7702
J. Phys. Chem. 1994,98, 7702-7711
Site-Selection Spectroscopy of the Reaction Center Complex of Photosystem 11. 1. Triplet-minus-Singlet Absorption Difference: Search for a Second Exciton Band of P-6807 Stefan L. S. Kwa; Camiel Eijckelhoff, Rienk van Grondelle, and Jan P. Dekker Department of Physics and Astronomy and Institute for Molecular Biological Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands Received: December 17, 1993; In Final Form: March 22, 1994"
The Dl-D2-Cytb559 reaction center complex of photosystem I1 was studied at 4 K with triplet-minus-singlet (T-S) absorption difference spectroscopy using selective laser excitation. The T-S spectrum is mainly due to the bleaching of P-680 and significantly differs from the T-S spectrum of monomeric Chl a in detergent. The absorption spectrum ascribed to the primary electron donor, P-680, was interpreted in terms of two distinguishable distributions, around 68 1 and 684 nm, respectively. These spectral forms could be preferentially selected by relatively blue ,A(, = 661-677 nm) and extremely red excitation (Aexc = 691 nm), respectively. In addition to the bleaching at 681-684 nm, a small band at 667 nm was also observed, which had opposite polarization. We attributed the latter to an exciton band, not associated with a single pigment. Thus upon triplet formation a system consisting of N excitonically coupled pigments ( N > 1) is reduced by one due to localization of the triplet state on one of the pigments. The simplest, although not the only, case is given by N = 2, for which P-680 is a dimer with exciton bands at 681-684 and 667 nm. This interpretation immediately explains the positive and negative bands observed a t 679 and 669 nm in the circular dichroism spectrum of Dl-D2-CytbS59 (Braun, P.; Greenberg, B. M.; Scherz, A. Biochemistry 1990,29,10376-10387). The relatively small amplitude of the 667-nm band indicates that the angle between the two unperturbed Qytransitions of a putative Chl dimer (15-21O) is two times smaller than that of the special pair pigments of purple bacteria (38O). We do not rule out the case that N = 3 or larger.
as the primary and ultimate electron acceptors,respectively. The primary electron donor of the bacterial reaction center consists Conversion of light energy into an electrochemical potential of a dimeric pair of bacteriochlorophyll (BChl) molecules, called takes place in photosyntheticreaction centers. The reaction center the special pair.6 This arrangement causes strong excitonic of photosystem I1 has the ability to produce an unusually high coupling which is manifested by a large splitting of the lowestoxidizing potential which is used for the splitting of water. This energy absorption band (for a review see ref 9). reaction center was first isolated and identified as such in 1987l Structural information on P-680 has been obtained from and is known as the Dl-D2-Cytb559 complex. The D1 and D2 spectroscopic data. The CD spectrum of the Dl-D2-Cytb559 polypeptides of this complex serve as a framework to position six complex has been largely ascribed to the dimeric nature of P-68O.I0 chlorophyll a (Chl a ) and two pheophytin a (Pheo a ) molecules.* With ultrafast absorption difference measurements Durrant et These pigments can be excited by visible light, after which the al." observed that upon direct excitation of P-680 the oscillator energy is transferred to the primary electrondonor, P-680, which strength of two chlorins disappears, from which it was concluded is a Chl a species. This induces the primary charge separation that initially the excitation energy is delocalized between two resulting in the formation of the radical pair state P-680+Pheo-. pigments constituting P-680. The triplet yield of P-680 is high The Dl-D2-Cytb559 complex lacks the secondary electron and P-680T lives relatively long at low temperatures (- 1 ms3) acceptor QA,lost during the isolation procedure,' which prevents enabling accumulation of P-680T under continuous light excitafurther electron transport. In this isolated complex the chargetion. Transient hole-burning data