Structure of the Reaction Center of Photosystem I of Plants. An

Structure of the Reaction Center of Photosystem I of Plants. An Investigation with Linear-Dichroic Absorbance-Detected Magnetic Resonance ... Hydrogen...
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9960

J. Phys. Chem. 1996, 100, 9960-9967

Structure of the Reaction Center of Photosystem I of Plants. An Investigation with Linear-Dichroic Absorbance-Detected Magnetic Resonance J. Vrieze,† P. Gast, and A. J. Hoff* Department of Biophysics, Huygens Laboratory, Leiden UniVersity, P.O. Box 9504, 2300 RA Leiden, The Netherlands ReceiVed: August 24, 1995; In Final Form: April 1, 1996X

Photosystem I particles from spinach were studied with linear-dichroic absorbance-detected magnetic resonance in zero-magnetic field. The microwave-induced triplet-minus-singlet (T-S) spectra and the linear-dichroic (LD) T-S spectra were recorded for the |D| + |E| and the |D| - |E| microwave transitions of the triplet state of the primary donor, 3P700. From these data the directions of the optical transition moments contributing to the T-S spectra in the 600-750 nm region, with respect to the triplet x- and y-axes of 3P700 were obtained. The orientation of the optical QY-transition moment of P700 relative to the triplet x- and y-axis is found to be 35 ( 2° and 56 ( 1°, respectively. A comparison is made with data obtained for monomeric chlorophyll (Chl) a in two glasses. The orientation of the QY-transition moment with respect to the in-plane triplet xand y-axes of 3P700 differs from monomeric Chl a in the two glasses. This difference is ascribed to the different environments of P700 and Chl a, rather than to the dimeric structure of P700. In addition to the QY-absorption band of P700, the T-S and LD(T-S) spectra contain features that are ascribed to transitions involving accessory Chls. The contribution of the QY transition of the primary acceptor to a band at 687 nm in the T-S spectrum is discussed, and a comparison is made with the signal of the primary acceptor in the T-S spectrum of Heliobacterium chlorum.

Introduction The smallest functional unit of photosystem I (PSI) present in plants and cyanobacteria contains antenna chlorophylls (Chl), which act as light-harvesting pigments and transfer excitation energy to the reaction center, and the reaction center proper, where electron transport takes place (see for a recent review1). Excitation of the primary electron donor of PSI, P700, leads to charge separation, whereby an electron is transferred from P700 to the primary acceptor, A0, a Chl a molecule, and subsequently to the secondary acceptors, a phylloquinone and several ironsulfur centers. Blocking the electron transfer to the secondary electron acceptors leads, through recombination of the radical pair P700•+ A0•-, to the formation of the triplet state of the primary donor, 3P700. Recently, the structure of the photosynthetic unit of PSI was determined by X-ray crystallography with a resolution of 6 Å.2 In addition to a quinone and the iron-sulfur centers, pockets could be discerned of five chromophores presumably belonging to the reaction center proper. The precise orientation of the chromophores could not be determined. Since the antenna and reaction center chromophores of PSI are bound to the same protein, the reaction center itself cannot be isolated. The absorption bands of the antenna and reaction center chromophores overlap considerably, and for the optical characterization of the reaction center one therefore has to resort to difference spectroscopy techniques, such as (time-resolved) “oxidized-minus-reduced” and triplet-minus-singlet (T-S) absorbance-difference spectroscopy. The main feature of the oxidized-minus-reduced P700•+-P700 spectrum is a band at 700 nm, which has been attributed to bleaching of the QY absorption of the primary donor P700, named after this maximum.3 The red shift of the absorption, compared † Present address: Institut fu ¨ r Experimentalphysik, Freie Universita¨t Berlin, Arnimallee 14, 14195 Berlin, Germany. * Author to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, May 15, 1996.

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to the maximum of the QY absorption of monomeric Chl a (which is at 665-680 nm in organic solvents), generally is thought to result from P700 being a Chl a dimer, by analogy with the red shift of the primary donor band observed for purple bacteria. Because the triplet excited state of the primary donor has a different interaction with neighboring molecules than its singlet ground state, the T-S absorbance-difference spectrum provides information about the interaction between the primary donor and its environment. For example, excitation of the primary donor to its triplet state may alter the transition energies and oscillator strengths of the neighboring molecules, leading to the appearance of band shifts and intensity changes in the absorbance-difference spectrum. A triplet state is characterized by the zero-field splitting (zfs)parameters |D| and |E|, reflecting the energy splittings between the triplet sublevels x, y, and z, and by the populating probabilities and decay rates of the individual sublevels. These properties, which can be measured with high accuracy in zeromagnetic field with absorbance-detected magnetic resonance (ADMR), provide information about the structure of the molecule in its triplet state. In rigid media, in which the molecules are oriented at random, the orientation of an optical transition moment with respect to the triplet axes can be determined with linear-dichroic (LD) ADMR.4 Since the (LD) ADMR method is a double-resonance spectroscopy, one of its attractive features is that the triplet state of a particular species can be selected by its specific zero-field resonance frequencies, thus allowing the recording of its pure (LD) absorbancedifference spectrum. This is particularly useful for a study of PSI preparations, which contain many antenna Chls in addition to the reaction center pigments. In this paper we report (LD)ADMR experiments at 1.5 K on PSI particles lacking the iron-sulfur centers. The linear dichroism of the optical transitions contributing to the T-S spectrum has been determined for the |D| - |E| and |D| + |E| © 1996 American Chemical Society

