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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We compared the optical properties of the trimeric photosystem (PS) I complexes of the primordial cyanobacterium Gloeobacter violaceus PCC 7421 with those of Synechocystis sp. PCC 6803. Gloeobacter violaceus PS I showed (1) a shorter difference maximum of P700 by approximately 2 nm, (2) a smaller antenna size by approximately 10 chlorophyll (Chl) a molecules and (3) an absence of Red Chls. The energy transfer kinetics in the antennae at physiological temperatures were very similar between the two species due to the thermal equilibrium within the antenna; however, they differed at 77 K where energy transfer to Red Chls was clearly observed in Synechocystis sp. PCC 6803. Taken together with the lower P700 redox potential in G. violaceus by approximately 60 mV, we discuss differences in the optical properties of the PS I complexes with respect to the amino acid sequences of core proteins and further to evolution of cyanobacteria.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Most of the primary production on earth is carried out by oxygenic photosynthetic organisms using sunlight as a source of reducing energy and water as a source of electrons (1). Oxygen, a product of water cleavage by oxygenic photosynthesis, is in turn used as the terminal acceptor of electrons in oxygenic respiration, thus completing the energy cycle. In terms of the primary production, oxygenic photosynthetic organisms are referred to as “a safety net on the earth.” Cyanobacteria were the first organisms to perform oxygenic photosynthesis. Their reaction center (RC) complexes have been inherited from anoxygenic photosynthetic bacteria: a core of PS II was inherited from the RC of purple bacteria, and that of PS I from the RC of green sulfur bacteria or heliobacteria (2). However, many polypeptides associating with the core complexes in oxygenic photosynthesis were acquired after the evolution of cyanobacteria. The most significant evolutionary innovation was the development of the machinery for water oxidation in PS II (3); however, the origins of this machinery have not yet been resolved, although many approaches have been taken to attempt to address this question. The number of PS I subunits is increased in cyanobacteria compared to green sulfur bacteria; however, the electron transfer components are not very much changed (1,2). This conservation may arise from the requirement of a very low redox potential for the synthesis of NADPH (reduced form of nicotinamide adenine dinucleotide phosphate [NADP+]) for CO2 fixation. However, changing the pigment from bacteriochlorophyll a to chlorophyll (Chl) a induced a change in the redox potential of the primary electron donor and accompanying changes in the redox potentials of the electron transfer components.

There are more than 2000 species of cyanobacteria (4). Among these, certain species, including thermophilic cyanobacteria, are useful for structural biology studies based on the thermal stability of their components (5–7), and other species, such as Synechocystis sp. PCC 6803, are useful for molecular biology studies due to the ease with which these species can be transformed (8). However, to study bacterial evolution, other species may be more appropriate. Gloeobacter violaceus PCC 7421 is one candidate for use in evolutionary studies (9). This species appears to be part of the group of cyanobacteria that branched off at the earliest stage in the cyanobacteria lineage based on its small subunit rDNA sequence (10). Its branching point is earlier than that for primary symbiosis to chloroplasts, and therefore, this species might retain many primordial properties. This species does not have any intracellular membrane, such as a thylakoid membrane (11), and is the only cyanobacterium known to lack this characteristic. Its complete genome sequence was reported in 2003 (12); it revealed many unique properties of this organism, including differences in the amino acid sequences of many polypeptides. This organism, therefore, is a good model in which to study the progress of photosynthetic reaction components and processes from an evolutionary perspective.

