Furthermore, for the alternative interpretation to be consistent with the Stark, linear pressure shift and electron-phonon coupling results, it is necessary to postulate that the Chl a dimer responsible for the 708 nm absorption is unusually sensitive to its microenvironment. (We cannot exclude the possibility that the 708 nm band is due to say a trimer of coupled Chls.) Specifically, the monomers of the dimers that absorb at λB j708 nm would need to be considerably more weakly coupled than those that absorb at λB J710 nm. There are no precedents for this. We note that the λB-dependence of the hole-burned spectra of P870 of the bacterial reaction center (Rb. sphaeroides) was well accounted for under the assumption that the electronphonon coupling parameters are constant across the inhomogeneous distribution of zero-phonon line frequencies.4 In summary, the available experimental data argue against the alternative interpretation that has the 714 nm hole seen in Figure 3 due to a single antenna Qy-state with an absorption maximum at 708 nm. Thus, we favor the interpretation that has the 714 nm hole due to a new Qy-state at 714 nm. The Stokes Shift. On the basis of their 5 K site-selected fluorescence spectra for PS I of Synechocystis PCC 6803, Gobets et al.18 concluded that the fluorescence, with an origin band at 722 nm (Figure 9), is due to a single Qy-state responsible for the 708 nm absorption band. Thus, and as they pointed out, the Stokes shift is unusually large for an antenna state, 280 cm-1 (14 nm). This shift is due to low-frequency modes. Hole profiles, such as those shown in Figure 4 but obtained at lower burn fluences, establish that the total Huang-Rhys factor (S) approaches 2 for λB J710 nm. They also show that the peak frequency (ωm) of the coupling phonons is 16 cm-1. Let us assume that only 16 cm-1 phonons couple, so that the Stokes shift 2Smωm ) 280 cm-1, where Sm is the Huang-Rhys factor for the 16 cm-1 phonons. The result is Sm ) 12, which is clearly at odds with experiment since the Franck-Condon factor for the ZPH would be exp(-24). However, the λB ) 714 and 718 nm hole spectra of Figure 4 suggest that there are higher frequency phonons that couple with a frequency ωh ≈ 100 cm-1 (Huang-Rhys factor Sh), see upward dashed arrows. For our purposes it suffices to take 2 as the experimental value of S which equals Sm + Sh. The Stokes shift of 280 cm-1 is now equal to 2(Smωm + Shωh). The hole profiles indicate that Sh < Sm. With this restriction, an acceptable solution to the above equations for an absorption maximum at 708 nm does not exist. This is not the case, however, for an absorption maximum at

846 J. Phys. Chem. B, Vol. 104, No. 4, 2000 714 nm. The Stokes shift is then 160 cm-1, which leads to Sm ) 1.4 and Sh ) 0.6. Preliminary simulations (using the theory of ref 29) of the hole profiles at longer burn wavelengths (λB g 714 nm) indicate that these values are reasonable. The results of detailed simulations will be reported on elsewhere.51 To conclude this subsection we consider the temperature dependence of the fluorescence intensity, Figure 9. As discussed in ref 19, thermal population of P700* from the lower energy red antenna state is expected to lead to a reduction in fluorescence intensity. The integrated intensities of the fluorescence origin bands in Figure 9 for T ) 4.2, 40, 57, 77, and 93 K lead to an activation energy of ≈ 70 cm-1. As mentioned, the hole-burning results of Gillie et al.51 indicate that the average zero-point level of P700* is at ≈710 nm. This wavelength and the 70 cm-1 activation energy lead to λ ) 713.5 nm for the corresponding level of the emissive antenna state which, within experimental uncertainty, is in agreement with the position of the broad hole at 714 nm, Figure 3. This provides additional support for the existence of a 714 nm state. At temperatures >93 K, the fluorescence intensity decreases much more rapidly than predicted with the above activation energy. This may be due, in part, to a dependence of the energy gap on temperature above about 100 K. Nature of the Red-Absorbing Antenna Chlorophylls. The results led us