Reaction Center of Photosystem I of Plants zero-field transitions, from which the orientations of the optical yT of transition moments relative to the principal axes b xT and b the triplet zeo-field splitting tensor are obtained. The results for P700 are compared with those for monomeric Chl a in two different environments, a toluene/pyridine glass and an ethanol/ methanol glass. A change in relative orientation of the in-plane B Y, of tripet axes with respect to the QY-transition moment, Q P700, compared to the corresponding relative orientations for Chl a in the two glasses, is ascribed to a change in the environment. The absorbance changes in the T-S spectrum in the region 660-690 nm are discussed in terms of the involvement of the higher-energy exciton component of the QY transition of P700, the QY transitions of “accessory” Chl a pigments and the QY transition of the primary acceptor A0. The T-S spectrum is interpreted qualitatively, based on the linear dichroism of the absorption bands, and compared with literature data and with the T-S spectrum of the reaction center of purple bacteria and of Heliobacterium chlorum. Experimental Section Sample Preparation. PSI particles lacking the iron-sulfur centers (CP1 particles) were prepared from spinach chloroplasts as described in refs 5 and 6 and showed a single 65 kDa band with SDS-PAAGE.5 They contained 30 Chls/P700. We have chosen these PSI particles as they give rise to a high triplet yield without the addition of reducing agents.5 The optical density of the samples, containing glycerol (65% v/v) to prevent cracking upon cooling, was ∼0.5 at 675 nm in a 2 mm cuvette. The samples were frozen in the dark. All measurements were performed at ∼1.5 K in a home-built bath cryostat. Experimental Setup. The (LD)ADMR setup was basically the same as that described previously.4 Light from a tungsteniodine lamp (250 W) was used for continuous excitation and probing the transmittance. The transmittance was detected by a Peltier-cooled photodiode (RCA 30842), while the detection wavelength was selected with a monochromator (Jobin-Yvon, 1200 lines/mm, blaze 750 nm). The microwave-induced absorbance-difference spectra were recorded by amplitude modulation (37 Hz) of the microwaves at a fixed resonant frequency, while slowly varying the wavelength of absorption (600-750 nm) with a resolution of 3-4 nm and a step size of 1 nm per point. The difference in transmittance was obtained by phase-sensitive detection with a lock-in amplifier (Stanford SR 510). The sample cuvette was placed in a tunable loopgap cavity, such that the direction of the microwave magnetic field was perpendicular to the direction of the light beam. For the linear-dichroic absorbance-difference spectra, a photoelastic modulator (PEM), operating at 50 kHz, and a GlanThompson polarizing crystal were placed between the sample and the monochromator for analyzing the change in transmitted intensity of the light polarized parallel and perpendicular to the direction of the microwave field. The signal from the photodiode was doubly demodulated with two lock-in amplifiers in series. The first lock-in amplifier (EG&G 5209) was phaselocked at twice the frequency of the PEM and the second lockin amplifier (PAR 5101) at the modulation frequency of the microwaves. A selective amplifier (EG&G 189) was placed between the two lock-in amplifiers to increase the signal-tonoise ratio. The isotropic and linear-dichroic T-S spectra were measured simultaneously. Interpretation of the Linear-Dichroic T-S Spectra. When the frequency of the microwaves is set within one of the |D| + |E| or |D| - |E| transitions, those molecules are preferentially affected which have their microwave transition moment parallel to the direction of the microwave magnetic field. Thus, the triplet concentration of this particular distribution is changed

J. Phys. Chem., Vol. 100, No. 23, 1996 9961 and an anisotropy of the transmittance is induced in the sample. The effect of resonant microwaves on the transmittance is always small, so that the microwave-induced relative change in transmittance is approximately equal to the change in absorbance, ∆A ∝ -∆I/I. The change in absorbance, for light polarized parallel and perpendicular to the direction of the microwave field (∆A| and A⊥, respectively), is expressed as an anisotropy ratio R, which is related to the angle Ri between the optical transition moment and the triplet i-axis according to7,8

Ri )

2 LD(T - S) ∆A| - ∆A⊥ (3 cos Ri - 1) ) ) T-S ∆A| + ∆A⊥ (3 + cos2 R ) i

i ) x, y, z (1) To avoid saturation effects, the LD(T-S) and T-S signals are extrapolated to zero microwave power to obtain the value of Ri. In general, the Ri values obtained for two (or three) different perpendicular microwave transitions yield eight possible solutions for the orientation of the optical transition moment in the y T, b zT) triplet axes frame and hence lead to four possible (x bT, b relative orientations (within 0-90°) of two different optical transition moments. When both optical transition moments are yT plane, there are only two possible polarized in the triplet b xTb relative orientations. Results Zero-Field Splitting Parameters and Sublevel Decay Rates of 3P700. At a detection wavelength of 700 nm, microwave transitions are found centered at 725 and 955 MHz (data not shown), which are assigned as the |D| - |E| and the |D| + |E| transitions of 3P700, respectively, in agreement with earlier results for 3P700 of plants and cyanobacteria.9,10 Contributions of antenna Chl a triplets are estimated to be less than a few percent, based on earlier ADMR experiments on LHC II preparations.11 The exact frequencies of the zero-field transitions depend slightly on the detection wavelength: a range of |D| values was found, from 835 to 845 MHz with decreasing detection wavelength, whereas |E| is little wavelength dependent, having a value of 115 ( 2 MHz. The wavelength dependence of the zfs parameters is ascribed to heterogeneity of the primary donor, resulting from slightly different conformations of the constituting chromophores and/or different protein environments. This heterogeneity presumably arises partly from the isolation process, which for reaction centers of purple bacteria leads to a notable heterogeneity of the zfs parameters of the primary donor triplet (Vrieze, J., unpublished results), partly from frozenin differences in the (native) microenvironment (Owen, G., unpublished results). We also determined the decay rates of the individual triplet sublevels, kx ) 1050 ( 100, ky ) 1100 ( 100, and kz ) 110 ( 20 s-1, in good agreement with values reported previously.9 The decay rates did not change significantly when measured at different frequencies within the ADMR transitions. Microwave-Induced T-S Spectrum. Figure 1a shows the microwave-induced T-S spectrum for the |D| + |E| transition at 955 MHz. The T-S spectrum for the |D| - |E| transition at 725 MHz is practically identical (not shown). The negative features in the T-S spectrum correspond to a decrease in transmittance (increase in ground-state absorption), due to an increase in the ground-state population resulting from irradiation with resonant microwaves. The main features of the T-S spectrum are a negative band at 700 nm with a shoulder around 687 nm, a broad positive band absorbing above 715 nm, a positive band at 674 nm, and a broad negative band centered at 644 nm, in agreement with results reported previously.9