suggest that the light-induced separated state will therefore recombine to form the singlet and triplet state is indeed located on P-680.12-lS With light-induced triplet excited state of P-680 (P-680(*)and P-680T,r e s p t i ~ e l y ) . ~ ~ FT-IR absorptiondifferencespectroscopy Noguchi et a1.I6found This mechanism of triplet-state formation is called the radicalthat two bands at 1669 and 1707 cm-1, assigned to the ketoQC pair mechanism. stretching modes of the twochlorophylls that contribute to P-680, The exact structure of the Dl-D2-Cytb559 complex has not and which are in slightly different chemical environments, shift been resolved, but its central part most likely resembles the wellto the red upon formation of P-680T at 80 K. They concluded known structure of the reaction center of purple bacteria.6 that the observed molecule in the triplet state was exclusively Obvious similarities include the amino acid sequences of the D1 formed via the radical pair mechanism (not by intersystem and the D2 proteins, which are homologous to the L and M crossing) and thermally equilibrated between the two Chls of subunits of bacterial reaction centers,- and the organization of P-680. From the temperature dependence of the ratio of these the electron acceptor chain, where both systems use a (bacteria)bands they estimated an energy difference of 8.4 meV between pheophytin molecule and very similar quinoneiron complexes the lowest triplet states of the two Chls, which means that at 4 K the triplet state is completely localized on one Chl.17-19 t Abbreviations: BChl, bacteriochlorophyll; BisTris, 2-[bis(Z-hydroxyIn the visible region until now only a single Qyband, found at ethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;BPheo, bacteriopheophytin, 680-684 nm, could be ascribed to P-680, even at low temperaCD, circular dichroism; Chl, chlorophyll; CP47, core antenna complex CP47 tures.5919 In this study we have attempted to characterize in detail of photosystem 11; LHC-11, light-harvesting complex I1 of photosystem 11; Pheo, pheophytin; PS-11, photosystem 11; P-680, primary electron donor of the absorption properties of P-680 and specifically searched for photosystem 11;P-960, primary electrondonor of Rps. viridis; Rb. sphaeroides, a second exciton band in the absorption spectrum of P-680. By Rhodobacter sphaeroides; Rps. viridis, Rhodopseudomonas viridis; Rs. selective excitationwith a tunable, continuous laser we measured rubrum, Rhodospirillum rubrum; T S ,triplet-minus-singlet. triplet-minus-singlet ( T S ) absorption difference spectra of P-680 @Abstractpublished in Advance ACS Abstracts, June 1, 1994.
Introduction
0022-365419412098-7702%04.50/0 0 1994 American Chemical Society
The Journal of Physical Chemistry, Vol. 98, No. 31, 1994 7703
Reaction Center Complex of Photosystem 11. 1
scattering light of the laser could be filtered out electronically, except at low probe light intensities. The latter occurred when a spectral bandwidth of < 1 nm was used. The small scattering/ fluorescence contribution to the AZsignal was determined before each measurement by blocking the probe beam, and was subtracted from Al. Since the monochromator was placed before the sample, and not between the sample and the photodiode, the scattering/fluorescence signal was wavelength independent and thus no artificial laser-scattering peak was present in the T-S spectra. We define the anisotropy r of the T S difference absorption AA as
0
I
'
660
I
670
I
BE0
I
N O
I
700
Wavelength (nm) Figure 1. Steady-state absorption spectrum of Dl-D2-Cytb559 at 4 K (-) and its sccond derivative spectrum multiplied by -1 (- - -). (P-680T-minus-P-680) in the visible region at 4 K. Since the putative second exciton transition of P-680 should have a different orientation as the transition at 680-684 nm, polarized photoselection measurements were performed to identify such a band. Materials and Methods
