Function of chlorophyll d in reaction centers of photosystems I and II of the oxygenic photosynthesis of Acaryochloris marina

Reaction center chlorophylls (Chls) in photosystems II and I were studied in the isolated thylakoid membranes of a cyanobacterium, Acaryochloris marina, which contains Chls d and a as the major and minor pigments, respectively. The membranes contained PS I and II complexes at a 1.8:1 molar ratio on the basis of the spin densities on the tyrosine D radical and the photo-oxidized PS I primary donor (P740+). In the presence of ferricyanide, laser excitation induced bleach at 725 nm that recovered with time constants of 25 micros and 1.2 ms. The signal, designated P725, was suppressed by PS II inhibitors DCMU and hydroxylamine. The P725 spectrum was tentatively assigned to the absorption changes of the special pair Chl d, the accessory Chl d, and the acceptor pheophytin a in PS II. The addition of ascorbate induced the additional signal with a slow decay time constant of 4.5 ms. This signal showed a broad bleach at 740 nm and shift-type absorption changes at around 707 and 685 nm, which were assigned to the absorption changes of PS I special pair of Chl d (P740), the accessory Chl d, and the primary acceptor Chl a (A0), respectively. Mechanisms and the evolution of the Chl-d based reaction centers using far-red light are discussed together with the amino acid sequences of PS II D1 and D2 proteins.     

Chlorophyll d and Acaryochloris marina: current status

The discovery of the chlorophyll d-containing cyanobacterium Acaryochloris marina in 1996 precipitated a shift in our understanding of oxygenic photosynthesis. The presence of the red-shifted chlorophyll d in the reaction centre of the photosystems of Acaryochloris has opened up new avenues of research on photosystem energetics and challenged the unique status of chlorophyll a in oxygenic photosynthesis. In this review, we detail the chemistry and role of chlorophyll d in photosynthesis and summarise the unique adaptations that have allowed the proliferation of Acaryochloris in diverse ecological niches around the world.     

The major light-harvesting pigment protein of Acaryochloris marina

The major light-harvesting protein complex containing chlorophyll (Chl) d was isolated from Acaryochloris marina thylakoid membranes. Isolation was achieved by detergent solubilisation followed by separation on 6-40% sucrose gradients using ultracentrifugation. The best Chl d yield (70%) used 0.3% dodecyl maltoside, 0.15% octyl glucoside, 0.05% zwittergent 3-14 with the detergent:total Chl d ratio around 10:1 (w/w). Characterisation of the light-harvesting pigment protein complex (lhc) involved non-denaturing electrophoresis, SDS-PAGE, absorbance and fluorescence spectroscopy. The main polypeptide in the lhc was shown to be ca. 34 kDa and to contain Chl d and Chl a, indicating that the Acaryochloris lhc is similar to that of prochlorophytes. The Chl a level varied with the culture conditions, which is consistent with previous findings.     

Raman spectroscopy of chlorophyll d from Acaryochloris marina

The Raman spectroscopy of chlorophyll (Chl) d isolated from Acaryochloris marina has been measured in the range of 250-3200 cm(-1) at 77 K following excitation of its B(x) band at 488 nm. A peak at 1659 cm(-1) of medium intensity arising from Cz=O stretching vibration in the formyl group 3(1) specific to Chl d was observed clearly. Peaks due to other Cz=O stretching vibrations of the 13(1) keto-, 13(3) ester- and 17(3) groups have also been observed with much weaker intensities. Intense Raman peaks in the range of 1000-1800 cm(-1) are reported and homologous comparison with corresponding Raman shifts of Chl a, Chl b and BChl a are presented.     

Spectroscopic studies of photosystem II in chlorophyll d-containing Acaryochloris marina

Photosystem II (PSII) electron transfer (ET) in the chlorophyll d-containing cyanobacterium Acaryochloris marina (A. marina) was studied by time-resolved electron paramagnetic resonance (EPR) spectroscopy at room temperature, chlorophyll fluorescence, and low-temperature optical spectroscopy. To maximize the ability to measure PSII ET in the intact cells of this organism, growth conditions were optimized to provide the highest specific O(2) activity and the instrumental parameters for the EPR measurements of tyrosine Z (Y(Z)) reduction were adjusted to give the best signal-to-noise over spectral resolution. Analysis of the Y(Z)(*) reduction kinetics revealed that ET to the oxygen-evolving complex on the donor side of PSII in A. marina is indistinguishable from that in higher plants and other cyanobacteria. Likewise, the charge recombination kinetics between the first plastoquinone acceptor Q(A) and the donor side of PSII monitored by the chlorophyll fluorescence decay on the seconds time scale are not significantly different between A. marina and non-chlorophyll d organisms, while low-temperature optical absorption spectroscopy identified the primary electron acceptor in A. marina as pheophytin a. The results indicate that, if the PSII primary electron donor in A. marina is made up of chlorophyll d instead of chlorophyll a, then there must be very different interactions with the protein environment to account for the ET properties, which are similar to higher plants and other cyanobacteria. Nevertheless, the water oxidation mechanism in A. marina is kinetically unaltered.     