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Vrieze et al. TABLE 1: Orientation (deg) of the Optical Transition Moments of Chl a with Respect to the Triplet Axes According to Ref 14 solvent

toluene/pyridine (85/15% v/v)

ethanol/methanol (65/35% v/v)

λdet

|Rx|

|Ry|

|Rx|

|Ry|

assignment

620 678/675a >700

47 ( 10 52 ( 2 56 ( 5

47 ( 10 36 ( 3 33 ( 5

49 ( 10 48 ( 3 53 ( 2

50 ( 10 45 ( 2 41 ( 3

vibronic QY QY Tn r T0b

a The detection wavelength was set at 678 nm for toluene/pyridine and 675 nm for ethanol/methanol. b Tn r T0: transitions between the lowest triplet state and higher excited triplet states.

Figure 1. Microwave-induced T-S absorbance-difference spectrum of PSI recorded at 955 MHz, corresponding with the |D| + |E| transition (a). The LD(T-S) spectrum recorded at 955 MHz (|D| + |E|) (b) and 725 MHz (|D| - |E|) (c). Microwave power at the source, 1 mW; wavelength resolution, 3-4 nm. Temperature ∼1.5 K.

The absorption band in the T-S spectrum centered at 700 nm is slightly asymmetric. A similar asymmetry observed for the P700•+-P700 absorbance-difference spectrum has been attributed by Schaffernicht and Junge12 to the presence of a relatively wide, negative dimer band at 700 nm and a positive band centered at 695 nm, which carried half of the oscillator strength of the 700 nm band and which was almost completely cancelled by the 700 nm band. It is likely, however, that the asymmetry of the 700 nm band, or at least a large part of it, is due to the heterogeneity of P700, reflected by the spread in zfs parameters (see above). Because the microwave transitions due to the components of the heterogeneous distribution overlap considerably, a single microwave frequency is resonant with several components and will give rise to overlapping components in the T-S spectrum. A slight asymmetry was also observed by hole-burning spectroscopy.13 Microwave-Induced LD(T-S) Spectra. In previous work, we have determined the orientations of the optical transition moments for Chl a in two different glasses, viz. toluene/pyridine and ethanol/methanol, for the |D| - |E| (725 MHz) and |D| + |E| (955 MHz) transitions, which are polarized along b xT and b yT, respectively.14 (Conforming to the literature,15 we choose a positive sign of D and a negative sign of E.) Some of the results from ref 14, which we will use for a comparison with P700, are summarized in Table 1, together with an assignment of the relevant absorption bands in the T-S spectrum of Chl a. The triplet-triplet absorption, which shows up in the T-S

Figure 2. LD(T-S) signal vs the isotropic T-S signal at a detection wavelength of 700 nm, measured as a function of the microwave power (0.01-100 mW at the microwave source). The microwave frequency was set at 725 MHz (dots) and at 955 MHz (open circles). The R value for vanishing microwave power is given by the slope of the straight line using a linear least-squares fit through the data points obtained for low microwave powers (inset). For high microwave powers the R values decrease because the probability of microwave absorption then is similar for all orientations.

spectrum as a broad structureless absorption above 700 nm (not shown, see ref 14) that is well isolated from the ground-state absorbance, has almost the same polarization as the QY transition. Both the QY and the triplet-triplet transitions show a small but significant difference, 5-8°, in orientation for the two solvent mixtures, which we think is due to different ways of ligation of ethanol/methanol and pyridine to the Chl molecule, or to differences in hydrogen bonding to the ring substituents. For the PSI particles, the LD(T-S) spectra for the |D| + |E| and the |D| - |E| transition of 3P700 are shown in Figure 1, b and c, respectively. The values of R at 700 nm for the |D| |E| and the |D| + |E| transition follow from a series of LD(T-S) and T-S signals recorded as a function of the microwave power (Figure 2). The R values for low microwave power, where LD(T-S) and T-S are linearly dependent, yield angles yT of 35 ( between the optical transition moment and b xT and b 2° and 56 ( 1°, respectively, using the same signs of D and E as for Chl a. The LD(T-S) spectra recorded at the |D| - |E| and |D| + |E| transitions are normalized on the corresponding R values for the absorption at 700 nm. The precise orientations of the individual transition moments contributing to the 665-690 nm region cannot be determined because of the strong overlap of the absorption bands. The T-S spectrum shows some structure around 674 nm, which may indicate that more than two Chls contribute to the T-S

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TABLE 2: Wavelengths (λmax) of the Extrema of the Gaussian Bands Used for a Devonvolution of the T-S Spectrum of PSI, and the Orientation (r) of the Corresponding Transition Moments with Respect to the x- and y-Triplet Axesa λmax (nm)

width (cm-1)

sign in T-S

635 644 657 662 665 669 674 682 687 692 700 >720 P680b

300 300 300 130 130 130 130 130 130 130 250 1500

+ + + + + +

|Rx| (|D| - |E|) (deg)

|Ry| (|D| + |E|) (deg)

65 ( 5 38 ( 5 57 ( 5 54 ( 5 53 ( 5 54 ( 5 65 ( 5 65 ( 10 66 ( 5 50 ( 20 35 ( 2 39 ( 4 32 ( 4

32 ( 5 57 ( 5 33 ( 5 40 ( 10 44 ( 10 38 ( 10 25 ( 5 30 ( 10 25 ( 5 45 ( 15 56 ( 1 52 ( 2 49 ( 2

a The angles R are estimated from a Gaussian deconvolution (see text). The errors in R result from both the variations between the different Gaussian deconvolutions and the experimental errors in the R values. b Values for P680 in the D1D2 complex of Photosystem II taken from ref 21.

