Theoretical study on the thermodynamic properties of chlorophyll d-peptides coordinating ligand

The chlorophyll d containing cyanobacterium, Acaryochloris marina has provided a model system for the study of chlorophyll replacement in the function of oxygenic photosynthesis. Chlorophyll d replaces most functions of chlorophyll a in Acaryochloris marina. It not only functions as the major light-harvesting pigment, but also acts as an electron transfer cofactor in the primary charge separation reaction in the two photosystems. The Mg-chlorophyll d-peptide coordinating interaction between the amino acid residues and chlorophylls using the latest semi-empirical PM5 method were examined. It is suggested that chlorophyll d possesses similar coordination ligand properties to chlorophyll a, but chlorophyll b possesses different ligand properties. Compared with other studies involving theoretical correlation and our prior experiments, this study suggests that the chlorophyll a-bound proteins will bind chlorophyll d without difficulty when chlorophyll d is available.     

Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803

The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA⁺, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.     

Excitation energy transfer in intact cells and in the phycobiliprotein antennae of the chlorophyll d containing cyanobacterium Acaryochloris marina

The cyanobacterium Acaryochloris marina is unique because it mainly contains Chlorophyll d (Chl d) in the core complexes of PS I and PS II instead of the usually dominant Chl a. Furthermore, its light harvesting system has a structure also different from other cyanobacteria. It has both, a membrane-internal chlorophyll containing antenna and a membrane-external phycobiliprotein (PBP) complex. The first one binds Chl d and is structurally analogous to CP43. The latter one has a rod-like structure consisting of three phycocyanin (PC) homohexamers and one heterohexamer containing PC and allophycocyanin (APC). In this paper, we give an overview on the investigations of excitation energy transfer (EET) in this PBP-light-harvesting system and of charge separation in the photosystem II (PS II) reaction center of A. marina performed at the Technische Universität Berlin. Due to the unique structure of the PBP antenna in A. marina, this EET occurs on a much shorter overall time scale than in other cyanobacteria. We also briefly discuss the question of the pigment composition in the reaction center (RC) of PS II and the nature of the primary donor of the PS II RC.     

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.     

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.     

Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina

We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P⁺Q⁻, between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (Q_A). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, Chl_D1. It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.     

Quest for minor but key chlorophyll molecules in photosynthetic reaction centers – unusual pigment composition in the reaction centers of the chlorophyll d-dominated cyanobacterium Acaryochloris marina

A short overview, based on our own findings, is given of the minor pigments that function as key components in photosynthesis. Recently, we found the presence of chlorophyll a, chlorophyll d′ and pheophytin a as minor pigments in the chlorophyll d-dominated cyanobacterium Acaryochloris marina. This revised version was published online in June 2006 with corrections to the Cover Date.     

Minor but key chlorophylls in Photosystem II

A ‘metal-free’ chlorophyll (Chl) a, pheophytin (Phe) a, functions as the primary electron acceptor in PS II. On the basis of Phe a/PS II = 2, Phe a content is postulated as an index for estimation of the stoichiometry of pigments and photosystems. We found Phe a in a Chl d-dominant cyanobacterium Acaryochloris marina, whereas Phe d was absent. The minimum Chl a:Phe a ratio was 2:2, indicating that the primary electron donor is Chl a, accessory is Chl d, and the primary electron acceptor is Phe a in PS II of A. marina. Chl d was artificially formed by the treatment of Chl a with papain in aqueous organic solvents. Further, we will raise a key question on the mechanisms of water oxidation in PS II.     

Cationic state distribution over the chlorophyll d-containing PD1/PD2 pair in photosystem II

Most of the chlorophyll (Chl) cofactors in photosystem II (PSII) from Acaryochloris marina are Chld, although a few Chla molecules are also present. To evaluate the possibility that Chla may participate in the P_D1/P_D2 Chl pair in PSII from A. marina, the P_D1^?+/P_D2^?+ charge ratio was investigated using the PSII crystal structure analyzed at 1.9-Å resolution, while considering all possibilities for the Chld-containing P_D1/P_D2 pair, i.e., Chld/Chld, Chla/Chld, and Chld/Chla pairs. Chld/Chld and Chla/Chld pairs resulted in a large P_D1^?+ population relative to P_D2^?+, as identified in Chla/Chla homodimer pairs in PSII from other species, e.g., Thermosynechococcus elongatus PSII. However, the Chld/Chla pair possessed a P_D1^?+/P_D2^?+ ratio of approximately 50/50, which is in contrast to previous spectroscopic studies on A. marina PSII. The present results strongly exclude the possibility that the Chld/Chla pair serves as P_D1/P_D2 in A. marina PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella

We assessed the microbial diversity and microenvironmental niche characteristics in the didemnid ascidian Lissoclinum patella using 16S rRNA gene sequencing, microsensor and imaging techniques. L. patella harbors three distinct microbial communities spatially separated by few millimeters of tunic tissue: (i) a biofilm on its upper surface exposed to high irradiance and O₂ levels, (ii) a cloacal cavity dominated by the prochlorophyte Prochloron spp. characterized by strong depletion of visible light and a dynamic chemical microenvironment ranging from hyperoxia in light to anoxia in darkness and (iii) a biofilm covering the underside of the animal, where light is depleted of visible wavelengths and enriched in near-infrared radiation (NIR). Variable chlorophyll fluorescence imaging demonstrated photosynthetic activity, and hyperspectral imaging revealed a diversity of photopigments in all microhabitats. Amplicon sequencing revealed the dominance of cyanobacteria in all three layers. Sequences representing the chlorophyll d containing cyanobacterium Acaryochloris marina and anoxygenic phototrophs were abundant on the underside of the ascidian in shallow waters but declined in deeper waters. This depth dependency was supported by a negative correlation between A. marina abundance and collection depth, explained by the increased attenuation of NIR as a function of water depth. The combination of microenvironmental analysis and fine-scale sampling techniques used in this investigation gives valuable first insights into the distribution, abundance and diversity of bacterial communities associated with tropical ascidians. In particular, we show that microenvironments and microbial diversity can vary significantly over scales of a few millimeters in such habitats; which is information easily lost by bulk sampling.     

