Photosynthesis Research 49: 237-244, 1996. © 1996 KluwerAcademic Publishers. Printed in the Netherlands.
Regular paper
Antenna organization in the purple sulfur bacteria Chromatium tepidum and
Chromatium vinosum Hans Kramer& Jan Amesz* Department of Biophysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands; *Authorfor correspondence Received 20 March 1996; accepted in revised form 23 July 1996
Key words: light harvesting, singlet-singlet annihilation, domain size, fluorescence (C. tepidum, C. vinosum)
Abstract Structural aspects of the core antenna in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum were studied by means of fluorescence emission and singlet-singlet annihilation measurements. In both species the number of bacteriochlorophylls of the core antenna between which energy transfer can occur corresponds to one core-reaction center complex only. From measurements of variable fluorescence we conclude that in C, tepidum excitation energy can be transferred back from the core antenna (B920) to the peripheral B800--850 complex in spite of the relatively large energy gap, and on basis of annihilation measurements a model of separate core-reaction center units accompanied by their own peripheral antenna is suggested. C. vinosum contains besides a core antenna, B890, two peripheral antennae, B800-820 and B800-850. Energy transfer was found to occur from the core to B800-850, but not to B800-820, and it was concluded that in C. vinosum each core-reaction center complex has its own complement of B800-850. The results reported here are compared to those obtained earlier with various strains and species of purple non-sulfur bacteria.
Abbreviations: BChl-bacteriochlorophyll; B800-820 and B800-850- antenna complexes with Qy-band absorption maxima near 800 nm and 820 or 850 rim, respectively; B890 and B920-antenna complexes with Qy-band absorption maxima near 890 and 920 nm, respectively; L H l - l i g h t harvesting 1 or core antenna; LH2-1ight harvesting 2 or peripheral antenna Introduction The purple sulfur bacteria Chromatium (C.) vinosum and C. tepidum show clear differences with respect to their spectral properties and antenna composition. C. vinosum resembles species like Rhodopseudomonas (Rps.) cryptolactis (Kramer et al. 1995a) and Rps. acidophila (Deinum et al. 1991) with respect to the location of the Qy absorption bands of the peripheral antenna BChls. Its absorption spectrum is shown in Figure 1A. In addition to a core antenna, B890, it possesses two peripheral antennae, B800-850 and B800-820 (Cogdell and Thornber 1979; Hayashi et al. 1981). It was earlier observed that the organization of the antenna in Rps. cryptolactis (Kramer et al. 1995a) differs
from that of Rps. acidophila (Deinum et al. 1991) although they both contain a B880, a B800-820 and a B800--850 complex. Therefore it was of interest to investigate the antenna organization in C. vinosum. The more recently discovered C. tepidum (Figure 1B) contains only one peripheral antenna, B800-850. Its core antenna, B920, absorbs at the unusually long wavelength of about 915 nm (Gareia et al. 1986; Nozawa et al. 1986), which makes it an interesting subject for studying energy transfer (Kennis et al. 1995) and antenna organization. In cultures ofRps, acidophila that contain a B800850 antenna it was observed that excitations could he transferred over a large number of core BChls (Deinum et al. 1991). Therefore the B800-850 complexes were
238 0.50
reaction center and its own complement of peripheral antenna.
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Materials and methods
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thought to connect several core units (each containing two reaction centers) resulting in a large domain size for transfer of excitations in the core antenna. For Rps. cryptolactis the situation is different. The presence of a B800-850 antenna did not result in a large domain size of the core antenna and it was assumed that the antenna consisted of separate core units (containing 2-3 reaction centers) each associated with its own peripheral antenna without an extensive interconnecting network (Kramer et al. 1995a). The same model was also appropriate for describing the organization in some LH2 mutants of Rb. sphaeroides (Kramer et al. 1995b). However, for a mutant with blue-shifted absorption in which both tyrosines in the a-subunit of LH2 had been replaced by other amino acids, a model similar to that proposed by Monger and Parson (1977) with large arrays of interconnected core-reaction center complexes was obtained. The results to be reported in this paper show that the situation in C. vinosum and C. tepidum is different from that in the above-mentioned species: in both Chromatium species domain sizes of the core units of only about 25 BChls were found, indicating that each reaction center-core complex contains only one
Cells of C. tepidum and C. vinosum were grown at a light intensity of 3500 lux at 50 °C and 30 °C, respectively (Madigan 1984; Cohen-Bazire et al. 1957). Membrane fragments, prepared by sonification, were suspended in a buffer containing 10 mM riffs (pH = 8.0). All measurements were performed under anaerobic conditions, obtained by addition of oxygen scavengers (glucose oxidase, catalase and glucose) and by purging the sample with N2. Fluorescence was measured by means of the spectrofluorimeter described elsewhere (Bakker et al. 1983). The sample was excited either by a frequencydoubled Nd:YAG laser with a maximum pulse energy of about 5 mJ/cm 2 at 532 nm and a pulse width of approximately 25 ps, or by a low intensity xenon flash with filters providing a band width of 40 nm, centered at 520 nm. Neutral density filters were used for varying the energy of the incident laser flash. For some measurements the reaction centers were kept in the oxidized state (P+) by means of continuous background illumination with a tungsten halogen lamp provided with a Schott BG-18 filter. The time integrated fluorescence at a single wavelength was detected by a RCA-30810 photodiode after passing a monochromator with a resolution set at 2 or 4 nm for fluorescence emission spectra and annihilation measurements, respectively. For fluorescence measurements upon laser excitation the incident laser energy was measured by splitting of part of the excitation light onto a calibrated RCA-30810 photodiode. Fluorescence excitation and absorption spectra were measured as described by Rijgersberg et al. (1980).