spectrum. By analogy with the 780-850 nm region in the T-S spectra of reaction centers of purple bacteria,16-19 we assume that the signal in the 660-690 nm region results from overlap of adjacent positive and negative bands. The sharp oscillations observed in the T-S spectra of reaction centers of purple bacteria are not observed in the T-S spectrum of PSI, probably because of a stronger overlap of the absorption bands of PSI. Approximate orientations were obtained by deconvoluting the bands in the T-S and LD(T-S) spectra with Gaussian bands and measuring the R values at wavelengths corresponding to the center of the component bands. To this end, the T-S spectrum was first deconvoluted with Gaussian bands with maxima as in Table 2. Note that the P700 absorption does not tail appreciably on the blue side, so that a Gaussian approximation to the true line shape (see e.g., ref 20) is warranted. Then, the LD(T-S) spectra were deconvoluted keeping the widths and positions of the Gaussians fixed. The widths of all Gaussians were chosen equal except for the Gaussian at 700 nm, which was taken larger to account for the site heterogeneity (see above). The angles between the optical transition moments and the triplet axes, and the uncertainties in the angles were estimated by varying the widths between 120 and 150 cm-1, and for 700 nm between 230 and 275 cm-1. We take the values of Table 2 as approximations for the actual orientation of the transition moments giving rise to the stronger bands, for instance the 674 and 687 nm bands. For comparison, Table 2 also shows the R values of the absorption of the primary donor, P680, of Photosystem II.21 For the long-wavelength band in the T-S spectrum of PSI, centered at 700 nm, the orientation of the optical transition moment with respect to the triplet axes does not change for wavelengths between 697 and 715 nm within the experimental error, indicating that the absorbance at these wavelengths does not overlap with other positive or negative absorption bands. The broad, structureless absorption above 715 nm resembles that of the T-S spectrum of Chl a14 and is ascribed to transitions between the lowest triplet state of P700 and higher excited triplet states. Similar to Chl a in the above-mentioned glasses, the polarization of these triplet-triplet absorptions is close to that of the Qy transition at 700 nm. The band at 687 nm, visible as a shoulder in the T-S spectrum, is well resolved in the LD(T-S) spectra, for both the |D| - |E| and |D| + |E| zero-field transitions. Its R value

is considerably different from that of the 700 nm transition. The choice of a positive band at 692 nm in the Guassian fit is based on the somewhat better resolved T-S spectrum reported by den Blanken and Hoff.9 We note that the maximum and width of the 700 nm band are somewhat different in the LD(T-S) spectra at the blue side, compared to the T-S spectrum. Around 665 nm, a feature is observed with can be deconvoluted with positive bands at 662 and 669 nm, and a negative band at 665 nm. This feature is observed in the T-S spectrum and in both LD(T-S) spectra. The observed low intensities correspond to small band shifts upon 3P700 formation. Discussion Many attempts have been made to extract the structure of P700 by comparing spectroscopic data for PSI with similar data for Chl a in Vitro. The discussion has been focused mainly on the dimeric or monomeric character of P700, with arguments based on a simple exciton model in which the QY-transition moments of the dimer halves are coupled by dipole-dipole interaction.22 Because of its simplicity, the exciton model is a useful framework for discussing the results of the present work, with regard to both the structure of P700 and the interactions between P700 and adjacent Chl molecules. Below we will confront quite general predictions of the exciton treatment with regard to band shifts and band polarizations with our experimental results. It will be shown that serious discrepancies exist, which do not depend on the details of the exciton calculation. We conclude that for an adequate interpretation of the available optical data the exciton model is insufficient and that interactions between the cofactor pigments other than dipole interactions between the transition moments must contribute. Orientation of the QY Transition Moment of P700. The red shift of the QY absorption of P700 compared to that of monomeric Chl a was attributed to delocalization of the singlet excitation over two Chl a molecules.22 From the circulardichroic P700•+-P700 spectrum it was concluded that the QY transitions of the dimer halves are excitonically coupled, though with much less interaction between the two monomer QY transitions than in the dimer of reaction centers of purple bacteria, the energy gap between the high- and low-energy band being approximately 150 cm-1 for P70023,24 vs 600-900 cm-1 for the dimer in purple bacteria.25 Recently, the dimeric character of P700 has been confirmed with resonance-Raman spectroscopy by Moe¨nne-Loccoz et al.,26 who reported that Raman bands characteristic for two Chl a molecules are bleached upon 3P700 and P700•+ formation, indicating that two Chl a molecules are involved in the ground state of P700. The similarity between 3P700 and 3Chl a in Vitro, however, as regards their zfs parameters and triplet-sublevel decay rates, suggests that at cryogenic temperatures the triplet state is localized on a single monomer of the Chl a dimer9,10 (see also below). Let us now turn to our main point of interest, the orientation of Q B Y and of the near-infrared triplet-triplet transition moment. These orientations with respect to the in-plane triplet axes are depicted in Figure 3 for P700 and for Chl a in the two different glasses (see also Tables 1 and 2). We first note that delocalization of the triplet state over two Chl a molecules cannot explain the angles reported here. This is because for observing zfs parameters similar to those of monomeric 3Chl a, the triplet axes of the two partners should coincide and consequently the transition moment of the most intense dimer exciton transition would either coincide with one of the in-plane triplet axes or the angles between this dimer yT of the delocalized triplet state would transition and b xT and b be the same as the angles between Q B Y of monomeric Chl a and its in-plane triplet axes. Neither of these situations obtains for

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Figure 3. Orientation of the QY and triplet-triplet (Tn r T0) transition moments with respect to b xT and b yT of the triplet-carrying molecule of P700 (a), Chl a in ethanol/methanol (b), and Chl a in toluene/pyridine (c). Absorption wavelengths (in nm) of the transitions are given between brackets.