Structure of a large photosystem II supercomplex from Acaryochloris marina

Acaryochloris marina is a prochlorophyte-like cyanobacterium containing both phycobilins and chlorophyll d as light harvesting pigments. We show that the chlorophyll d light harvesting system, composed of Pcb proteins, functionally associates with the photosystem II (PSII) reaction center (RC) core to form a giant supercomplex. This supercomplex has a molecular mass of about 2300 kDa and dimensions of 385 A x 240 A. It is composed of two PSII-RC core dimers arranged end-to-end, flanked by eight symmetrically related Pcb proteins on each side. Thus each PSII-RC monomer has four Pcb subunits acting as a light harvesting system which increases the absorption cross section of the PSII-RC core by almost 200%.     

Effect of Iron on Growth and Ultrastructure of Acaryochloris marina

The cyanobacterial genus Acaryochloris is the only known group of oxygenic phototrophs that contain chlorophyll d rather than chlorophyll a as the major photosynthetic pigment. Studies on this organism are still in their earliest stages, and biochemical analysis has rapidly outpaced growth optimization. We have investigated culture growth of the major strains of Acaryochloris marina (MBIC11017 and MBIC10697) by using several published and some newly developed growth media. It was determined that heavy addition of iron significantly enhanced culture longevity. These high-iron cultures showed an ultrastructure with thylakoid stacks that resemble traditional cyanobacteria (unlike previous studies). These cultures also show a novel reversal in the pigment ratios of the photosystem II signature components chlorophyll a and pheophytin a, as opposed to those in previous studies.     

Effect of iron on growth and ultrastructure of Acaryochloris marina

The cyanobacterial genus Acaryochloris is the only known group of oxygenic phototrophs that contain chlorophyll d rather than chlorophyll a as the major photosynthetic pigment. Studies on this organism are still in their earliest stages, and biochemical analysis has rapidly outpaced growth optimization. We have investigated culture growth of the major strains of Acaryochloris marina (MBIC11017 and MBIC10697) by using several published and some newly developed growth media. It was determined that heavy addition of iron significantly enhanced culture longevity. These high-iron cultures showed an ultrastructure with thylakoid stacks that resemble traditional cyanobacteria (unlike previous studies). These cultures also show a novel reversal in the pigment ratios of the photosystem II signature components chlorophyll a and pheophytin a, as opposed to those in previous studies.     

Pigment composition and adaptation in free-living and symbiotic strains of Acaryochloris marina

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.     

Ultrastructure of Acaryochloris marina, an oxyphotobacterium containing mainly chlorophyll d

We present a detailed investigation of the ultrastructure of the chlorophyll a/d-containing unicellular oxyphotobacterium Acaryochloris marina, combining light and transmission electron microscopy and showing freeze fractures of this organism for the first time. The cells were 1.8-2.1 microm x 1.5-1.7 microm in size. The cell envelope consisted of a peptidoglycan layer of approximately 10 nm thickness combined with an outer membrane. Cell division was intermediate between the constrictive and the septum type. The nucleoplasm, which contained several carboxysomes, was surrounded by 7-11 concentrically arranged thylakoids, which were predominantly stacked, with the exception of distinct areas where phycobiliproteins were located. The thylakoids were perforated by channel-like structures connecting the central and peripheral portions of the cytoplasm and not yet observed in other organisms. In freeze fractures, the protoplasmic fracture faces of thylakoid membranes were densely covered with particles of inhomogenous size. The particle size histogram peaked at 10-11, 13 and 18 nm. The 18-nm particles are assumed to represent photosystem I trimers. The particles on exoplasmic fracture faces, proposed to represent photosystem II complexes, were significantly larger than the corresponding particles of cyanobacteria and clustered to form large aggregates. This kind of arrangement is unique among photosynthetic organisms.     