Results
Like in Rps. cryptolactis (Kramer et al. 1995a), Rps. acidophila (Deinum et al. 1991) and the double Tyr LH2 mutant of Rb. sphaeroides (Kramer et al. 1995b), measurements of variable fluorescence with C. vinosum revealed no increase of the fluorescence yield at wavelengths below 840 nm upon oxidation of the primary electron donor (Figure 2A). This indicates that there is no back transfer of energy to B800-820. The fluorescence increase in the B800-850 region was
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Figure 2. Fluorescence emission spectra of C. vinosum (A) and C. tepidum (B) with the primary donor in the reduced state (solid
Figure 3. Fluorescence emission spectra of C. vinosum (A) and C. tepidum (B) obtained with a weak xenon flash (solid lines) and with
lines) and in the oxidized state (broken lines), respectively. The dotted lines represent the ratios of the two spectra. For C. vinosum the ratio drops for wavelengths below 890 nm, indicating limited back transfer from the core to B800-850. No increase is observed for fluorescence originating from the B800-820 complex, indicating that back transfer of energy from the core to B 800-820 does not occur in C. vinosum. For C. tepidura the ratio is approximately independent of wavelength between 860 and 980 nm, indicating extensive back transfer from the core to B800-850.
somewhat lower than that of the core antenna, where it reached a maximum of about a factor 2.2. This might be an indication that energy equilibration between the B800-850 antenna and the core is not very fast. For C. tepidum the situation is different since the energy gap between B920 and B800-850 is considerably larger than that of B890 and B800-850 in C. vinosum. Nevertheless, the spectra in Figure 2B show that the increase of fluorescence in the B800-850 complex was not significantly different from that of B920. In spite of the large energy gap between peripheral and core antenna the increase with about a factor of 1.4 over the entire spectrum indicates that uphill energy transfer from the core to BChl 850 does occur. The lower fluorescence increase upon oxidation of the primary electron donor in C. tepidum compared to that obtained with C. vinosum is consistent with the observation that trapping of excitation energy by the reaction center in
an intense laser flash (broken lines), normalized at 940 and 975 rim, respectively. The dotted lines represent the difference of the two spectra. The primary donor was kept in the oxidized state by means of continuous background illumination.
C tepidum is slower than in C vinosum (Kennis et al. 1994; 1995). Fluorescence measurements obtained with an intense laser flash are shown in Figure 3. For both species annihilation was much more pronounced in the core antenna than in the peripheral antenna. The difference spectra suggest that annihilation effects in the core antennae are somewhat stronger in C. tepidum than in C. vinosum indicating a longer lifetime of excitations in the core antenna of C. tepidum compared to that of C. vinosum. This is in agreement with the results of Kennis et al. (1994, 1995) who observed time constants of 300 ps and 230 ps for trapping by closed reaction centers in C. tepidum and C. vinosum, respectively. For the purpose of obtaining information about the domain sizes in C. vinosum and C. tepidum we measured the fluorescence originating from the core antenna as a function of the incident laser intensity. Since part of the excitations will reach the core antenna via the peripheral antennae we also measured the annihilation curves for the peripheral antennae and cor-
240 rected the core annihilation curves for excitations lost by annihilation in the peripheral antennae. The corrected curves were obtained as described elsewhere (Kramer et al. 1995a, b). The distribution of excitations over the carotenoids associated with the core and peripheral antenna, respectively, was estimated from the absorption spectra. Fluorescence excitation spectra provided us with the energy transfer efficiencies from the carotenoid absorbing at 532 nm and from the peripheral antennae to the core (data not shown), used to analyze the annihilation data. BChl concentrations were calculated using extinction coefficients at the Qy absorption maximum of 140 m M - lcm- 1 for the core antenna (Clayton 1963; Francke and Amesz 1995) and of 184 m M - i c m - i for LH2 (Clayton and Clayton 1981). The number of BChl molecules per domain (ND) was calculated from the intensity at which the average number of excitations per domain equals 1. The efficiencies for energy transfer from the carotenoids absorbing at 532 nm to the BChl of the core were 30% and 25% for C. tepidura and C. vinosum, respectively. Energy transfer from B800--850 to B920 in C. tepidum occurred with an efficiency of about 90%. For C. vinosum relatively low efficiencies of 67% and 76% were obtained for energy transfer from B800-820 and B800-850 to B890, respectively. However, as will be shown below, the outcome of the analyses did not critically depend on these numbers. Figure 4A shows the annihilation curves of C. tepidum. The data were fitted with the equation of Paillotin et al. (1979). The curves at 870 nm and 940 nm could be fitted with r _> 5 and r -- 1, respectively, where r is the ratio of the mono-excitation decay rates for loss and trapping and the hi-excitation decay rate for annihilation. The uncorrected curve for 940 um resulted in a domain size of the core of 132 BChls. After correction, the 940 nm curve could be fitted with r = 0.1 and the number of BChls per domain decreased to 33 BChls (Figure 4B). For this correction it was estimated from the absorption spectrum that 70% of the excitations at 532 nm were absorbed by the carotenoids of the peripheral antenna. Measurements on C. vinosum were performed at 830 run, 860 nm and 920 nm. The annihilation curves are shown in Figure 5A. The curve at 830 nm could not be fitted adequately but a best fit with r = 5 is shown. The curves at 860 nm and 920 nm could be fitted with r >_ 5 and r - 0.7, respectively. The latter curve resulted in a domain size for the core antenna of about 52 BChls. For the purpose of correcting the data, a deconvolution of the absorption spectrum in the Qy
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Figure 4. (A) Fluorescence yield as function of the incident laser energy in C. tepidum, measured at 940 nm (closed circles) and 870 nm (open circles), fitted with r - 1 and r = 5, respectively. (I3) Fluorescence yield as function of the incident laser energy in C. tepidum, measured at 940 nm (dosed circles), and the same curve but now corrected for annihilation effects in B800-850 (open circles), fitted with r - 1 and r - 0.1 (see text), respectively. Continuous background illumination was used for keeping the primary donor in the oxidized state.