P700 and we conclude that at liquid helium temperature the triplet state of P700 is localized on a single Chl a molecule. At first sight, a localized triplet state might seem at variance with the observation that Q B Y (at 700 nm) and the triplet-triplet transition moment (>715 nm) are “rotated”, by equal amounts and in the same direction, as compared to Chl a (at 675/678 nm and >700 nm, respectively) in the solvent mixtures (Figure 3). For, if we assume that in any environment the triplet axes have a fixed orientation in the molecular frame of the Chl a molecule, and the two uncoupled monomer QY-transition moments of the dimer halves are not parallel, then the orientation of the exciton dimer QY-transition moments in the (monomeric) triplet axes system would be different from the monomer QYtransition moment in the same axes system. By contrast, the triplet-triplet transition moment should have the same orientation in the axes system of the localized triplet state as found for monomeric Chl a. The observed simultaneous rotation of the triplet-triplet transition moment and Q B Y, therefore, would not be expected for a localized triplet state. Thus, we have to conclude that the assumption that the orientation of the triplet axes in the molecular frame of Chl a is independent of the environment is untenable and that the observed change in orientation with respect to the in-plane triplex axes of both Q BY and the triplet-triplet transition moment of P700, compared to Chl a in the solvents studied, is due to the change in environment. That a change in orientation may result from a change in environment is supported by the observation of a difference between the orientations of these two transition moments for Chl a in ethanol/methanol and toluene/pyridine (Figure 3, b and c). A similar change in orientation is observed for the QY-vibronic transition of P700 at 644 nm (compare 644 nm in Table 2 with 620 nm for Chl a in Table 1). Taken together, these observations make it unlikely that dipolar coupling is responsible for the difference in orientation of Q BY of P700 compared to that of monomeric Chl a. The angles between the triplet-triplet transition moment of xT and b yT, 40° and 50°, respectively, should (localized) 3P700 and b be taken as representative for the triplet-triplet transition moment of monomeric 3Chl a in the protein environment of P700. For monomeric Chl a, the orientation of the triplet-triplet and QY-transition moments is practically the same (to within 5°). Therefore, Q B Y of monomeric Chl a in the protein environment is expected to be aligned at an angle of about 5° (clockwise in Figure 3) to the monomeric triplet-triplet transition. Because Q B Y of P700 subtends an equal angle (4° clockwise in Figure 3a) with its triplet-triplet transition moment, the polarization of the QY transition of the Chl a monomer and of P700 must practically coincide. From the nearcollinearity of Q B Y of the P700 dimer and the triplet-triplet transition moment of a monomeric (uncoupled) 3Chl a molecule in the P700 environment, we conclude that application of the exciton model discussed above implies either that the QYtransition moments of the dimer halves of P700 are (anti-)parallel, giving rise to a similarly aligned QY-transition moment of the

Vrieze et al. coupled dimer or that the dipolar coupling between the dimer halves is very weak. The latter possibility is in better agreement with the structure of the primary donor at 6 Å resolution, which suggests that the molecular in-plane axes of the dimer halves make a significant angle.2,27 Recent results on the primary donor of Photosystem II, P680, show that the QY and triplet-triplet transition moments of P680 also are approximately parallel and have an orientation similar to that of P700 with respect to the in-plane triplet axes (Table 2, and Figure 5 in ref 21). Thus, for both P700 and P680, the polarization of the main QY transition and the triplet-triplet transition in the coordinate frame spanned by the triplet axes is changed compared to the in Vitro value. Triplet State of P700. The observed rotation of the triplettriplet transition moment and Q B Y of P700 (and of P680 of Photosystem II) compared to that of Chl a in organic glasses can be due to a change of the orientation in the molecular frame of Chl a of either the optical transition moments or the in-plane triplet axes, because with LD-ADMR only relative angles are measured. From Figure 3 it is clear that if the optical transition moments rotate with respect to a fixed triplet axes system, then both optical transition moments (that of the QY and the triplet-triplet transition) rotate by the same amount. This would be surprising because the electron configuration of the two states is quite different. The simplest explanation for the observed corotation of the optical transition moment is that the frame of reference rotates. Possible causes for a rotation of the in-plane triplet axes are discussed below. At first sight, comparison of the angles for Q B Y of P700 and of yT are Chl a in toluene/pyridine might suggest that b xT and b interchanged on going from 3Chl a to 3P700 or that in a fixed triplet axes system the sign of E is reversed. If a sign inversion of E indeed occurs, then the orbital configuration of the triplet state in toluene/pyridine should be different from that in ViVo, since triplet states with opposite signs of E stem from different configurations in the “four-orbital” model of the porphin nucleus.28 It has been shown, however, that for different configurations the absolute value of E is in general not equal.28 In contrast, for Chl a in the different solvents and for P700 we observed approximately the same absolute E values. Furthermore, the orientation of the triplet axes of Chl a in ethanol/ methanol (45° and 48° with respect to Q B Y, see Table 1) cannot be explained by a sign inversion of E. Thus, we conclude that a sign inversion of E does not take place and that indeed the angle between the in-plane triplet axes and Q B Y is changed by 10-20° for P700 with respect to Chl a in the two glasses. This change is apparently caused by the difference in environment. A rotation of the in-plane triplet axes may be caused by a slightly different electronic structure owing to differences in the coordination of the central Mg atom. The triplet state of Chl a with a five-coordinated central Mg atom, such as in P700,27 may be different from that with a six-coordinated Mg atom, as for Chl a in toluene/pyridine. Chl a in ethanol/methanol, which at low temperature contains both six-coordinated and fivecoordinated Mg,29,30 reflects an intermediate case. On the other hand, hydrogen bonding of the solvent to ring substituents may also influence the electronic states.31 The orientation of the triplet axes of P700 may reflect a distortion of the π-electron density, compared to Chl a in the two glasses, due to the presence of either amino acids or a chromophore close to the ring substituents (or to the central Mg atom of the triplet-carrying Chl a molecule). This chromophore may be the dimer half not carrying the triplet state. For the cation P700•+ it has been proposed that two closelying doublet configurations with different symmetry are