Functional characteristics of chlorophyll d-predominating photosynthetic apparatus in intact cells of Acaryochloris marina

Functional organization of the photosynthetic apparatus in the unique chlorophyll d-predominating prokaryote, Acaryochloris marina, was studied using polarographic measurements of single-turnover flash yields, action spectra and optical cross sections for PS-specific reactions. O₂ evolution was indicative of PS II activity, while reversible photoinhibition of respiratory O₂ uptake under aerobic conditions in the presence of DCMU and H₂ photoevolution by anaerobically adapted cells were the indicatives of PS I activity. O₂ evolution in the cells upon single-turnover flashes followed the normal S-state cycle with a period-4 oscillation. Analysis of action spectra for the partial reactions of photosynthesis revealed that: (1) distinct spectral forms of Chl d are nonuniformly distributed between PS I and PS II, e.g. Chl d-695 and Chl d-735 are preferentially located in PS II and PS I, respectively; (2) a minor fraction of Chl a in the cells belongs mostly to PS II; (3) biliproteins transfer excitation energy both to PS II and, with a lower efficiency, PS I; (4) the efficiency of energy transfer from biliproteins to PS II depends on the light quality growth conditions and is larger in white light (WL)-grown cells compared to the red light (RL)-grown cells. Content of functional O₂ evolving PS II centers decreases 2 times in the RL-grown cells relative to the WL-grown cells, whereas content of competent PS I centers involved in photoinhibition of respiration remains almost the same in both the cultures. The effective antenna size of PS I was estimated to be 80–90 Chl d including 3–10 molecules absorbing at 735 nm. The effective optical cross-section of PS II corresponded to 90–100 Chl d and, presumably, 4 Chl a + 2 Pheo a [Mimuro et al. (1999) Biochim Biophys Acta 1412: 37–46]. Optical cross-section measurements indicated that the functional PS II units of A. marina attach one rod of four hexameric units of biliproteins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetics of phyllosemiquinone oxidation in the Photosystem I reaction centre of Acaryochloris marina

Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.     

Mapping the excitation energy migration pathways in phycobilisomes from the cyanobacterium Acaryochloris marina

In this study, we use ultrafast time-resolved absorption and fluorescence spectroscopies to examine A. marina phycobilisomes isolated from cells grown under light of different intensities and spectral regimes. Investigations were performed at room temperature and at 77?K. The study demonstrates that if complexes are stabilized by high phosphate (900?mM) buffer, there are no differences between them in temporal and spectral properties of fluorescence. However, when the complexes are allowed to disassemble into trimers in low phosphate (50?mM) buffer, differences are clearly observed. The fluorescence properties of intact or disassembled phycobilisomes from cells grown in low intensity white light are unresponsive to variation in phosphate concentration. This antenna complex was further studied in detail with application of femtosecond time-resolved absorption at room temperature. Combined spectroscopic and kinetic analysis of time-resolved fluorescence and absorption data of this antenna allowed us to identify spectrally different forms of phycocyanobilins and to propose a simplified model of how they could be distributed within the phycobilisome structure.     

Uphill energy transfer in a chlorophyll d-dominating oxygenic photosynthetic prokaryote, Acaryochloris marina

The steady-state fluorescence properties and uphill energy transfer were analyzed on intact cells of a chlorophyll (Chl) d-dominating photosynthetic prokaryote, Acaryochloris marina. Observed spectra revealed clear differences, depending on the cell pigments that had been sensitized; using these properties, it was possible to assign fluorescence components to specific Chl pigments. At 22 degrees C, the main emission at 724 nm came from photosystem (PS) II Chl d, which was also the source of one additional band at 704 nm. Chl a emissions were observed at 681 nm and 671 nm. This emission pattern essentially matched that observed at -196 degrees C, as the main emission of Chl d was located at 735 nm, and three minor bands were observed at 704 nm, 683 nm, and 667 nm, originating from Chl d, Chl a, and Chl a, respectively. These three minor bands, however, had not been sensitized by carotenoids, suggesting specific localization in PS II. At 22 degrees C, excitation of the red edge of the absorption band (which, at 736 nm, was 20 nm longer than the absorption maximum), resulted in fluorescence bands of Chl d at 724 nm and of Chl a at 682 nm, directly demonstrating an uphill energy transfer in this alga. This transfer is a critical factor for in vivo activity, due to an inversion of energy levels between antenna Chl d and the primary electron donor of Chl a in PS II.     

Response of chlorophyll d-containing cyanobacterium Acaryochloris marina to UV and visible irradiations

We have previously investigated the response mechanisms of photosystem II complexes from spinach to strong UV and visible irradiations (Wei et al J Photochem Photobiol B 104:118–125, 2011). In this work, we extend our study to the effects of strong light on the unusual cyanobacterium Acaryochloris marina, which is able to use chlorophyll d (Chl d) to harvest solar energy at a longer wavelength (740 nm). We found that ultraviolet (UV) or high level of visible and near-far red light is harmful to A. marina. Treatment with strong white light (1,200 μmol quanta m^?2 s^?1) caused a parallel decrease in PSII oxygen evolution of intact cells and in extracted pigments Chl d, zeaxanthin, and α-carotene analyzed by high-performance liquid chromatography, with severe loss after 6 h. When cells were irradiated with 700 nm of light (100 μmol quanta m^?2 s^?1) there was also bleaching of Chl d and loss of photosynthetic activity. Interestingly, UVB radiation (138 μmol quanta m^?2 s^?1) caused a loss of photosynthetic activity without reduction in Chl d. Excess absorption of light by Chl d (visible or 700 nm) causes a reduction in photosynthesis and loss of pigments in light harvesting and photoprotection, likely by photoinhibition and inactivation of photosystem II, while inhibition of photosynthesis by UVB radiation may occur by release of Mn ion(s) in Mn₄CaO₅ center in photosystem II.     