to the conclusion that in PS I of Synechocystis PCC 6803 there is an antenna Qy-state at 714 nm that is responsible for the fluorescence origin band at 722 nm, Figure 9. Its strong electron-phonon coupling, large linear pressure shifting rate and large permanent dipole moment change indicate that it is a state of a strongly coupled Chl a dimer. (The possibility that it is a state of, say, a strongly coupled trimer cannot be excluded.) The 714 nm state contributes to the low energy side of the 708 nm absorption band seen in frame A of Figure 2. As judged by the hole burned spectra shown in Figure 3, the absorption bandwidth of the 714 nm state is ≈230 cm-1. To account for the band shape of the absorption centered near 708 nm requires that there be another antenna Qy-state whose absorption maximum lies slightly to the blue of 708 nm. Unfortunately, our results do not provide a bandwidth for its absorption. Thus, a precise determination of the energy of the 708 nm state is not possible. An important question is what the percentage contribution of the 708 nm absorption band (frame A of Figure 2) to the entire origin region of the Qy-absorption spectrum is. Various fitting procedures led to a percentage of 4-5. (They took into account the contribution of P700 to the absorption. Estimation of this contribution was based on the results of Gillie et al.49 and that, under the conditions of our experiments, about half of the P700 dimers are oxidized, as discussed earlier.) Taking the total number of Chls per monomer of WT to be 90, we estimate that the number of Chls that contribute to the 708 nm absorption band is ≈4. These Chls are to be associated with the ≈708 and 714 nm Qy-states. It is possible that the 708 nm state is associated with a Chl a dimer but, if so, our results indicate that its monomers are weakly coupled relative to the Chls of the 714 nm state. The results of Figure 2, and others not shown, bear on the question of where or where not the red Chls are located in the overall structure of PS I depicted in Figure 1. First, the absorption spectra of the trimeric F and K mutants show that the red Chls are not bound to the F and K subunits. (The K subunit is not expected to bind Chls while the F subunit may bind 2-3 Chls, cf. Introduction.) However, the spectra of the monomeric L mutant and trimeric M mutant show that deletion of the L and M subunits leads to a 30% decrease in the intensity

Ra¨tsep et al. of the WT trimer 708 nm absorption band. The WT trimer-L and WT trimer-M difference absorption spectra in the region of the 708 nm band are very similar. This is puzzling since the M subunit is not expected to bind Chls, cf. Introduction. Adding to the puzzle is that the WT trimer-WT monomer 708 nm difference profile spectrum is very similar to the two just mentioned (the L subunit is present in WT monomer but not in the L mutant). Formation of the WT monomer leads again to a 30% decrease in the intensity of 708 nm absorption band. As is the case for the L and M mutants, this decrease appears to be about the same for the absorptions due to the ≈708 and 714 nm states. We offer the following explanation for the above results: the red absorbing Chls at ≈708 and 714 nm are not directly bound to the L or M subunits. Rather, they are located close to the interfacial regions between the PsaA/B subunits and the L and M subunits. It is likely that they are bound to the PsaA or PsaB subunits. The partial (30%) loss of absorption intensity of the 708 nm band in the WT monomer, L and M mutants does not necessarily signal the actual loss of Chls since the structural changes introduced by formation of the WT monomer and deletion of the L and M subunits may simply perturb the excitonic interactions between the Chls responsible for the red absorptions. This could result in a redistribution of oscillator strength and blue-shifting of transition frequencies. The current 4 Å resolution of the PS I structure of Synechococcus is too low to permit a meaningful search for the coupled Chls of the 708 and 714 nm states. Even if a structure of sufficient resolution was available, there is the