Reaction Center of Photosystem I of Plants mixed32 under the influence of the environment, possibly involving vibronic coupling through an asymmetric mode. Although for the cation of P700 this idea has recently been refuted,33 for the triplet state of Chl a it still seems a viable option. Comparitive measurements of the temperature dependence from 1 to 300 K of the E value of 3P700 and 3Chl a might shed light on this matter, as the value of E may depend on the temperature via the distribution over vibronic states.34 It is interesting to note that Sieckman et al.10 observed a large increase in the value of E of 3P700 on cooling from room temperature to liquid helium temperature, although the authors explained this by triplet delocalization at room temperature. We note that the strong coupling of 3P700 with a charge-transfer triplet of the type P700•+ A•-, where A is an intermediate acceptor, such as suggested by Small35 for the excited singlet state of the primary donor, is highly unlikely, as the value of |D| of 3P700 is virtually the same as that of monomeric 3Chl a. The LD-ADMR data presented here can be compared with the EPR data on oriented PSI particles of Rutherford and Se´tif,36 who found an orientation of b zT parallel to the membrane, and yT and the membrane of 20-30° and angles between b xT and b 70-60°, respectively. When assuming an orientation of Q B Y of P700 within 20° parallel to the membrane plane,37 the values xTb yT plane reported here, 35° for the orientation of Q B Y in the b and 56°, correspond well with the results of Rutherford and yT Se´tif.36 Sieckman et al.10 calculated for the angle between b and the membrane two possible values, viz., 27.5° or 62.5°, by combining their EPR results with data from linear-dichroic absorption spectroscopy.37 They noted that their results agreed yT makes an angle with those of Rutherford and Se´tif36 only if b of 62.5° with the membrane, but they rejected this idea because only the complementary angle of 27.5° was compatable with B Y of monomeric Chl a quoted the parallel orientation of b yT and Q in the literature.15 However, our results on Chl a in Vitro show B Y are far from parallel. Thus, there conclusively that b yT and Q is no reason to reject the alternative angle of 62.5°, which is close to the angle of 60-70° found by Rutherford and Se´tif.36 “Monomer” Absorption in the T-S Spectrum. When, on excitation, two Chl a molecules are strongly excitonically coupled in the singlet state, and relatively weakly coupled in the triplet state (resulting in a localized triplet state), triplet formation should lead to the appearance of a “monomer” absorption of the dimer component that loses its interaction with the triplet-carrying molecule. This absorption then appears with a positive sign in the T-S spectrum at an energy close to the monomeric transition energy. If this monomer transition is not significantly coupled to other transitions, the orientation of its QY-transition moment with respect to that of the dimer QY transition should provide us with further information relevant to the problem of the dimer geometry (see above). It has been suggested earlier that the strong, positive feature at 674 nm in the T-S spectrum of PSI particles represents such an appearing monomer absorption.9 The 674 nm transition makes an angle of 65 ( 5° and 25 ( 5° with respect to b xT and xTb yT b yT, respectively. Therefore, it must be polarized in the b plane of the triplet-carrying Chl a molecule of P700. According to Table 2, the angle between the 674 nm transition moment and that of P700 then is ∼30° or ∼80°. The “perpendicular” orientation with an angle of ∼80° between the QY-transition moment of one dimer half and the dimer transition moment can be rejected, as it would not give rise to a dimer transition with high intensity and a polarization perpendicular to the transition moment of a dimer half. If we assume a symmetrically delocalized singlet excitation of the dimer, then the value of 30° leads to the QY-transition moment of the triplet-carrying Chl a molecule making an angle of ∼5° with its own triplet

J. Phys. Chem., Vol. 100, No. 23, 1996 9965 x-axis. We think this situation can be rejected also, as the angle between the QY and triplet-triplet transition moment of the triplet-carrying molecule then would be excessive compared to what is expected on the basis of our results for Chl a in the two glasses (Figure 3). Alternatively, when speculating about the possibility of an asymmetrically delocalized singlet excitation, we would need an almost totally localized singlet excitation to explain the orientation of the 674 nm “monomer” transition moment, in clear contrast with the assumption of strong coupling in the singlet state, needed for the appearance of a triplet “monomer” transition in the T-S spectrum. We conclude that the 674 nm transition cannot be assigned to a “monomer” absorption of the triplet state of an excitonically coupled dimer. The LD-ADMR data suggest two conceivable candidates for the “monomer” absorption, namely the positive absorption bands with relatively low dipolar strengths at 682 and 692 nm (Table 2). In principle, a weak positive band could be due to a “monomer” absorption, the low intensity being explained by an asymmetric delocalization of the QY-singlet excitation over the two dimer molecules, due to a relatively large difference in transition energy of the dimer halves compared to their QYsinglet exciton interaction (so-called site splitting). Triplet formation would then lead to a relatively small band shift, a strong overlap between the appearing “monomer” absorption and the bleaching of the dimer exciton band, and therefore a weak “monomer” positive absorption in the T-S spectrum. Unfortunately, we were unable to determine the (accurate) polarizations of these weak transitions. For reaction centers of purple bacteria, a pure “monomer” absorption has not been observed in the T-S spectra,18,19 and the positive bands in the T-S spectra have been ascribed to band shifts of chromophores located close to the primary donor.18,19,38 Similarly, the PSI a pure “monomer” absorption may be absent. The positive band at 674 nm may be ascribed to a transition of a Chl a located close to P700, which shifts upon 3P700 formation. The relatively large positive amplitude in the T-S difference spectrum indicates that the shift is important, i.e., that it has a relatively strong coupling with the QY transition of the triplet-carrying molecule of P700, similar to that observed for the QY transitions of the accessory bacteriochlorophylls (BChls) in reaction centers of purple bacteria.18,19 Also, the orientation of the 674 nm transition moment with respect to that of P700 (∼30°) is similar to that observed for the QYtransition moments of the accessory BChls in the T-S spectrum of purple bacteria.16,18,19 We exclude a blue shift of the 687 nm absorption to 674 nm upon 3P700 formation, as such a strong blue shift of 250-300 cm-1 seems too high in view of the shifts of ∼50 cm-1 observed for the QY absorptions of the accessory BChls in the T-S spectra of purple bacteria. High-Energy Absorption of P700. In the literature, various bands at wavelengths ranging from 644 to 690 nm have been assigned to the high-energy QY-exciton component of P700. The broad 644 nm transition has a polarization close to that of the 700 nm band and is more likely a 1200 cm-1 QY-vibronic transition of P700, because its energy and its orientation with respect to the QY transition of P700 agree with those found for the vibronic transition of Chl a at 620 nm (see Table 1). By analogy, the broad 635 nm signal in the T-S spectrum of PSI can be ascribed to a vibronic transition related to the 687 nm transition. Shubin et al.24 concluded from the circular-dichroic P700•+-P700 spectrum that an absorption around 690 nm is the high-energy component. We find that the polarization of the 687 nm transition in the T-S spectrum makes an angle of ∼30° or of ∼80° with the polarization of the 700 nm band (Table 2). Because the angle of 30° is in better agreement with that found from the linear-dichroic P700•+-P700 spectrum,37 we may reject