Samplehparntion. Dl-D2-Cytb559 was isolated as described elsewhere." For the measurements the sample was in a buffer containing 20 mM BisTris, pH 6.5,20 mM NaCl, 0.03 96 (w/v) n-dodecylb-D-maltoside, and 70 96 (w/v) glycerol and was placed in a perspex cuvette (1 .O X 1.O X 4.0 cm) which was cooled to 4 K in a He-flow cryostat (Oxford). In Figure 1 the steady-state absorption spectrum of Dl-DZ-Cytb559 at 4 K is shown in the Qyregion (solid line). The spectrum reveals only two maxima: the band at 671 nm is due to accessory chlorophyll a (Chl a) molecules, while the band at 679 nm is mainly due to the primary donor, P-680.5J5J9 The presence of a weak shoulder around 684 nm is clear from the second derivativeof the absorption spectrum (dashed line) and confirms earlier 4 K spectra of stabilized D1D2-Cytb559 preparations (see refs 5 , 15, and 21). The width of spectralbands and line shapes, whether symmetric or asymmetric, is defined here as the full width measured at half height. Triplet-minus-SingIet Absorption Difference Spectra. Revenible light-inducedabsorptiondifferencespectra, also called lightinduced triplet-minus-singlet ( T S ) absorption difference spectra, or transient hole-burning spectra with the triplet state acting as a populationbottleneck,were measured as described elsewhere.22 Briefly, the excitation light (A = 610-710 nm, 5-100 mW/cm2) was provided by a C W dye laser (Coherent 599, DCM dye, spectral bandwidth -2 cm-1) pumped by an Ar+ laser (Coherent Innova 310). Theexcitationintensitywashighenought tocreatea steadystate triplet population of pigment molecules during illumination. Probe light was provided by a 150-W halogen lamp in combination with a 1/4 monochromator (Oriel 77200) and was detected after the sample with a photodiode (Centronics OSD 5-3T). Since the laser intensitywas chosen such that the absorptiondifferenceL4 was small ( 691 nm) arises from Chl a molecules different from P-680 with their zero-phonon line at equal or longer wavelengths than 691 nm,34 the SDF of which could not be neglected in this case, because of the absence of a dominating competing process. Regardless of the interpretation of the absorption at 689-691 nm, we will base our further conclusions only on the observation that the transition at 689-691 nm is parallel to the Qytransition of P-680 at 684 nm. Real and Pseudo Phonon Side Hole. A second clear difference between measurement and simulation can be seen in a region where the SDF is significantly larger than zero, but only when measuring at a higher spectral resolution (Figure 6, upper lines, measurement; lower line, simulation). While the real phonon side hole, due to bleaching of the phonon side wing, is prominent in the simulation (on the high-energy side of the zero-phonon hole), it seemsto be absent in the measured spectrum. The pseudophonon side hole (on the low-energyside of the zero-phononhole), due to bleaching of sites excited in their phonon side wing, can be observed in the measurement. Similarly, in all published transient hole spectra of Dl-D2-Cytb559, identified to be arising from P-680 by the 6-cm-l width of the zero-phonon hole, the pseudo-phonon side hole is always more prominent than the real phonon side Because of this discrepancy we conclude that, although a large variety of site profiles can be generated by choosing different parameter sets, we have reached a limitation of the theory of hole profiles by Hayes et al.23 The theory only accounts for the spectra due to the disappearanceof the molecules from the ground-state, but not for the spectra of the appearance of these molecules in the excited state. Therefore we ascribe the absence of the real phonon side hole in the measured spectrum to a positive contribution which cancels a significant part of the high-energy side of the negative band. Although it would lead too far to speculate on the specificshape of this positive spectrum for every excitation wavelength, we will present below a deconvolution of the nonselectively excited T S spectrum that is consistent with this interpretation. We conclude that the site-selected T S spectra show various spectral features ( 1 4 , several of which are not trivial. For most excitationwavelengths(661-687 nm) the spectra can beexplained by simulating the absorption spectrum for P-680 with only one site spectrum, which is inhomogeneously broadened with a site distribution function (SDF) consisting of two Gaussians at 68 1 and 684 nm. However at 689 and 691 nm additional absorption