complication that Synechococcus contains significantly more red Chls than Synechocystis, cf. Introduction. On the basis of the results of ref 19 on Synechococcus and our estimate that ≈4 Chl molecules contribute to the entire 708 nm absorption band of Synechocystis, the number of Chls contributing to the 708 and 719 nm absorption bands of PS I of Synechococcus is about 8. Acknowledgment. Research at the Ames Laboratory was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, U.S. Department of Energy. Ames Laboratory is operated for USDOE by Iowa State University under Contract W-7405-Eng-82. Research at the Department of Biochemistry, Biophysics, and Molecular Biology was supported by NSF Grants MCB 9696170 and MCB 9723001. References and Notes (1) Topics in Current Physics, Persistent Spectral Hole Burning: Science and Applications; Moerner, W. E., Ed.; Springer-Verlag: New York, 1987; Vol. 44. (2) Jankowiak, R.; Hayes, J. M.; Small, G. J. Chem. ReV. 1993, 93, 1471. (3) Jankowiak, R.; Small, G. J. In Photosynthetic Reaction Centers; J. Deisenhofer, J., Norris, J., Eds.; Academic Press: New York, 1993; Vol. 2, p 133. (4) Lyle, P. A.; Kolaczkowski, S. V.; Small, G. J. J. Phys. Chem. 1993, 97, 6926. (5) Small, G. J. Chem. Phys. 1995, 197, 239. (6) Hayes, J. M.; Gillie, J. K.; Tang, D.; Small, G. J. Biochim. Biophys. Acta 1988, 932, 287. (7) Wu, H.-M.; Ra¨tsep, M.; Jankowiak, R.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1998, 102, 4023. (8) Small, G. J. Photochem. Photobiol. In preparation. (9) Chang, H.-C.; Jankowiak, R.; Yocum, C. F.; Picorel, R.; Alfonso, M.; Seibert, M.; Small, G. J. J. Phys. Chem. 1994, 98, 7717. (10) Jankowiak, R.; Tang, D.; Small, G. J.; Seibert, M. J. Phys. Chem. 1989, 93, 1649. (11) Tang, D.; Jankowiak, R.; Seibert, M.; Yocum, C. F.; Small, G. J. J. Phys. Chem. 1990, 94, 6519. (12) den Hartog, F. Ph.D. Dissertation, University of Leiden, Leiden, 1988. (13) Kruip, J.; Bald, D.; Boekema, E.; Rogner, M. Photosynth. Res. 1994, 40, 279.

Chlorophyll a Antenna States (14) Krauss, N.; Schubert, W.-D.; Klukas, O.; Fromme, P.; Witt, H. T.; Saenger, N. Nat. Struct. Biol. 1996, 3, 965. (15) Schubert, W.-D.; Klukas, O.; Krauss, N.; Saenger, W.; Fromme, P.; Witt, H. T. J. Mol. Biol. 1997, 272, 741. (16) Manna, P.; Chitnis, P. R. In Concepts in Photobiology: Photosynthesis and Photomorphogenesis; Singhal, G. S., Renger, G., Sapory, S. K., Irrgang, K.-D., Govindjee, Eds.; Kluwer Academic: Dordrecht, 1999; p 212. (17) van der Lee, J.; Bald, D.; Kwa, S. L. S.; van Grondelle, R.; Rogner, M.; Dekker: J. P. Photosynth. Res. 1993, 35, 311. (18) Gobets, B.; van Amerongen, H.; Monshouwer, R.; Kruip, J.; Rogner, M.; van Grondelle, R.; Dekker: J. P. Biochim. Biophys. Acta 1994, 1188, 75. (19) Pålsson, L.-O.; Flemming, C.; Gobets, B.; van Grondelle, R.; Dekker: J. P.; Schlodder, E. Biophys. J. 1998, 74, 2611. (20) Soukalis, V.; Savikhin, S.; Xu, W.; Chitnis, P. R.; Struve, W. S. Biophys. J. 1999, 76, 2711. (21) Sun, J.; Ke, A.; Jin, P.; Chitnis, V. P.; Chitnis, P. R. Methods Enzymol. 1998, 297, 124. (22) Reddy, N. R. S.; Kolaczkowski, S. V.; Small, G. J. J. Phys. Chem. 1993, 97, 6934. (23) Chang, H.-C.; Jankowiak, R.; Reddy, N. R. S.; Small, G. J. Chem. Phys. 1995, 197, 307. (24) Reddy, N. R. S.; Small, G. J. In Biophysical Techniques in Photosynthesis; Amesz, J., Hoff, A. J., Eds.; Kluwer Academic Publishers: Dordrecht, 1996; p 123. (25) Ra¨tsep, M.; Wu, H.-M.; Hayes, J. J.; Blankenship, R. E.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1998, 102, 4035. (26) Lee, I.-J.; Hayes, J. M.; Small; G. J. J. Chem. Phys. 1989, 91, 3413. (27) Shu, L.; Small, G. J. Chem. Phys. 1990, 147, 447. (28) Shu, L.; Small, G. J. J. Opt. Soc. Am. B 1992, 9, 724. (29) Hayes, J. M.; Lyle, P. A.; Small, G. J. J. Chem. Phys. 1994, 98, 7337 and references therein. (30) Middendorf, T. R.; Mazzola, L. T.; Lao, K.; Steffen, M. A.; Boxer, S. G. Biochim. Biophys. Acta 1993, 1143, 223. (31) Johnson, S. G.; Small, G. J. J. Phys. Chem. 1991, 95, 471. (32) Beekman, L. M. P.; Steffen, M.; van Stokkum, I. H. M.; Olson, J.; Hunter, C. N.; Boxer, S. G.; van Grondelle, R. J. Phys. Chem. B 1997, 101, 7284.