The properties of PS I in G. violaceus are very different from those in other cyanobacteria; for example, the G. violaceus PS I consists of nine subunits (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaL, PsaM and PsaZ), and four subunits commonly found in other species (PsaI, PsaJ, PsaK and PsaX) are missing (13). In their stead, a new subunit, PsaZ, is found in this bacterium (13). The secondary electron acceptor, a quinone molecule, is menaquinone (MQ-4) (14), instead of phylloquinone, which is found in other oxygenic photosynthetic organisms. MQ-4 is found in photosynthetic bacteria, and G. violaceus may have a novel biosynthetic pathway for this molecule, because several genes for its synthetic enzymes in other bacteria are missing in G. violaceus. Another significant difference is the redox potential of the primary electron donor, i.e. P700. Nakamura et al. have recently designed an opto-electrical electrode, which allows for accuracy within a few mV, and measured the potential of P700 (15). The redox potential of P700 in G. violaceus was reported to be 398 ± 4 mV at room temperature, significantly lower than that of Synechocystis sp. PCC 6803 (455 ± 4 mV) (16). Furthermore, the typical PS I fluorescence observed at approximately 725 nm at 77 K was missing in G. violaceus (17,18). These differences might be a result of the evolutionary position of this organism, and may be linked to the development of photosynthetic reaction processes.

Based on these findings, we sought to assess the basic properties of PS I in G. violaceus, particularly its optical properties related to the development of photosynthetic reaction systems and the reaction mechanisms from bacterial photosynthesis. We also performed a comparative study using Synechocystis sp. PCC 6803.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Algal culture. Gloeobacter violaceus PCC 7421 and Synechocystis sp. PCC 6803 were grown photosynthetically in BG11 medium (19) at 298 K. The light source was a fluorescent lamp with intensities of 5 μmole photon m−2 s−1 and 20 μmole photon m−2 s−1, respectively. Air was continuously supplied through an air filter (Myrex, Millipore).

Isolation of photosystem I complexes.  Photosystem I complexes were isolated according to previously described methods (13). Briefly, PS I complexes were solubilized from membranes with dodecyl-β-d-maltoside (final concentration, 0.75% [wt/vol]) and purified by sucrose density gradient centrifugation; the trimer fraction was selectively collected.

Steady state optical measurements.  Absorption spectra at 80 K were measured with a Hitachi 557 spectrophotometer (Hitachi, Japan) equipped with a commercially available Dewar bottle attachment. Fluorescence spectra were measured at 77 K with a Hitachi 850 spectrofluorometer. For low-temperature spectra, a custom-made Dewar system was used (18). Polyethylene glycol (average molecular weight 3350; Sigma-Aldrich) was added to obtain homogeneous ice at 77 K (final concentration 15% [wt/vol]). The spectral sensitivity of the fluorometer was corrected by using a substandard lamp (Hitachi) with a known radiation profile.

Time-resolved fluorescence spectrum.  The time-resolved fluorescence spectrum (TRFS) was measured by a time-correlated single photon counting method at 295 K and 77 K (18,20). For low-temperature measurements, a custom-made Dewar bottle was used. The light source was a Ti:sapphire laser with an excitation wavelength of 400 nm. The time interval of data acquisition was controlled at between 2.6 and 51.2 ps per channel, using a time-to-amplitude converter. Time zero was set as the time when the amplitude of the excitation beam was highest on the time-to-amplitude converter. Excitation pulses were detected as a pulse with a given time width, typically 30 ps, and therefore times with negative values resulted. We measured TRFS for a given wavelength region and decay curves at several discrete wavelengths.

Data processing.  Fluorescence lifetimes were estimated by a convolution calculation (21). The low-temperature fluorescence spectra were resolved to components by global analysis. Prior to global analysis, single value decomposition (SVD) analysis (22) was applied to the whole set of decay datasets to estimate an appropriate number of components. On the basis of the SVD results, global analysis was then applied (23).