region (Kennis et al. 1995) was used to estimate the relative amounts of the three antenna complexes. From this, it was estimated that the excitations were initially distributed over the carotenoids of B800-820, B800850 and B890 in a ratio of 1 : 0.5 : 0.7. We further assumed that excitations on B800-820 are transferred solely to B800-850 and not directly to B890 (Kennis et al. 1995). The corrected curve (Figure 5B) could now be fitted with r - 0 and a very small domain size of about 18 BChls was obtained. This number was essentially independent of the efficiency of energy transfer from the peripheral antennae to B890 in the range of 50% to 100% and a significant increase was only obtained at lower efficiencies. Since the estimates of the initial distributions of excitations over the carotenoids associated with the peripheral and core antennae from the absorption spectra may not be entirely accurate, we in addition calculated and analyzed corrected curves for various distributions. The results are summarized in Table 1. The domain sizes obtained with C. tepidum showed no sig-
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Figure 5. (A) Fluorescenceyield as functionof the incidentlaser energy in C. vinosum, measured at 920 nm (closed circles), 860 nm (open circles) and 830 nm (triangles),fittedwith r = 0.7, r = 5 and r = 5, respectively.(B) Fluorescenceyield as functionof the incident laser energy in C. vinosum, measuredat 920 nm (closedcircles) and the same curve but now corrected for annihilationeffects in B800820 and B800-850 (open circles), fitted with r = 1 and r = 0 (see text), respectively. Continuousbackground illuminationwas used for keeping the primarydonor in the oxidized state.
nificant deviation with the 33 BChls mentioned earlier, provided that the LH2 antenna contributes for more than 60% to the initial excitation distribution, which seems to be a reasonable assumption. For C. vinosum we assumed that the ratio of the amount of carotenoids associated with B 8 0 0 - 8 2 0 to that of 800-850 was 2 : 1. Virtually the same results, i.e. the same domain sizes of about 20 BChls, were obtained when the number of excitations that initially arrive at the carotenoids associated with the LH2 antennae was varied between 50 and 80%.
Discussion
From our measurements we conclude that the B890 units in C. vinosum and the B920 units in C. tepidum appear to correspond to one photosynthetic unit of about 25 BChls (Francke and Amesz 1995) only. Since in both C. tepidum and C. vinosum back transfer of energy from the core BChls to B800-850 was observed
The data were obtainedfrom analysesof the annihilationcurvesobtainedwith C tepidum at 940 nm and C vinosum at 920 tun after correctionfor annihilationin the peripheral antennae.The corrections were calculatedfor differentinitialdistributionsof excitationsoverthe carotenoidsassociatedwith the peripheral (LH2) and core (LH1) antenna,respectively. We assumed energy transfer efficienciesof 30 and 90% for energytransferfrom carotenoidand from B800-850 to B920 in C tepidum and of 25, 67 and76% for energytransferfromthe carotenoid, B800-820 and B800-850 to B890 in C vinosum, respectively.. For C vinosum we assumedthat the ratio of the amountof carotenoidsassociated with B800-820 to that of B800-850 was 2 : 1. The bold numbers refer to the curves in Figures 4B and 5B for C tepidum and C vinosum, respectively.
to occur we have to assume that these peripheral antennae do not form an interconnecting network. This would imply that in both species the antenna consists of separate photosynthetic units surrounded by their own complement of B800-850 antenna. For C. vinosum, however, it cannot be ruled out that the B 8 0 0 - 8 2 0 antenna is energetically connected to several reaction center-core units. The results reported here, in earlier work (Kramer et al. 1995a, b), and by Deinum et al. (1991) indicate that the organization of the antenna system in various species of purple bacteria may be quite different, although the basic components are very similar. The various types of organization encountered in these studies are shown schematically in Figure 6. The recent X-ray studies of McDermott et al. (1995) have made it possible to draw somewhat more realistic pictures than was earlier possible (Deinum et al. 1991). Determination of the three-dimensional structure of crystals of the B800-850 complex of Rps. acidophila have shown a ring-like structure containing 18 BChls 850, 9 BChls 800 and 9 a and ~-polypeptides. Electron diffraction of 2-dimensional crystals indicates a similar structure for the LH2 complex of Rhodovulum
242
A
B
C
D
Figure 6. Models describing schematically the different antenna configurations of the various species studied so far. Reaction centers are represented by black circles, core complexes by dark tings and LH2 complexes by white tings. Model (A) represents C. tepidum, model (B) Rps. cryptolactis, grown at high light intensity (Kramer et al. 1995a), model (C) Rps. acidophila strain 7750HT (Deinum et al. 1991) and model (D) the double Tyr LH2 mutant of Rb. sphaeroides (Kramer et al. 1995b). The amount of peripheral BChls was estimated from the absorption spectra. As discussed in the text, these models, with various core-LH2 ratios, also apply to other species and strains studied.
sulfidophilum (Savage et al. 1995), and optical studies of Kennis et al. (1996) indicate a similar structure for the solubilized B800-850 complex of C. tepidum. These observations suggest that a structure identical or similar to that observed in the crystal (McDermott et al. 1995) is also present in the membrane of various species of purple bacteria. It should be noted, however, that the number of BChl 800 molecules in the C. tepidum complex must be even (Kennis et al.