-
Measured I
I
v
681
I
I
-
I
Detection wavelength (nm) Figure 9. (Upper panel) measured isotropic T S absorption difference spectra of Dl-D2-Cytb559at 4 K excited at 679,681,683,685,and 687 nm. The spectra were normalized to the same incident laser intensity (30 mW/cm2). OD679 was 0.8/cm at 4 K, and the spectral bandwidth in the detection was 1.O nm. The absoluteamplitudescan be compared to those in Figure 5 . (Lower panel) simulations of the measured spectra using the parameters given in the caption of Figure 8. is comparable to the amplitude roughly estimated on the basis of the number of absorbed photons (AA = 3 X 1V; using incident laser intensity 30 mW/cm* at 691 nm; OD691 == 0.01 (see Figure 1); @T 0.8 and triplet lifetime 1 m ~ illuminated ; ~ volume == 1 cm3; tCh1680 105 M-1 cm-I). Thus there is a measurable steady-state absorption at 691 nm (see Figure l), which also leads to efficient triplet formation (see Figure 5 ) . The properties of the transition associated with this absorption can be summarized as (1) there is no strict correlation between site energiesof this transitionat 69 1 nm and the bleachedQ, transitions at 684 nm (see Figure 5 , no clear site selection or line narrowing is observed), and (2) the transition at 691 nm is parallel to the Qytransition at 684 nm since the anisotropy is high (r = 0.33). We will discuss several interpretations of the absorption at 691 nm. One explanation is that a fraction of the zero-phonon lines centered around 684 nm has a width 10-20 times larger than y = 3 cm-1 used in the simulation, because then the amplitude of the simulated spectra excited at 689 and 691 nm would increase with roughly the same factor. Thermal broadening by local heating can be ruled out, since the absorption at 691 nm is also seen in the absence of laser light (see Figure 1). Therefore a width of y = 30-60 cm-1 would yield an excited-state lifetime of P-680of 0.1-0.2 ps. Such times have been observed additionally to 3-pslifetimeswith transient absorption measurements.11 Since a hole width of 2y = 6 cm-l (corresponding to a 2-ps lifetime)
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Reaction Center Complex of Photosystem 11. 1
The Journal of Physical Chemistry, Vol. 98, No. 31, I994 7709
is observed which arises from a transition which is parallel to the Qy transition of P-680. The Band at 667 nm. To investigate the origin of the bands at 667 nm, we have performed anisotropy measurements in this wavelength region (Figure 5). From these and earlier reported datal9 we conclude that the orientations of transitions at 667 and at 681-684 nm areclearly different. Therefore the 667-nm band cannot be a vibronic band of the Qy(m) band at 681-684 nm. Assigning this band to a red shift of an accessory pigment upon breaking of excitonic coupling due to the formation of P-680T is not likely since usually the opposite is observed. For example upon the formation of P-960Tin the reaction center of the purple bacteria Rps. viridis, the bands of the accessory BChl molecules shift to the blue.35 In view of the different orientation of the 667-nm transition s' 860 860 870 880 890 it can be interpreted as an exciton band, not associated with one 0 specific pigment. This means that two or more excitonically coupled pigments are involved in the T S spectrum. The simplest Detection wavelength (nm) excitonic coupled system consists of N = 2 pigments. In this case Figure 10. Average of the isotropic T-S absorption difference spectra the bands at 681-684 and 667 nm can be assigned to be the two of D1 -D2-Cytb559 at 4 K excited nonselectively at 661,667, and 67 1 nm perpendicular exciton transitions of the P-680 dimer. This (-) taken from Figure 2, and the T-S absorption difference spectrum interpretationis inspired by the oppositepolarization of theexciton of Chl u in Triton X-100 at 4 K taken from ref 22 (- -). The T-S bandsat 100~1014and850nmoftheBChldimerin