P700 difference spectrum.  A content of P700 was calculated as the difference in the ferricyanide-oxidized spectrum minus the ascorbate-reduced spectrum using a Hitachi 557 spectrophotometer at 298 K (24). The extinction coefficient of P700, ΔA700–730 = 64 mm−1 cm−1, was used for quantification as reported by Hiyama and Ke (25).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Absorption and fluorescence spectra of photosystem I at 80 K

The low-temperature absorption spectra of the PS I complexes are shown in Fig. 1. For G. violaceus (heavy line), there were at least two peaks located at 670 and 681 nm in the Qy region of Chl a. Cryptic peaks were resolved by the second derivative spectrum at 685 and 694 nm (data not shown). At wavelengths longer than 700 nm, there was no band; the same features have been observed in intact cells at 80 K (M. Mimuro, T. Tomo, K. Koyama and T. Tsuchiya, unpublished data). The PS I complexes of Synechocystis sp. PCC 6803 showed a similar pattern for the major Chl forms at low temperatures (fine line). Contrary to what is seen in G. violaceus, obvious bands were observed beyond 700 nm and were determined to be at 701, 706 and 712 nm. As the absorption maximum of P700 was reported to be 696 nm at 80 K (26), these bands were determined to be so-called “Red chlorophylls” (Red Chls) (27–32), indicating the presence of multiple forms of Red Chls. The 706 and 712 nm components were consistent with those detected by other methods (31,32). The presence of Red Chls in Synechocystis sp. PCC 6803 was clearly shown by the low-temperature absorption spectrum; at the same time, the absence of Red Chls in G. violaceus was also evident.

image

Figure 1.  Absorption spectra of PS I complexes at 80 K. Complexes isolated from Gloeobacter violaceus (heavy line) and Synechocystis sp. PCC 6803 (fine line) were measured.

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The low-temperature fluorescence spectra of the PS I complexes are shown in Fig. 2B for the same amount of Chl a. Gloeobacter violaceus showed a far lower intensity, approximately 15% of that of Synechocystis sp. PCC 6803. A peak was detected at 693 nm, but there was no fluorescence at wavelengths longer than 700 nm (Fig. 2B, heavy line). After magnification, small shoulders were discernible at approximately 672 and 657 nm (Fig. 2B, broken line). These components might have resulted from Chl a in detergent micelles (33). In contrast, the Synechocystis sp. PCC 6803 PS I complexes showed a single typical fluorescence maximum at 723 nm at 77 K (Fig. 2B, faint line); this peak was located at slightly shorter wavelengths than that observed in intact cells, and its bandwidth was narrower than that of intact cells by 25% (data not shown). At wavelengths shorter than 700 nm, fluorescence bands were not seen. A low fluorescence yield reflected the fluorescence lifetimes of the individual components, as described below.

image

Figure 2.  Steady state fluorescence spectra of PS I complexes isolated from Gloeobacter violaceus and Synechocystis sp. PCC 6803. The excitation wavelength was 440 nm. Fluorescence spectra of PS I complexes (A) measured at 298 K (G. violaceus, heavy line; Synechocystis sp. PCC 6803, fine line) and (B) spectra at 77 K (line types are the same as in A). Intensity was normalized on the basis of fluorescence yields under the condition of the same amount of Chl a. Broken line in (B) represents a magnified spectrum after normalization to the maximum intensity of the two spectra.

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The fluorescence spectrum was also measured at 298 K (Fig. 2A). Gloeobacter violaceus showed a single peak at 683 nm, and its band shape was nearly symmetrical (heavy line). In contrast, Synechocystis sp. PCC 6803 showed a single peak at 683 nm; however, there was a significant intensity in the long wavelength region of the maximum (faint line), indicating the presence of a cryptic band in this region. This fluorescence band might come from the Red Chls detected at room temperature, and represented a distinct difference in the fluorescence properties of the PS I complexes between the two species. Contrary to what was seen in the measurements at 77 K (Fig. 2B), the actual intensities on a Chl a base were almost identical between the two preparations.