1996; Nozawa et al. 1986), implying that the number of BChls 850 must be a multiple of four, e.g. 16, as in Rhodospirillum molischianum (Kleinekofort et al. 1992) and Ectothiorhodospira sp. (Oling et al. 1996). There is evidence, though less detailed, that the core complex surrounds the reaction center with a similar ring-like structure (Meckenstock et al. 1994; Boonstra et al. 1994; Karrash et al. 1995).
243 In d i s t i n g u i s h i n g the various structures depicted in Figure 6 one should keep in m i n d that these structures m a y not be solely d e p e n d e n t on the species and strains b u t also on the growth conditions applied, which were widely different for the various samples studied. Nevertheless it is o f interest to note that the relatively simple m o d e l o b t a i n e d for Rps. acidophila, with interc o n n e c t i n g L H 2 networks and m o r e or less isolated core-reaction center complexes ( D e i n u m et al. 1991), does not apply to any o f the other strains and species studied. The original m o d e l of M o n g e r and Parson (1977), with large arrays o f connected core-reaction center complexes, appears to apply only to the double Tyr L H 2 m u t a n t o f Rb. sphaeroides, which has a relatively small a m o u n t of peripheral a n t e n n a BChls ( K r a m e r et al. 1995b) and to Rhodospirillum rubrum which has no peripheral a n t e n n a at all (Vos et al. 1986). Isolated arrays o f core-reaction center-LH2 complexes c o n t a i n i n g one or m o r e reaction centers are found in both Chromatium species, in Rps. cryptolactis (Kramer et al. 1995a) and in the pseudo wild type and single Tyr L H 2 m u t a n t s of Rb. sphaeroides ( K r a m e r et al. 1995b).
Acknowledgments The investigation was supported by the Netherlands F o u n d a t i o n for Scientific Research ( N W O ) via the F o u n d a t i o n s for Life Sciences (SLW) and for Chemical Research (SON) and by the E u r o p e a n C o m m u n i t y .
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mons DW (eds) Chlorophyll Organization and Energy Transfer in Photosynthesis, pp 61-73. CIBA Symp 61, new series. Excerpta Medica, Amsterdam Cohen-Bazire G, Sistrom WR and Stanier RY (1957) Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J Cell Comp Physiol 49:25-68 Deinum G, Otte SCM, Gardiner AT, Aartsma TJ, Cogdell 1LI and Amesz J (1991) Antenna organization of Rhodopseudomonas acidophila: a study of the excitation migration. Biochim Biophys Acta 1060:125-131 Francke C and Amesz J (1995) The size of the photosynthetic unit in purple bacteria. Photosynth Res 46:347-352 Garcia D, Parot P, Vermtglio A and Madigan MT (1986) The lightharvesting complexes of a thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. Biochim Biophys Acta 850:390-395 Hayashi H, Nozawa T, Hatano M and Morita S (1981) Circular dichroism of bacteriochlorophyll a in light harvesting bacteriochlorophyll protein complexes from Chromatium vinosum. J Biochem (Japan) 89:1853-1861 Karrash S, Bullough PA and Ghosh R (1995) The 8.5 /~ projection map of the light harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 14: 631-638 Kennis JTM, Aartsma TJ and Amesz J (1994) Energy trapping in the purple sulfur bacteria Chromatium vinosum and Chromatium tepidum. Biochim Biophys Acta 1188:278-286 Kennis JTM, Aartsma TJ and Amesz J (1995) Energy transfer between antenna complexes in the purple sulfur bacteria Chromatium tepidumand Chromatiumvinosum. Chem Phys 194: 285289 Kennis JTM, Streltsov AM, Aartsma TJ, Nozawa T and Amesz J (1996) Energy transfer and exciton coupling in isolated B800850 complexes of the photosynthetic purple sulfur bacterium C. tepidum. The effect of structural symmetry on bacteriochlorophyU excited states. J Phys Chem 100:2438-2442 Kleinekofort W, Germeroth L, van den Brock JA, Schubert D and Michel H (1992) The light-harvesting complex II (B800/850) from RhodospiriUummolischianum is an octamer. Biochim Binphys Acta 1140:102-104 Kramer H, Deinum G, Gardiner AT, Cogdell RJ, Aartsma TJ and Amesz J (1995a) Energy transfer in the photosynthetic antenna system of the purple non-sulfur bacterium Rhodopseudomonas cryptolactis. Biochim Biophys Acta 1231:33--40 Kramer H, Jones MR, Fowler GJS, Francke C, Aartsma TJ, Hunter CN and Amesz J (1995b) Energy migration in Rhodobacter sphaeroides mutants altered by mutagenesis of the peripheral LH2 complex or by removal of the core LH1 complex. Biochim Biophys Acta 1231: 89-97 Madigan MT (1984) A novel photosynthetic purple bacterium isolated from a Yellowstone hot spring. Science 225:313-315 McDermott G, Prince SM, Freer AA, Hawthornthwaite-Lawless AM, Papiz MZ, Cogdell ILl and Isaacs NW (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374:517-521 Meckenstock RU, Krusche K, Staehelin LA, Cyrklaff M and Zuber H (1994) The six fold symmetry of the B880 light harvesting complex and the structure of the photosynthetic membranes of Rhodopseudomonas marina. J Biol Chem 375:429-438 Monger TG and Parson WW (1977) Singlet-triplet fusion in Rhodopseudomonassphaeroideschromatophores. A probe of the organization of the photosynthetic apparatus. Biochim Biophys Acta 460:393--407