thereaction spectrum of Chl a was excited at 672 nm but shifted on the computer to match the band position with that of the Dl-D2-Cytb559 spectrum. center of Rps. viridis.15.35 Note that the wavelengths of the 681684 and 667-nm bands correspond closely to those in the CD spectrum assigned by Braun et al. to P-680.10 I I I I hac = 661 - 671 nm Theobservationthat the position of the 667-nm band is constant (within f1 nm) in the site-selectedT-S spectra and not (positively) .,,......... correlated with that of the band at 68 1-684 nm (Figure 3) favors its assignment to an exciton band of a Chl a dimer. Positive correlation is only expected for vibronic bands (see e.g. ref 22). For exciton bands correlation is lost since the site energies of the unperturbed Qy transitions of the constituting Chls are inhomogeneously broadened. The latter means that the two pigments of, for example, an individual dimer will not always be in resonance, which leads to inhomogeneity in the amount of exciton splitting,'6 i.e. loss of correlation. Furthermore, if the interaction energy between the constituting pigments is also inhomogeneously broadened (e.g. if the distance between the Chls is inhomogeneously broadened), even a negative correlation could be expected. This is seen for example for the two exciton bands of P-960 in the reaction center of Rps. viridis: upon shifting the low-energy exciton band at 1000 nm to the red by persistent hole burning, the high-energy exciton component at -850 nm shifted to the blue (negative c o r r e l a t i ~ n ) . ~ ~ P-680Monomer, Dimer or N-mer? T-S Data. On the basis of T S absorption difference measurements Van der Vos et al. concluded that P-680 is a monomer.19 However they did not 2nd Der. explain the absorption changes below 675 nm. We have measured T S spectra of monomericChl a in Triton X-100detergent, using I I I I the same apparatus as in the present study.22 In Figure 10 we 660 670 680 690 700 plotted this spectrum, taken from ref 22 (dashed line). This spectrum was excited at 672 nm and was shifted to the red on Detection wavelength (nm) the computer to align the peak with that of the Dl-D2-Cytb559 Figure 11. Average of the isotropic T-S absorption difference spectra spectrum (solid line) taken from Figure 2. Although the QY(0)of D1 -D2-Cytb559 at 4 K excited nonselectively at 661,667, and 671 nm and its second derivative (-, upper and lower curve) taken from Figure band in the Chl a spectrum is rather narrow (6.5 nm) it completely 2, a fit of these spectra by assuming that P-680 is a dimer (- - -) and the lacks the relatively narrow positive and negative features as components of the fit These components were the simulated 681observed in the Dl-D2-Cytb559 spectrum at 673 and 667 nm 684-nm band (excited nonselectively, see lowest curve of Figure 8), a (Figures 2,3 and 5). Therefore we rule out that P-680 is a simple negative Gaussian band at 667.7 nm (width, 7.0 nm; height, -0.055), and monomer. Weak excitonic interactionsof a P-680 monomer with a positive band at 678 nm, which was calculated from the simulated other pigments, which would be diminished upon triplet formation, 681-684-nm band by multiplication with a factor -0.5, broadening the could account for the extra spectral features. s e r u m (which we arbitrarily did by convolution with a 5-nm wide triangular function) and shifting by 2.2 nm to the blue (see text for the A special case of the latter interpretation is that monomeric interpretation). The fourth component (an offset of 0.026) is not shown. P-680 is coupled to only one other monomer. However, this situation is spectroscopically identical to the interpretation that P-680 is a dimer, and again the band at 667 am can be assigned (non-site-selected) T-S spectrum of Dl-D2-Cyt6559. We stress to the high-energy exciton band of the dimer. Only to illustrate that there are several ways to successfully decompose the T-S this interpretation we present in Figure 11 a deconvolution of the ~pectrum,~J9 so a good fit cannot be considered as evidence for
2 -
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(.e.).