Time-resolved fluorescence spectrum at 295 K

The TRFS of the PS I complexes is shown after normalization to the maximum intensities of the individual spectra (Figs. 3 and 4). The magnification factors of individual spectra are also shown for comparison. At 295 K, G. violaceus showed a single peak with a maximum at 686 nm just after excitation, which was discernible up to several hundred ps (Fig. 3A); a measurable intensity was no longer observed after 900 ps, indicating a very short lifetime of this component. The fluorescence lifetime, estimated by a convolution calculation on the decay at 686 nm, was 10 ps with an amplitude of 99.4%; a second component was also resolved, with a lifetime of 67 ps and an amplitude of 0.6%. We obtained essentially the same results for the decay curves at 670 and 700 nm, indicating that the spectra reflected relaxation from the thermally equilibrated state of the pigment pools. A very long-lived component (τ = 2100 ps, amplitude = 0.1%) was observed only at 670 nm, and was attributed to an uncoupled Chl a in detergent micelles.

image

Figure 3.  Time-resolved fluorescence spectra of PS I complexes measured at 295 K. The excitation wavelength was 400 nm. Individual spectra were normalized to the maximum intensity of the respective spectra, and the numbers shown on the individual spectra indicate magnification factors relative to that of the most intense spectrum. Spectra of (A) Gloeobacter violaceus and (B) Synechocystis sp. PCC 6803.

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image

Figure 4.  Time-resolved fluorescence spectra of PS I complexes measured at 77 K. The excitation wavelength was 400 nm. Individual spectra were normalized to the maximum intensity of the respective spectra, and the numbers shown on the individual spectra indicate the magnification factors relative to that of the most intense spectrum. Spectra of (A) Gloeobacter violaceus and (B) Synechocystis sp. PCC 6803.

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The TRFS of PS I complexes isolated from Synechocystis sp. PCC 6803 (Fig. 3B) showed a significant difference in the presence of one component in the long wavelength of the maximum. The peak was observed at 683 nm, and shifted to 686 nm at 210 ps after excitation. The second component appeared at approximately 708 nm at 16 ps; this corresponded to the Red Chls, and was consistent with a steady state fluorescence spectrum (Fig. 2A). A convolution calculation gave a lifetime for the main decay component of 11 ps with an amplitude of 99.0%, and for the second component, approximately 55 ps (amplitude, 1.0%). These values were obtained from the decay at 670, 686, 700 and 710 nm, indicating that the antenna pigments were thermally equilibrated. A third long-lived component (τ > 1000 ps) was also discernible, but its amplitude was less than 0.1%, and therefore it was attributed to the uncoupled Chl a molecules in preparations generated using a detergent. The presence of a short-lived PS I fluorescence component is consistent with PS I fluorescence observed in other samples (34–36).

Time-resolved fluorescence spectrum at 77 K

At 77 K (Fig. 4A), the TRFS of G. violaceus was different from that observed at 295 K (Fig. 3A). The 689 nm component appeared a short time after excitation, and it shifted to 692 nm after 50 ps (Fig. 4A). At approximately 200 ps, the bandwidth became broader, indicating the appearance of a new component, which was shown to be a 710 nm component; this band persisted for up to 2.2 ns but at a very low intensity. At 0.9 ns after excitation, two additional bands were discernible at 673 and 683 nm; however, their intensities were very low. Individual fluorescence bands were also characterized by their lifetimes.

In contrast, Synechocystis sp. PCC 6803 showed two components at 689 and 712 nm soon after excitation, and a subsequent redshift of the 712 nm component to 724 nm was clearly observed (Fig. 4B). The 689 nm component disappeared within 50 ps after excitation, indicating a fast energy flow from this component to the 712 nm component. The redshift of the maximum wavelength was consistent with TRFS observations in intact cells at 77 K (data not shown). This time-dependent process clearly showed an energy transfer to Red Chls in this species.