244 Nozawa T, Fukada T, Hatano M and Madigan MT (1986) Organization of intracytoplasmic membranes in a novel thermophilic purple photosynthetic bacterium as revealed by absorption, circulax dichroism and emission spectra. Biochim Biophys Acta 852: 191-197 Oling E Boekema EJ, de Zarate IO, Visschers R, van Gmndelle R, Keegstra W, Bdsson A and Picorel R (1996) Two-dimensional crystals of LH2 light-harvesting complexes from Ectothiorhodospira sp. and Rhodobacter capsulatus investigated by electron microscopy. Biochim Biophys Acta 1273:44-50 Paillotin G, Swenberg CE, Breton J and Geacintov NE (1979) Analysis of picosecond laser-induced fluorescence phenomena in photosynthetic membranes utilizing a master equation approach. Biophys J 25:512-533
Rijgersherg CE van Grondelle R and Amesz J (1980) Energy transfer and bacteriochlorophyU fluorescence in purple bacteria at low temperature. Biochim Biophys Acta 592:53--64 Savage H, Cyrklaff M, Montoya G, K01dbrandt W and Sinning I (1995) Two dimensional crystallization and structure determination of LHII from Rhv. sulfutophilum at 7A, resolution. In: Mathis P (ed) Photosynthesis: From Light To Biosphere, Vol I, pp 239242. Kluwer Academic Publishers, Dordrecht Vos M, van Gmndelle R, van der Kooij FW, van de Pol D, Amesz J and Duysans LNM (1986) Singlet-singlet annihilation at low temperatures in the antenna of purple bacteria. Biochim Biophys Acta 850:501-512
Regular paper
Antenna organization in the purple sulfur bacteria Chromatium tepidum and
Chromatium vinosum Hans Kramer& Jan Amesz* Department of Biophysics, University of Leiden, P.O. Box 9504, 2300 RA Leiden, The Netherlands; *Authorfor correspondence Received 20 March 1996; accepted in revised form 23 July 1996
Key words: light harvesting, singlet-singlet annihilation, domain size, fluorescence (C. tepidum, C. vinosum)
Abstract Structural aspects of the core antenna in the purple sulfur bacteria Chromatium tepidum and Chromatium vinosum were studied by means of fluorescence emission and singlet-singlet annihilation measurements. In both species the number of bacteriochlorophylls of the core antenna between which energy transfer can occur corresponds to one core-reaction center complex only. From measurements of variable fluorescence we conclude that in C, tepidum excitation energy can be transferred back from the core antenna (B920) to the peripheral B800--850 complex in spite of the relatively large energy gap, and on basis of annihilation measurements a model of separate core-reaction center units accompanied by their own peripheral antenna is suggested. C. vinosum contains besides a core antenna, B890, two peripheral antennae, B800-820 and B800-850. Energy transfer was found to occur from the core to B800-850, but not to B800-820, and it was concluded that in C. vinosum each core-reaction center complex has its own complement of B800-850. The results reported here are compared to those obtained earlier with various strains and species of purple non-sulfur bacteria.
Abbreviations: BChl-bacteriochlorophyll; B800-820 and B800-850- antenna complexes with Qy-band absorption maxima near 800 nm and 820 or 850 rim, respectively; B890 and B920-antenna complexes with Qy-band absorption maxima near 890 and 920 nm, respectively; L H l - l i g h t harvesting 1 or core antenna; LH2-1ight harvesting 2 or peripheral antenna Introduction The purple sulfur bacteria Chromatium (C.) vinosum and C. tepidum show clear differences with respect to their spectral properties and antenna composition. C. vinosum resembles species like Rhodopseudomonas (Rps.) cryptolactis (Kramer et al. 1995a) and Rps. acidophila (Deinum et al. 1991) with respect to the location of the Qy absorption bands of the peripheral antenna BChls. Its absorption spectrum is shown in Figure 1A. In addition to a core antenna, B890, it possesses two peripheral antennae, B800-850 and B800-820 (Cogdell and Thornber 1979; Hayashi et al. 1981). It was earlier observed that the organization of the antenna in Rps. cryptolactis (Kramer et al. 1995a) differs
from that of Rps. acidophila (Deinum et al. 1991) although they both contain a B880, a B800-820 and a B800--850 complex. Therefore it was of interest to investigate the antenna organization in C. vinosum. The more recently discovered C. tepidum (Figure 1B) contains only one peripheral antenna, B800-850. Its core antenna, B920, absorbs at the unusually long wavelength of about 915 nm (Gareia et al. 1986; Nozawa et al. 1986), which makes it an interesting subject for studying energy transfer (Kennis et al. 1995) and antenna organization. In cultures ofRps, acidophila that contain a B800850 antenna it was observed that excitations could he transferred over a large number of core BChls (Deinum et al. 1991). Therefore the B800-850 complexes were
238 0.50
reaction center and its own complement of peripheral antenna.