7710 The Journal of Physical Chemistry, Vol. 98, No. 31, 1994
a specific model. The deconvolution we present is based on an interpretation suggested by Van Kan et al.5 and using the result of the simulations of the site-selected T S spectra in Figure 3. We show the measured AA spectrum (upper solid line) and its second derivative (upper and lower solid line, respectively; both taken from Figure 2), the fits of these spectra (dashed lines) and the components constructing the fit of the AA spectrum (dotted lines). The fit is described by four components. The 681484nm band, which is the inhomogeneous broadened OD spectrum obtained from the simulations of the site-selected spectra (see lowest spectrum of Figure 8), and a small Gaussian band at 667.7 nm account for the bleaching of the two exciton bands of P-680. Since we know that the triplet state of P-680 is localized on one Chl,16J7J9a positive spectrum from the other Chl should appear in the spectrum, which has exactly half the oscillator strength of the 681-684-nm band in the simulation.5 This contribution was calculated from the 681-684-nm band, by shifting it 2.2 nm to the blue, broadening it on computer, and multiplying it with a factor -0.5. A small offset (0.026, not shown) accounted for the triplet-triplet absorption of the Chl a molecule in the triplet state. Examples of bacteriochlorophyll (BChl) dimers with T S absorption difference spectra that have been described by a bleaching of the dimer exciton bands and the appearance of a broader monomer band, are the special pair in the reaction centers of Rps. viridisg5 and Rhodobacter sphaeroides (Rb. sphaeroides),37 and the BChl dimers in B820 antenna subunits of Rhodospirillum rubrum (Rs. rubrum).38 The fit shows that our measurements can easily be explained by assuming that P-680 is a dimer and is not in contradiction with this interpretation. On the basis of simple exciton theory the position of the appearing monomer band should be at an energy between and equidistant from both exciton bands. The latter is not the case for P-680, but is also not the case for T S spectra of the special pair dimer in Rps. viridis,35 where the monomer band appears even to the blue of the high-energy exciton band. The ratio of the oscillator strengths of the 681- and the 667-nm exciton band (- 30:1, Figure 11) corresponds to an angle of -21° between the Qytransitions of the two Chl a molecules of a dimeric P-680. However, because no vibronic bands have been incorporated in the deconvolution of Figure 11, the already very small amplitude of the 667-nm could have easily been overestimated by a factor of -2, which introduces an uncertainty in the angle (15O-2l0). The almost parallel orientation of the Chl Qytransitions confirms earlier conclusions by Van Gorkom and c o - w ~ r k e r sTherefore .~~~~ the ADMR and LDMR data by Van der Vos et al.I9 can also be explained by assuming that P-680 is a dimer. In this respect the low value for the fluorescence anisotropy we found at 77 Kzocan only be explained by the presence of another pigment at around 680 nm which is differently oriented than P-680, e.g. Pheo a2’ or Chl a (see also the accompanying manuscript, ref 34). Other Data. The assignment of the bands at 681-684 and 667 nm to the exciton bands of a P-680 dimer explains circular dichroism spectra of D 1-D2-Cytb559, since these spectra are basically composed of two bands at 68 1 and 666 nm which have opposite rotational strengths.10.28-41 This is also observed in the circular dichroism spectrum of the reaction center of Rps. viridis at 100 K, where the exciton bands of P-960 at 980 and 850 nm have positive and negative rotational strength, respectively.42 This interpretation is in agreement with conclusions based on T-S FT-IR data of Noguchi et a1.I6 and ultra-fast pump-probe measurements of Durrant et al.11 As has already been pointed out by Noguchi et al., the experimental results from many different techniques are not in disagreement. The question of whether P-680 is dimeric or monomeric is only a matter of definition. The ground-state absorption properties of P-680 resemble those of a Chl a dimer and can be listed as follows: with one photon two pigments are excited,” and there are two exciton bands with opposite polarization (this workand ref 19) andoppositerotational