The TRFS of the two species clearly showed time-dependent changes in the spectra at 77 K, indicating that thermal equilibrium was not established at this temperature. Therefore, we performed decay analysis using a combination of SVD and global analysis; the former determined the number of decay components and the latter, the decay-associated fluorescence spectra. In the case of G. violaceus, three components, with lifetimes of 12, 32 and 650 ps, were sufficient to fit the spectral components, and the resolved decay-associated spectra are shown in Fig. 5. The 12 ps component (Fig. 5, left, A) showed a positive peak at 682 nm and a negative peak at 696 nm, indicating energy transfer to the long wavelength component. The 32 ps component (Fig. 5, left, B) showed a maximum at 692 nm, which corresponded to the decay of the main fluorescence component. The 650 ps component (Fig. 5, left, C) showed multiple peaks at 682 and 706 nm, with a shoulder at 670 nm. This spectrum was similar to that observed at 2.2 ns after excitation (Fig. 3A), corresponding to the long-lived component. This is most likely a result of uncoupled Chl a molecule(s) in the preparations.

image

Figure 5.  Global analysis on time-resolved fluorescence spectra of the PS I complex at 77 K. The number of components was estimated by SVD analysis and the component spectra were obtained by global analysis (22). Left: Spectra of Gloeobacter violaceus and right: of Synechocystis sp. PCC 6803. In left, lifetimes of the individual components were 12 ps (A), 32 ps (B) and 650 ps (C), respectively, and in right, they were shorter than 10 ps (A), 32 ps (B), 140 ps (C) and 660 ps (D), respectively.

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In contrast, four decay components were necessary to fit the decay profile of Synechocystis sp. PCC 6803; those lifetimes were shorter than 10, 32, 140 and 660 ps, and the decay-associated spectra of the individual components are shown in Fig. 5. The component with the shortest lifetime (τ < 10 ps) (Fig. 5, right, A) showed a maximum at 686 nm, and the second component (τ = 32 ps) (Fig. 5, right, B) showed an energy transfer-type spectrum with a positive peak at 696 nm and a negative peak at 715 nm. The third and fourth components (Fig. 5, right, C and D) showed single maxima at 714 and 722 nm, respectively. The rise component was resolved in the 32 ps lifetime component, and this showed plural bands, leading us to conclude that one energy output was connected to the 714 and 722 nm components in Synechocystis sp. PCC 6803. The presence of Red Chls and an energy transfer pathway to these molecules are clearly different from what is seen in G. violaceus.

In G. violaceus, the energy transfer time from Chl a682 to Chl a692 was 12 ps; the corresponding transfer pathway in Synechocystis sp. PCC 6803 was from Chl a686 to Chl a696 with a constant of shorter than 10 ps. In the latter organism, there was an additional pathway from Chl a696 to the Red Chls (Chl a714 and Chl a724) with a constant of 32 ps; the presence of this transfer pathway was another clear difference between the two species.

P700 difference spectrum and contents

The photochemical activity of our preparations was high, consistent with our previous study (14). The difference maximum for the P700 spectrum of the G. violaceus PS I complexes (Fig. 6, heavy line) was located at 699 nm; this was shifted to the blue by 2 nm relative to that of Synechocystis sp. PCC 6803 (fine line), even though the spectral bandwidths were almost identical in the two species. The peak wavelength of the P700 in Synechocystis sp. PCC 6803 was consistent with that observed for other cyanobacteria and a green alga (37,38). The P700 content in G. violaceus was estimated to be one P700 per 90-100 Chl a, slightly higher than that seen in Synechocystis sp. PCC 6803 (one P700 per 100-110 Chl a).

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Figure 6.  Absorption difference spectra of P700 at 298 K. The difference spectrum was measured as the ferricyanide-oxidized spectrum minus the ascorbate-reduced spectrum in complexes isolated from Gloeobacter violaceus (heavy line) and Synechocystis sp. PCC 6803 (fine line).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Differences in photosystem I properties

A subunit composition in PS I of G. violaceus is much simpler than that of other cyanobacteria due to a lack of four subunits (10). In the C-terminal region of PsaB, the extension consisting of 155 amino acids was found; its sequences were similar to the peptidoglycan binding domain of eubacteria (10). This feature was known only on G. violaceus.