807
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Materials and methods
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Wavelength (nm) Figure 1. Absorption spectra of membrane fragments of C vinosum (A) and C. tepidum (B) measured at room temperature.
thought to connect several core units (each containing two reaction centers) resulting in a large domain size for transfer of excitations in the core antenna. For Rps. cryptolactis the situation is different. The presence of a B800-850 antenna did not result in a large domain size of the core antenna and it was assumed that the antenna consisted of separate core units (containing 2-3 reaction centers) each associated with its own peripheral antenna without an extensive interconnecting network (Kramer et al. 1995a). The same model was also appropriate for describing the organization in some LH2 mutants of Rb. sphaeroides (Kramer et al. 1995b). However, for a mutant with blue-shifted absorption in which both tyrosines in the a-subunit of LH2 had been replaced by other amino acids, a model similar to that proposed by Monger and Parson (1977) with large arrays of interconnected core-reaction center complexes was obtained. The results to be reported in this paper show that the situation in C. vinosum and C. tepidum is different from that in the above-mentioned species: in both Chromatium species domain sizes of the core units of only about 25 BChls were found, indicating that each reaction center-core complex contains only one
Cells of C. tepidum and C. vinosum were grown at a light intensity of 3500 lux at 50 °C and 30 °C, respectively (Madigan 1984; Cohen-Bazire et al. 1957). Membrane fragments, prepared by sonification, were suspended in a buffer containing 10 mM riffs (pH = 8.0). All measurements were performed under anaerobic conditions, obtained by addition of oxygen scavengers (glucose oxidase, catalase and glucose) and by purging the sample with N2. Fluorescence was measured by means of the spectrofluorimeter described elsewhere (Bakker et al. 1983). The sample was excited either by a frequencydoubled Nd:YAG laser with a maximum pulse energy of about 5 mJ/cm 2 at 532 nm and a pulse width of approximately 25 ps, or by a low intensity xenon flash with filters providing a band width of 40 nm, centered at 520 nm. Neutral density filters were used for varying the energy of the incident laser flash. For some measurements the reaction centers were kept in the oxidized state (P+) by means of continuous background illumination with a tungsten halogen lamp provided with a Schott BG-18 filter. The time integrated fluorescence at a single wavelength was detected by a RCA-30810 photodiode after passing a monochromator with a resolution set at 2 or 4 nm for fluorescence emission spectra and annihilation measurements, respectively. For fluorescence measurements upon laser excitation the incident laser energy was measured by splitting of part of the excitation light onto a calibrated RCA-30810 photodiode. Fluorescence excitation and absorption spectra were measured as described by Rijgersberg et al. (1980).
Results
Like in Rps. cryptolactis (Kramer et al. 1995a), Rps. acidophila (Deinum et al. 1991) and the double Tyr LH2 mutant of Rb. sphaeroides (Kramer et al. 1995b), measurements of variable fluorescence with C. vinosum revealed no increase of the fluorescence yield at wavelengths below 840 nm upon oxidation of the primary electron donor (Figure 2A). This indicates that there is no back transfer of energy to B800-820. The fluorescence increase in the B800-850 region was
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Figure 2. Fluorescence emission spectra of C. vinosum (A) and C. tepidum (B) with the primary donor in the reduced state (solid
Figure 3. Fluorescence emission spectra of C. vinosum (A) and C. tepidum (B) obtained with a weak xenon flash (solid lines) and with
lines) and in the oxidized state (broken lines), respectively. The dotted lines represent the ratios of the two spectra. For C. vinosum the ratio drops for wavelengths below 890 nm, indicating limited back transfer from the core to B800-850. No increase is observed for fluorescence originating from the B800-820 complex, indicating that back transfer of energy from the core to B 800-820 does not occur in C. vinosum. For C. tepidura the ratio is approximately independent of wavelength between 860 and 980 nm, indicating extensive back transfer from the core to B800-850.
somewhat lower than that of the core antenna, where it reached a maximum of about a factor 2.2. This might be an indication that energy equilibration between the B800-850 antenna and the core is not very fast. For C. tepidum the situation is different since the energy gap between B920 and B800-850 is considerably larger than that of B890 and B800-850 in C. vinosum. Nevertheless, the spectra in Figure 2B show that the increase of fluorescence in the B800-850 complex was not significantly different from that of B920. In spite of the large energy gap between peripheral and core antenna the increase with about a factor of 1.4 over the entire spectrum indicates that uphill energy transfer from the core to BChl 850 does occur. The lower fluorescence increase upon oxidation of the primary electron donor in C. tepidum compared to that obtained with C. vinosum is consistent with the observation that trapping of excitation energy by the reaction center in
an intense laser flash (broken lines), normalized at 940 and 975 rim, respectively. The dotted lines represent the difference of the two spectra. The primary donor was kept in the oxidized state by means of continuous background illumination.