9966 J. Phys. Chem., Vol. 100, No. 23, 1996 the latter solution. The remaining low value of 30° precludes assignment of the 687 nm band to the high-energy component of a symmetric dimer, for which a value close to 90° is expected. In principle, (part of) the 687 nm band might still represent the high-energy exciton component of an asymmetric dimer, i.e., a dimer in which the constituting monomers absorb at energies differing by more than the interaction energy. The dimer transition moments of the low- and high-energy exciton components then would have a relative orientation that depends on the strength of the coupling between the QY-transition dipoles of the two monomers. In the present case this would imply that the interaction energy is much smaller than ∼150 cm-1, the difference in energy between 687 and 700 nm. Such an interpretation stands in contrast with the interpretation of the ciricular-dichroic P700•+-P700 spectrum,23,24 which is far better explained by assuming an interaction of P700 with an adjacent Chl a molecule absorbing around 687 nm, which gives rise to two bands with opposite rotational strength. (Note that the resulting transition moments (at 687 and 700 nm) need not be perpendicular for exhibiting opposite rotational strengths.) In conclusion, there is no evidence for an isolated high-energy exciton component in the T-S spectrum. At most, the 687 nm band could contain some contribution of a high-energy transition heavily admixed with transition moments of chromophores other than the dimer chromophores. We note that even for the dimer in reaction centers of purple bacteria, in which the coupling undoubtedly is stronger than in P700, the high-energy transition is not observed as an isolated transition.18,19 Primary Acceptor A0 and the Accessory Chromophores. A Comparison with Heliobacterium chlorum. The T-S and (circular-dichroic) P700•+-P700 spectra23,24,37 of PSI all point to a relatively strong coupling between the species absorbing at 687 nm and P700. From the A0•--A0 absorbance-difference spectrum it was concluded that the primary acceptor A0 absorbs at ∼690 nm.39,40 Therefore, it is tempting to conclude that the 687 nm band in the T-S spectrum is associated with A0, which is coupled strongly (located close) to the primary donor P700. Such a strong coupling, however, would be totally different from that in reaction centers of purple bacteria, where the primary acceptor I (a bacteriopheophytin, BPh) is separated by ∼17 Å from the primary donor,41,42 resulting in a very weak coupling between the two, in agreement with the low intensities (small band shifts) of the bacteriopheophytin signals in the T-S spectra.16,18,19 Furthermore, the orientation of the 687 nm yT plane of P700 and oriented at 30° transition, lying in the b xTb with respect to Q B Y of P700, is quite different from that found for the primary acceptor in reaction centers of purple bacteria. We think it therefore unlikely that the 687 nm absorption represents a transition from A0. An alternative explanation for the 687 nm transition in the T-S spectrum of PSI, contrasting with the above interpretation involving A0, assumes an analogous chromophore composition of the reaction center of PSI and those of the purple bacteria.43 This analogy can be extended to Heliobacterium (H.) chlorum and will be illustrated by interpreting the T-S spectra and the “reduced primary acceptor difference spectra” (i.e., the A0•-A0 spectra of PSI and H. chlorum, and the I•--I spectrum of purple bacteria) for each of these species, by introducing additional accessory Chl(s) that are coupled to the primary acceptor molecule(s). Here, the “accessory” and “acceptor” Chl in the reaction center of PSI correspond to the accessory BChl and the acceptor BPh in the reaction center of purple bacteria, respectively.41,42 In this model, the primary acceptor and the accessory Chl represent a coupled system, with the coupling weak compared to the site-splitting between the transition energies of the uncoupled chromophores. Thus, they give rise

Vrieze et al.