The optical and electrochemical properties of the trimeric PS I complexes isolated from G. violaceus were different from those of Synechocystis sp. PCC 6803 in four ways: (1) P700 in G. violaceus had a lower redox potential by approximately 60 mV (16), (2) the P700 difference maximum of G. violaceus had a shorter wavelength by approximately 40 cm−1, (3) G. violaceus had a smaller PS I antenna size by approximately 10 Chl a molecules, and (4) Red Chls are absent in G. violaceus. These characteristics might be closely related to the evolutionary history of G. violaceus. Since it is known that G. violaceus is assigned to the earliest branch of the cyanobacteria phylogenetic tree (10), the development of photosynthetic systems in later cyanobacteria might be an adaptation to higher environmental oxygen concentrations. The lower redox potential of G. violaceus may be indicative of this species’ placement in the evolutionary pathway in that an oxygen concentration might be lower when this species appeared than that in the later time when other developed type cyanobacteria appeared. A smaller number of antenna pigments in G. violaceus and the acquisition of a more efficient antenna system in developed cyanobacteria also may be indicative of an evolutionary pathway. The G. violaceus PS I consists of a smaller number of subunits than other cyanobacteria; two of the missing subunits (PsaJ and PsaK) are part of a Chl a-binding protein (37). This suggests that the acquisition of PsaJ and PsaK corresponded to an increase in antenna size.

A larger antenna size might also be related to changes in the P700 difference maximum. There was an energetic difference of approximately 40 cm−1 between G. violaceus (699 nm) and Synechocystis sp. PCC 6803 (701 nm). The same phenomenon has been observed on cell membranes (M. Mimuro, K. Koyama and T. Tsuchiya, unpublished), indicating that the blueshift was not due to the isolation of PS I complexes, but rather to an intrinsic property of G. violaceus. At physiological temperatures, the RC trap is usually thermally equilibrated with sensitizers (antennae), and therefore a lower energy level of the trap is equivalent to an increase in antenna pigments that can be equilibrated with the trap. The energy difference of approximately 40 cm−1 corresponds to the increase in the antenna size by approximately 20% at 298 K, assuming a Boltzmann distribution, and therefore, the shift of the maximum to the red enabled the inclusion of Red Chls in antenna pigments. Although it is not clear at this experimental stage whether the presence of Red Chls or the shift in the difference maximum is the primary event, these two modifications are closely related to each other and collectively facilitated an increase in antenna size.

Energy transfer from the antenna to the RC was also somewhat different between the two species. The fluorescence maximum of the Synechocystis sp. PCC 6803 antenna was 696 nm (Fig. 5), where the absorption maximum of P700 was located (26). In G. violaceus, the P700 difference maximum was shorter than that of Synechocystis sp. PCC 6803 by 2 nm, suggesting that the P700 absorption maximum of G. violaceus was 694 nm. However, the fluorescence maximum of antenna was observed at 692 nm (Fig. 5), not the same wavelength for the P700 absorption band. These differences might result in the differences in energy transfer time between the two species, i.e. 12 ps for G. violaceus, and shorter than 10 ps for Synechocystis sp. PCC 6803.