C tepidum is slower than in C vinosum (Kennis et al. 1994; 1995). Fluorescence measurements obtained with an intense laser flash are shown in Figure 3. For both species annihilation was much more pronounced in the core antenna than in the peripheral antenna. The difference spectra suggest that annihilation effects in the core antennae are somewhat stronger in C. tepidum than in C. vinosum indicating a longer lifetime of excitations in the core antenna of C. tepidum compared to that of C. vinosum. This is in agreement with the results of Kennis et al. (1994, 1995) who observed time constants of 300 ps and 230 ps for trapping by closed reaction centers in C. tepidum and C. vinosum, respectively. For the purpose of obtaining information about the domain sizes in C. vinosum and C. tepidum we measured the fluorescence originating from the core antenna as a function of the incident laser intensity. Since part of the excitations will reach the core antenna via the peripheral antennae we also measured the annihilation curves for the peripheral antennae and cor-
240 rected the core annihilation curves for excitations lost by annihilation in the peripheral antennae. The corrected curves were obtained as described elsewhere (Kramer et al. 1995a, b). The distribution of excitations over the carotenoids associated with the core and peripheral antenna, respectively, was estimated from the absorption spectra. Fluorescence excitation spectra provided us with the energy transfer efficiencies from the carotenoid absorbing at 532 nm and from the peripheral antennae to the core (data not shown), used to analyze the annihilation data. BChl concentrations were calculated using extinction coefficients at the Qy absorption maximum of 140 m M - lcm- 1 for the core antenna (Clayton 1963; Francke and Amesz 1995) and of 184 m M - i c m - i for LH2 (Clayton and Clayton 1981). The number of BChl molecules per domain (ND) was calculated from the intensity at which the average number of excitations per domain equals 1. The efficiencies for energy transfer from the carotenoids absorbing at 532 nm to the BChl of the core were 30% and 25% for C. tepidura and C. vinosum, respectively. Energy transfer from B800--850 to B920 in C. tepidum occurred with an efficiency of about 90%. For C. vinosum relatively low efficiencies of 67% and 76% were obtained for energy transfer from B800-820 and B800-850 to B890, respectively. However, as will be shown below, the outcome of the analyses did not critically depend on these numbers. Figure 4A shows the annihilation curves of C. tepidum. The data were fitted with the equation of Paillotin et al. (1979). The curves at 870 nm and 940 nm could be fitted with r _> 5 and r -- 1, respectively, where r is the ratio of the mono-excitation decay rates for loss and trapping and the hi-excitation decay rate for annihilation. The uncorrected curve for 940 um resulted in a domain size of the core of 132 BChls. After correction, the 940 nm curve could be fitted with r = 0.1 and the number of BChls per domain decreased to 33 BChls (Figure 4B). For this correction it was estimated from the absorption spectrum that 70% of the excitations at 532 nm were absorbed by the carotenoids of the peripheral antenna. Measurements on C. vinosum were performed at 830 run, 860 nm and 920 nm. The annihilation curves are shown in Figure 5A. The curve at 830 nm could not be fitted adequately but a best fit with r = 5 is shown. The curves at 860 nm and 920 nm could be fitted with r >_ 5 and r - 0.7, respectively. The latter curve resulted in a domain size for the core antenna of about 52 BChls. For the purpose of correcting the data, a deconvolution of the absorption spectrum in the Qy
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Figure 4. (A) Fluorescence yield as function of the incident laser energy in C. tepidum, measured at 940 nm (closed circles) and 870 nm (open circles), fitted with r - 1 and r = 5, respectively. (I3) Fluorescence yield as function of the incident laser energy in C. tepidum, measured at 940 nm (dosed circles), and the same curve but now corrected for annihilation effects in B800-850 (open circles), fitted with r - 1 and r - 0.1 (see text), respectively. Continuous background illumination was used for keeping the primary donor in the oxidized state.
region (Kennis et al. 1995) was used to estimate the relative amounts of the three antenna complexes. From this, it was estimated that the excitations were initially distributed over the carotenoids of B800-820, B800850 and B890 in a ratio of 1 : 0.5 : 0.7. We further assumed that excitations on B800-820 are transferred solely to B800-850 and not directly to B890 (Kennis et al. 1995). The corrected curve (Figure 5B) could now be fitted with r - 0 and a very small domain size of about 18 BChls was obtained. This number was essentially independent of the efficiency of energy transfer from the peripheral antennae to B890 in the range of 50% to 100% and a significant increase was only obtained at lower efficiencies. Since the estimates of the initial distributions of excitations over the carotenoids associated with the peripheral and core antennae from the absorption spectra may not be entirely accurate, we in addition calculated and analyzed corrected curves for various distributions. The results are summarized in Table 1. The domain sizes obtained with C. tepidum showed no sig-
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Figure 5. (A) Fluorescenceyield as functionof the incidentlaser energy in C. vinosum, measured at 920 nm (closed circles), 860 nm (open circles) and 830 nm (triangles),fittedwith r = 0.7, r = 5 and r = 5, respectively.(B) Fluorescenceyield as functionof the incident laser energy in C. vinosum, measuredat 920 nm (closedcircles) and the same curve but now corrected for annihilationeffects in B800820 and B800-850 (open circles), fitted with r = 1 and r = 0 (see text), respectively. Continuousbackground illuminationwas used for keeping the primarydonor in the oxidized state.
nificant deviation with the 33 BChls mentioned earlier, provided that the LH2 antenna contributes for more than 60% to the initial excitation distribution, which seems to be a reasonable assumption. For C. vinosum we assumed that the ratio of the amount of carotenoids associated with B 8 0 0 - 8 2 0 to that of 800-850 was 2 : 1. Virtually the same results, i.e. the same domain sizes of about 20 BChls, were obtained when the number of excitations that initially arrive at the carotenoids associated with the LH2 antennae was varied between 50 and 80%.