Figure 4. Microwave-induced T-S spectra of PSI (solid line) and of membranes of H. chlorum (dashed line). The microwaves were set at 955 MHz (|D| + |E|) for PSI and at 982 MHz (|D| + |E|) for H. chlorum. See also caption of Figure 1.

to two transitions, which manifest themselves in the A0•--A0 spectrum as a bleaching and a shift and in the T-S spectrum as band shifts (because the accessory Chls are assumed to interact with P700). When applying the above interpretation to the T-S spectra, the shoulders at 785 nm for H. chlorum44 and at 687 nm for PSI both are ascribed to a band shift of the QY transition of one or more accessory (B)Chls. Furthermore, the T-S spectra of PSI and H. chlorum can be compared in the 650-675 nm region (Figure 4). In this region, the T-S spectrum of PSI strongly resembles that of H. chlorum and Prostecochloris aestuarii (the spectrum of the latter species is not shown, see ref 45). In both organisms, a Chl a derivative functions as primary acceptor.46,47 We ascribe the two positive bands at 662 and 669 nm to band shifts of a composite absorption band centered at 665 nm, suggesting the involvement of two Chl a molecules. One of these may represent the true primary acceptor and the other the “inactive” acceptor (the latter by analogy with the inactive BPh in the M-branch of reaction centers of purple bacteria). For the bacteria H. chlorum and Prostecochloris austuarii, the absorption at ∼665 nm, due to the Chl a derivative, is distinct from the absorption of the BChls in the reaction center, absorbing around 790 nm, and therefore can be attributed unequivocally to the primary acceptor. For PSI we may, in view of the similarity of the T-S spectra, attribute the band shifts at 662 and 669 nm to small changes in the interaction of the active and inactive primary acceptor with P700 upon 3P700 formation, caused either by a change in a weak direct interaction with P700 or by an indirect change in interaction via accessory Chls. The above interpretation of the T-S spectrum is supported by comparison of the absorbance-difference spectra of the reduced-minus-neutral primary acceptor for the various species. The I•--I spectrum of Rb. sphaeroides shows two negative bands when I (the active BPh) is reduced, which are centered at 760 and 800 nm48,49 and ascribed to the BPh and the accessory BChl of the active L-branch, respectively.49 For H. chlorum similar features are observed when comparing the P•+A0•-PA0 and P•+X•--PX spectra, obtained by Kleinherenbrink et al.,50 where P is the primary donor absorbing at 794 nm and X is the secondary acceptor, an iron-sulfur cluster. The difference between these two spectra shows a band at 785 nm, attributed to an accessory BChl g, and a band at ∼670 nm, attributed to the primary acceptor. For PSI, two bands appear in the A0•-A0 spectrum,39 one intense band at 690 nm and a shoulder at ∼675 nm. By analogy with the above interpretations we can ascribe these bands to an accessory Chl a (which contributes most of its oscillator strength to the 690 nm absorption) and,

Reaction Center of Photosystem I of Plants close to this molecule, a Chl a functioning as primary acceptor (which contributes most of its oscillator strength to the 675 nm absorption). The above interpretation has recently been confirmed for H. chlorum by results of pump-probe experiments on a subpicosecond time scale.51 Summarizing, we propose that the signal in the T-S spectrum of PSI at 687 nm represents mostly the QY transition of an accessory Chl a, which is located close to P700 and coupled to the primary acceptor, the latter contributing mostly to the absorption at ∼665 nm. Conclusions The orientations of the transition moments contributing to the T-S spectra of PSI were determined with the use of (LD)ADMR. By comparing the orientations of the optical transition yT of 3P700 moments relative to the in-plane triplet axes b xT and b with those of Chl a in ethanol/methanol and toluene/pyridine glasses, we conclude that the observed differences in orientation are due to a change in environment, rather than to dipolar coupling between two Chl a chromophores of P700 or a change in the sign of the E zfs parameter. A strong positive signal at 674 nm in the T-S spectrum of PSI is probably due to an accessory Chl a chromophore located close to P700. The signal cannot be ascribed to the monomer absorption of the dimer half that does not carry the triplet state in a model assuming dipolarcoupled dimer halves, as suggested by den Blanken and Hoff.9 The T-S spectrum does not show evidence for an isolated highenergy band of P700. For the interpretation of the T-S spectrum in the 660-690 nm region, the involvement of accessory chromophores is needed. A negative absorption in the T-S spectrum at 687 nm is interpreted to originate from a Chl a adjacent to P700, which is coupled to the primary acceptor. The 662 and 669 nm signals in the T-S spectrum are attributed to Chl a molecules functioning as inactive and active primary acceptor, which both absorb around 665 nm in the singlet ground-state absorption spectrum. The interpretation of the 687 nm transition holds for all absorbance difference spectra of P700, including the T-S, P•+A0•--PA0, and P•+-X•--PX spectra. It corresponds well with our interpretation of the absorbance-difference spectra of reaction centers of H. chlorum and purple bacteria and suggests that the global organization of the chromophores close to P700 resembles that of the chromophores close to the primary donor in the reaction center of purple bacteria. Acknowledgment. B. M. Buel kindly provided the chloroplasts and S. J. Jansen expertly carried out the SDS-PAAGE of the PSI particles. J.V. thanks R. Louwe for useful discussion. We thank Prof. J. H. van der Waals for his interest and helpful suggestions. This work was supported by the Netherlands Foundation for Chemical Research (SON), financially assisted by the Netherlands Organization for Scientific Research (NWO). References and Notes (1) Golbeck, J. H.; Bryant, D. A. Curr. Top. Bioenerg. 1991, 16, 83. (2) Krauss, N.; Hinrichs, W.; Witt, I.; Fromme, P.; Pritzkow, W.; Dauter, Z.; Betzel, C.; Wilson, K. S.; Witt, H. T.; Saenger, W. Nature 1993, 362, 326. (3) Kok, B. Biochim. Biophys. Acta 1956, 22, 399. (4) Den Blanken, H. J.; Meiburg, R. F.; Hoff, A. J. Chem. Phys. Lett. 1984, 105, 336. (5) Rutherford, A. W.; Mullet, J. E. Biochim. Biophys. Acta 1981, 635, 225. (6) Gast, P.; Swarthoff, T.; Ebskamp, F. C. R.; Hoff, A. J. Biochim. Biophys. Acta 1983, 722, 163. (7) Hoff, A. J.; Den Blanken, H. J.; Vasmel, H.; Meiburg, R. F. Biochim. Biophys. Acta 1985, 806, 389. (8) Verme´glio, A.; Breton, J.; Paillotin, G.; Cogdell, R. Biochim. Biophys. Acta 1978, 501, 514.

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