Kwa et al. strength’o-28~41 which could be ascribed to P-680. Alternatively, the excited triplet state of P-680 is localized on a Chl a monomer.1619 We stress here that we cannot rule out that the absorption properties of P-680 are arising from interactions between more than two pigments ( N > 2). Van Mieghem et a1.18 concluded from ESR experiments that P-680 is structurally equivalent to one of the accessory BChls in the reaction center of Rps. viridis, except for a rotation in the Chl plane of 45O. However, recalculation with more recent and accurate angular values for Chl a learns that there is one possibility (out of two) that the equivalence between the orientation of accessory BChl and P-680T is almost exact (within the accuracy of 3°).43 This suggests even stronger that the Chl molecule on which the triplet state in Dl-D2-Cytb559 is localized is indeed the structural equivalent of an accessory BChl (ref 43, and A. W. Rutherford, personal communication), since the probability of finding a Chl in another binding pocket at exactly this orientation is extremely small. The consequence is that the exciton bands of P-680 are determined by the excitonic interaction of this Chl and, a t first approximation, its nearest neighbors. Van Gorkom and Schelvisu discussed the possibility that only one of the two “special pair binding pockets” in the D1-D2Cytb559 complex is occupied by a Chl, while the other is empty. Here the absence of a “special pair-like” Chl structure explains the absence of a large exciton splitting in the steady-state absorption spectrum. In a preliminary model proposed by Kwa43 P-680 would then consist of structural equivalents of BChlL (on which the triplet state is localized), and its nearest neighbors, PM and BPheoL ( N = 3). Such a model structure would perfectly explain the ESR data,18 the observed amount of splitting, relative dipole strengths, and the polarization of the exciton bands?’ and is by our knowledge not in contradiction with any data or MD simulation on the Dl-D2-Cytb559 complex so far. Acknowledgment. The authors thank Ing. F. Calkoen for expert technical assistance in isolating the D 1-D2-Cytb559 complex and Dr. H. van Amerongen for critically reading the manuscript. Dr. G. J. Small and Dr. H. J. van Gorkom are acknowledged for sending their manuscripts prior to publication and Prof. A. Freiberg for discussing the absorption at 689691 nm. The research was supported in part by the Dutch Foundations for Chemical Sciences (SON) and for Biophysics (SvB), with financial support of the Netherlands Organization for Scientific Research (NWO). J.P.D. was supported by a fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW).
References and Notes (1) Nanba, 0.;Satoh, K. Proc. Natl. Acad. Sci. U.S.A. 1987,84, 109112.
(2) Kobayashi, M.; Maeda, H.; Watanabe, T.; Nakane, H.; Satoh, K. FEES Lett. 1990, 260, 138-140. (3) Takahashi, Y.; Hansson,0.; Mathis, P.; Satoh, K. Eiochim. Eiophvs. . _ Acta 1987, 893, 49-59. (4) Danielius, R. V.; Satoh, K.; Van Kan, P. J. M.; Plijter, J. J.; Nuijs, A. M.; Van Gorkom, H. J. FEES Lett. 1987, 213,241-244, (5) VanKan,P. J. M.;Otte,S.C.M.;Kleinherenbrink,F.A.M.;Nievetn, M. C.; Aartsma, T. J.; Van Gorkom, H. J. Eiochim. Eiophys. Acta 1990,
1020, 146-152. (6) Michel, H.; Deisenhofer, J. Biochemistry 1988, 27, 1-7. , (7) Trebst, A. 2.Naturforsch. 1986, ~ I c 240-245. (8) Sayre, R. T.; Andersson, B.; Bogorad, L. Cell 1986.47, 601-608. (9) Friesner,R. A.; Won, Y.Eiochim. Biophys. Acta 1989,977,99-122. (10) Braun, P.; Greenberg, B. M.; Scherz. A. Biochemistry 1990, 29, 10376-10387. (1 1) Durrant, J. R.; Hastings, G.; Hong, Q.;Barber, J.; Porter, G.; Klug, D. R. Chem. Phys. Lett. 1992,188 (1,2), 54-60. (12) Jankowiak, R.; Tang, D.; Small, G. J.; Seibert, M. J. Phys. Chem. 1989, 93, 1649-1654. (1 3 ) Tang, D.; Jankowiak, R.; Seibert, M.; Small, G. J. Phorosynrh. Res. 1991,27, 19-29. (14) Johnson, S.G.; Lee, LJ.; Small, G. J. In Chlorophylls; Scheer, H., Ed.; CRC Press: Bcca Raton, 1991; pp 739-768 and references therein. (15) Jankowiak, R.; Small, G. J. In Photosynthetic Reaction Centers; Deisenhofer,J., Norris, J., Eds.;Academic Press: New York, 1993; p 133 and
references therein.
Reaction Center Complex of Photosystem 11. 1
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