J. Phys. Chem. B, Vol. 104, No. 4, 2000 847 (33) Beekman, L. M. P.; Frese, R. N.; Fowler, G. J. S.; Picorel, R.; Cogdell, R. J.; van Stokkum, I. H. M.; Hunter, C. N.; van Grondelle, R. J. Phys. Chem. B 1997, 101, 7293. (34) Kador, L.; Haarer, D.; Personov, R. J. Chem. Phys. 1987, 86, 5300. (35) Wu, H.-M.; Ra¨tsep, M.; Jankowiak, R.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1997, 101, 7641. (36) Reddy, N. R. S.; Wu, H.-M.; Jankowiak, R.; Picorel, R.; Cogdell, R. J.; Small, G. J. Photosynth. Res. 1996, 48, 277. (37) Wu, H.-M.; Reddy. N. R. S.; Small, G. J. J. Phys. Chem. 1997, 101, 651. (38) den Hartog, E. T. H.; Vacha, F.; Lock, A. J.; Barber, J.; Dekker: J. P.; Vo¨lker, S. J. Phys. Chem. B 1998, 102, 9174. (39) Jankowiak, R.; Ra¨tsep, M.; Picorel, R.; Small, G. J. J. Phys. Chem. B 1999, 103, 9759. (40) Kenney, M.; Jankowiak, R.; Small, G. J. Chem. Phys. 1990, 146, 47. (41) Kim, W.-H.; Reinot, T.; Hayes, J. M.; Small, G. J. J. Phys. Chem. 1995, 99, 7300 and references therein. (42) Vo¨lker, S. In Relaxation Processes in Molecular Excited States; Fu¨nfschilling, J., Ed.; Kluwer Academic Publishers: Dordrecht, 1989; p 113. (43) Narasimhan, L. R.; Littau, K. A.; Pack, D.; Bai, Y. S.; Elschner, A.; Fayer, M. D. Chem. ReV. 1990, 90, 439. (44) Wu, H.-M.; Ra¨tsep, M.; Lee, I.-J.; Cogdell, R. J.; Small, G. J. J. Phys. Chem. B 1997, 101, 7654. (45) Du, M.; Xie, X.; Jia, Y.; Mets, L.; Fleming, G. R. Chem. Phys. Lett. 1993, 201, 535. (46) Savikhin, S.; Wu, X.; Soukalis, V.; Chitnis, P. R.; Struve, W. S. Biophys. J. 1999, 76, 3278. (47) Tang, D.; Jankowiak, R.; Seibert, M.; Small, G. J. Photosyn. Res. 1991, 27, 191. (48) Ra¨tsep, M.; Wu, H.-M.; Hayes, J. M.; Small, G. J. Spectrochim. Acta A 1998, 54, 1279. (49) Gillie, J. K.; Lyle, P. A.; Small, G. J.; Golbeck, J. H. Photosyn. Res. 1989, 22, 233. (50) Brettel, K. Biochim. Biophys. Acta 1997, 1318, 322. (51) Hayes, J. M.; Small, G. J. J. Phys. Chem. B. In preparation. (52) Reddy, N. R. S.; Jankowiak, R.; Small, G. J. J. Phys. Chem. 1995, 99, 16168.

The Red-Absorbing Chlorophyll - American Chemical Society

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