Amino acid sequences as a possible factor in the observed difference in the difference maximum shift

In G. violaceus, P700 most likely consists of one Chl a and one Chl a′ molecule, because Chl a′ is stoichiometrically present in this species (14). The redox potential of G. violaceus P700 is lower than that of Synechocystis sp. PCC 6803 by approximately 60 mV (16). This difference may possibly result from differences in the amino acid sequences of one or more component polypeptides. Figure 7 shows the alignments of two major PS I polypeptides, PsaA and PsaB, in G. violaceus, Synechocystis sp. PCC 6803, and Thermosynechococcus elongatus. The ligands for P700 are supplied by histidine residues in the J-helices of PsaA and PsaB, and these residues are conserved across all three species. A replacement that might affect the orientation angle of the J-helix was found for both subunits; at the edge of PsaA J-helix, an alanine in Synechocystis sp. PCC 6803 is replaced by proline in G. violaceus (residue 696). Similarly, one glycine residue was inserted in the G. violaceus PsaB (residue 622) near the edge of J-helix on the periplasmic side. These unique modifications are likely to modify the properties of P700; site-directed mutagenesis experiments to determine the functions of these residues will be an important next step.

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Figure 7.  Primary structures of the two major subunits of PS I complex in Gloeobacter violaceus and Synechocystis sp. PCC 6803. The 11 transmembrane spanning regions and the sidedness of the loop structures were analyzed by sequence homology. Transmembrane regions are shaded, and loop regions localized in the periplasmic space for G. violaceus or lumenal space for Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus are underlined. Squares represent amino acids supplying ligands for Chl a.

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Factors involved in the presence of Red Chls

Contrary to the case of P700, the absence of Red Chls in G. violaceus is not necessarily attributable to differences in the PsaA and PsaB amino acid sequences. The number of Red Chls in G. violaceus was estimated to be zero, with 9–11 in Synechocystis sp. PCC 6803 (Fig. 1) (31,39). Two possible amino acid sequence-related reasons for this difference are (1) the absence of the four PS I subunits (PsaI, PsaJ, PsaK and PsaX) in G. violaceus and (2) differences in Chl a ligands. According to Jordan et al. (39), PsaJ and PsaK supply three and two ligands, respectively, to Chl a; however, none of these ligands are assigned to Red Chls. Therefore, the absence of the subunits PsaI, PsaJ and PsaK in G. violaceus is most likely not the reason for the absence of Red Chls. We therefore compared the primary structure of the two major PS I subunits (PsaA and PsaB) between G. violaceus and Synechocystis sp. PCC 6803 (Fig. 7). Ligands supplied from water molecules can only be compared by structural analysis at the atomic level, and since this level of resolution was not available to us, we could not consider these ligands. As shown in Fig. 7, among amino acid that provide ligands to Chl a (67 residues), one (A34) was different in PsaA in G. violaceus and Synechocystis sp. PCC 6803. In comparing G. violaceus and T. elongatus, we found three ligands that differed between the two species at the sites A34, B7 and B21, and one of these three (B7) was assigned to the Red Chl based on structural analysis (40). Differences in these ligands, however, do not explain the absence of Red Chls in G. violaceus. Based on the above considerations, we concluded that the absence of Red Chls was not due to any difference in the primary structures of the subunits. Therefore, we conclude that possible factors involved in the presence of Red Chls include (1) differences in molecular interactions between Chl a molecules and protein moieties and/or (2) differences in molecular interactions among plural Chl a molecules or differences in water ligands. We cannot exclude the possibility that other unknown factors are present. In order to prove the involvement of these factors, the three-dimensional structure will be needed for further study.

As indicated by the phylogenetic tree of cyanobacteria, G. violaceus is an early branching species in the cyanobacterial lineage (10), and thus it is logical to consider this species a primordial organism. Differences in PS I property are evident among cyanobacteria. The primordial properties of the G. violaceus PSs probably represent a starting point in the evolution of photosynthetic systems. Although our analysis here is largely descriptive, our findings provide a basis for future work. The development of a transformation system for this species will greatly facilitate progress in this field of inquiry.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Acknowledgements—  The authors thank Dr. T. Tsuchiya, Mr. M. Higuchi and Mr. H. Inoue, Kyoto University, for their help in the initial stage of this study. This work was supported in part by the Grant-in-Aid for Creative Research from the Japanese Society for the Promotion of Science (JSPS) to M.M. (Grant 17GS0314).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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