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c₆) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A₀, A₁, and three Fe₄S₄ clusters, F_X, F_A, and F_B. Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A₀ is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.     

Biofilm growth and near-infrared radiation-driven photosynthesis of the chlorophyll d-containing cyanobacterium Acaryochloris marina

The cyanobacterium Acaryochloris marina is the only known phototroph harboring chlorophyll (Chl) d. It is easy to cultivate it in a planktonic growth mode, and A. marina cultures have been subject to detailed biochemical and biophysical characterization. In natural situations, A. marina is mainly found associated with surfaces, but this growth mode has not been studied yet. Here, we show that the A. marina type strain MBIC11017 inoculated into alginate beads forms dense biofilm-like cell clusters, as in natural A. marina biofilms, characterized by strong O(2) concentration gradients that change with irradiance. Biofilm growth under both visible radiation (VIS, 400 to 700 nm) and near-infrared radiation (NIR, ～700 to 730 nm) yielded maximal cell-specific growth rates of 0.38 per day and 0.64 per day, respectively. The population doubling times were 1.09 and 1.82 days for NIR and visible light, respectively. The photosynthesis versus irradiance curves showed saturation at a photon irradiance of E(k) (saturating irradiance) >250 μmol photons m(-2) s(-1) for blue light but no clear saturation at 365 μmol photons m(-2) s(-1) for NIR. The maximal gross photosynthesis rates in the aggregates were ～1,272 μmol O(2) mg Chl d(-1) h(-1) (NIR) and ～1,128 μmol O(2) mg Chl d(-1) h(-1) (VIS). The photosynthetic efficiency (α) values were higher in NIR-irradiated cells [(268 ± 0.29) × 10(-6) m(2) mg Chl d(-1) (mean ± standard deviation)] than under blue light [(231 ± 0.22) × 10(-6) m(2) mg Chl d(-1)]. A. marina is well adapted to a biofilm growth mode under both visible and NIR irradiance and under O(2) conditions ranging from anoxia to hyperoxia, explaining its presence in natural niches with similar environmental conditions.     

Photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina

Fourier transform infrared spectroscopy (FTIR) difference spectroscopy in combination with deuterium exchange experiments has been used to study the photo-oxidation of P740, the primary electron donor in photosystem I from Acaryochloris marina. Comparison of (P740(+)-P740) and (P700(+)-P700) FTIR difference spectra show that P700 and P740 share many structural similarities. However, there are several distinct differences also: 1), The (P740(+)-P740) FTIR difference spectrum is significantly altered upon proton exchange, considerably more so than the (P700(+)-P700) FTIR difference spectrum. The P740 binding pocket is therefore more accessible than the P700 binding pocket. 2), Broad, "dimer" absorption bands are observed for both P700(+) and P740(+). These bands differ significantly in substructure, however, suggesting differences in the electronic organization of P700(+) and P740(+). 3), Bands are observed at 2727(-) and 2715(-) cm(-1) in the (P740(+)-P740) FTIR difference spectrum, but are absent in the (P700(+)-P700) FTIR difference spectrum. These bands are due to formyl CH modes of chlorophyll d. Therefore, P740 consists of two chlorophyll d molecules. Deuterium-induced modification of the (P740(+)-P740) FTIR difference spectrum indicates that only the highest frequency 13(3) ester carbonyl mode of P740 downshifts, indicating that this ester mode is weakly H-bonded. In contrast, the highest frequency ester carbonyl mode of P700 is free from H-bonding. Deuterium-induced changes in (P740(+)-P740) FTIR difference spectrum could also indicate that one of the chlorophyll d 3(1) carbonyls of P740 is hydrogen bonded.     

Identification of the primary electron donor in PS II of the Chl d-dominated cyanobacterium Acaryochloris marina

The primary electron donor of photosystem (PS) II in the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina was confirmed by delayed fluorescence (DF) and further proved by pigment contents of cells grown under several light intensities. The DF was found only in the Chl a region, identical to Synechocystis sp. PCC 6803, and disappeared following heat treatment. Pigment analyses indicated that at least two Chl a molecules were present per each two pheophytin a molecules, and these Chl a molecules are assigned to P(D1) and P(D2). These findings clearly indicate that Chl a is required for water oxidation in PS II.