Discussion
From our measurements we conclude that the B890 units in C. vinosum and the B920 units in C. tepidum appear to correspond to one photosynthetic unit of about 25 BChls (Francke and Amesz 1995) only. Since in both C. tepidum and C. vinosum back transfer of energy from the core BChls to B800-850 was observed
The data were obtainedfrom analysesof the annihilationcurvesobtainedwith C tepidum at 940 nm and C vinosum at 920 tun after correctionfor annihilationin the peripheral antennae.The corrections were calculatedfor differentinitialdistributionsof excitationsoverthe carotenoidsassociatedwith the peripheral (LH2) and core (LH1) antenna,respectively. We assumed energy transfer efficienciesof 30 and 90% for energytransferfrom carotenoidand from B800-850 to B920 in C tepidum and of 25, 67 and76% for energytransferfromthe carotenoid, B800-820 and B800-850 to B890 in C vinosum, respectively.. For C vinosum we assumedthat the ratio of the amountof carotenoidsassociated with B800-820 to that of B800-850 was 2 : 1. The bold numbers refer to the curves in Figures 4B and 5B for C tepidum and C vinosum, respectively.
to occur we have to assume that these peripheral antennae do not form an interconnecting network. This would imply that in both species the antenna consists of separate photosynthetic units surrounded by their own complement of B800-850 antenna. For C. vinosum, however, it cannot be ruled out that the B 8 0 0 - 8 2 0 antenna is energetically connected to several reaction center-core units. The results reported here, in earlier work (Kramer et al. 1995a, b), and by Deinum et al. (1991) indicate that the organization of the antenna system in various species of purple bacteria may be quite different, although the basic components are very similar. The various types of organization encountered in these studies are shown schematically in Figure 6. The recent X-ray studies of McDermott et al. (1995) have made it possible to draw somewhat more realistic pictures than was earlier possible (Deinum et al. 1991). Determination of the three-dimensional structure of crystals of the B800-850 complex of Rps. acidophila have shown a ring-like structure containing 18 BChls 850, 9 BChls 800 and 9 a and ~-polypeptides. Electron diffraction of 2-dimensional crystals indicates a similar structure for the LH2 complex of Rhodovulum
242
A
B
C
D
Figure 6. Models describing schematically the different antenna configurations of the various species studied so far. Reaction centers are represented by black circles, core complexes by dark tings and LH2 complexes by white tings. Model (A) represents C. tepidum, model (B) Rps. cryptolactis, grown at high light intensity (Kramer et al. 1995a), model (C) Rps. acidophila strain 7750HT (Deinum et al. 1991) and model (D) the double Tyr LH2 mutant of Rb. sphaeroides (Kramer et al. 1995b). The amount of peripheral BChls was estimated from the absorption spectra. As discussed in the text, these models, with various core-LH2 ratios, also apply to other species and strains studied.
sulfidophilum (Savage et al. 1995), and optical studies of Kennis et al. (1996) indicate a similar structure for the solubilized B800-850 complex of C. tepidum. These observations suggest that a structure identical or similar to that observed in the crystal (McDermott et al. 1995) is also present in the membrane of various species of purple bacteria. It should be noted, however, that the number of BChl 800 molecules in the C. tepidum complex must be even (Kennis et al.
1996; Nozawa et al. 1986), implying that the number of BChls 850 must be a multiple of four, e.g. 16, as in Rhodospirillum molischianum (Kleinekofort et al. 1992) and Ectothiorhodospira sp. (Oling et al. 1996). There is evidence, though less detailed, that the core complex surrounds the reaction center with a similar ring-like structure (Meckenstock et al. 1994; Boonstra et al. 1994; Karrash et al. 1995).
243 In d i s t i n g u i s h i n g the various structures depicted in Figure 6 one should keep in m i n d that these structures m a y not be solely d e p e n d e n t on the species and strains b u t also on the growth conditions applied, which were widely different for the various samples studied. Nevertheless it is o f interest to note that the relatively simple m o d e l o b t a i n e d for Rps. acidophila, with interc o n n e c t i n g L H 2 networks and m o r e or less isolated core-reaction center complexes ( D e i n u m et al. 1991), does not apply to any o f the other strains and species studied. The original m o d e l of M o n g e r and Parson (1977), with large arrays o f connected core-reaction center complexes, appears to apply only to the double Tyr L H 2 m u t a n t o f Rb. sphaeroides, which has a relatively small a m o u n t of peripheral a n t e n n a BChls ( K r a m e r et al. 1995b) and to Rhodospirillum rubrum which has no peripheral a n t e n n a at all (Vos et al. 1986). Isolated arrays o f core-reaction center-LH2 complexes c o n t a i n i n g one or m o r e reaction centers are found in both Chromatium species, in Rps. cryptolactis (Kramer et al. 1995a) and in the pseudo wild type and single Tyr L H 2 m u t a n t s of Rb. sphaeroides ( K r a m e r et al. 1995b).
Acknowledgments The investigation was supported by the Netherlands F o u n d a t i o n for Scientific Research ( N W O ) via the F o u n d a t i o n s for Life Sciences (SLW) and for Chemical Research (SON) and by the E u r o p e a n C o m m u n i t y .
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