Redox potential of chlorophyll d in vitro

Chlorophyll (Chl) d is a major chlorophyll in a novel oxygenic prokaryote Acaryochloris marina. Here we first report the redox potential of Chl d in vitro. The oxidation potential of Chl d was + 0.88 V vs. SHE in acetonitrile; the value was higher than that of Chl a (+ 0.81 V) and lower than that of Chl b (+ 0.94 V). The oxidation potential order, Chl b > Chl d > Chl a, can be explained by inductive effect of substituent groups on the conjugated π-electron system on the macrocycle. Corresponding pheophytins showed the same order; Phe b (+ 1.25 V) > Phe d (+ 1.21 V) > Phe a (+ 1.14 V), but the values were significantly higher than those of Chls, which are rationalized in terms of an electron density decrease in the π-system by the replacement of magnesium with more electronegative hydrogen. Consequently, oxidation potential of Chl a was found to be the lowest among Chls and Phes. The results will help us to broaden our views on photosystems in A. marina.     

Structural heterogeneity leads to functional homogeneity in A. marina phycocyanin

The major light harvesting antenna in all cyanobacterial species is the phycobilisome (PBS). The smallest PBS identified to date is that of Acaryochloris marina (A. marina), composed of a single four-hexamer rod. We have determined the crystal structure of phycocyanin (AmPC), the major component of the A. marina PBS (AmPBS) to 2.1?Å. The basic unit of the AmPC is a heterodimer of two related subunits (α and β), and we show that the asymmetric unit contains a superposition of two α and two β isoforms, the products of the simultaneous expression of different genes. This is the first time to our knowledge that isolated proteins crystallized with such identifiable heterogeneity. We believe that the presence of the different isoforms allows the AmPBS to have a significant bathochromic shift in its fluorescence emission spectrum, allowing, in the total absence of allophycocyanin, a better overlap with absorption of the chlorophyll d-containing reaction centers. We show that this bathochromic shift exists in intact AmPBS as well as in its disassembled components, thus suggesting that AmPC can efficiently serve as the AmPBS terminal emitter.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

Supramolecular organization of phycobiliproteins in the chlorophyll d-containing cyanobacterium Acaryochloris marina

Here we report the high-resolution detail of the organization of phycobiliprotein structures associated with photosynthetic membranes of the chlorophyll d-containing cyanobacterium Acaryochloris marina. Cryo-electron transmission-microscopy on native cell sections show extensive patches of near-crystalline phycobiliprotein rods that are associated with the stromal side of photosynthetic membranes. This supramolecular photosynthetic structure represents a novel mechanism of organizing the photosynthetic light-harvesting machinery. In addition, the specific location of phycobiliprotein patches suggests a physical separation of photosystem I and photosystem II reaction centres. Based on this finding and the known photosystem’s structure in Acaryochloris, we discuss possible membrane arrangements of photosynthetic membrane complexes in this species.     

ACARYOCHLORIS MARINA GEN. ET SP. NOV. (CYANOBACTERIA), AN OXYGENIC PHOTOSYNTHETIC PROKARYOTE CONTAINING CHL D AS A MAJOR PIGMENT1

The phylogenetic position of an oxygenic photosynthetic prokaryote containing chl d as a major pigment, which have been tentatively named “Acaryochloris marina,” was analyzed using small subunit rDNA sequences. Phylogenetic relationships inferred among A. marina, selected strains from the Cyanobacteria, and plastids showed that A. marina was within the cyanobacterial radiation. The A. marina lineage diverged independently from other phylogenetic subgroups of the Cyanobacteria. No organism was found to be identical or related closely to A. marina by a similarity search and phylogenetic analysis. Based on these results, in addition to the reported characteristics of the cell morphology, pigment composition, and photosynthesis, a new taxon, Acaryochloris marina Miyashita et Chihara gen. et sp. nov., is formally proposed for the oxy‐genic photosynthetic prokaryote.     

Isolation and characterization of biliprotein aggregates from Acaryochloris marina, a Prochloron-like prokaryote containing mainly chlorophyll d

Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment. In the electron microscope the biliprotein aggregates appear as rod-shaped structures of 26.0×11.3 nm, composed of four ring-shaped subunits 5.8 nm thick and 11.7 nm in diameter. Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin-type pigment, with very efficient energy transfer from the phycocyanin- to allophycocyanin-type constituent. The chromophore-binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.     

Endolithic chlorophyll d-containing phototrophs

Cyanobacteria in the genus Acaryochloris are the only known oxyphototrophs that have exchanged chlorophyll a (Chl a) with Chl d as their primary photopigment, facilitating oxygenic photosynthesis with near infrared (NIR) light. Yet their ecology and natural habitats are largely unknown. We used hyperspectral and variable chlorophyll fluorescence imaging, scanning electron microscopy, photopigment analysis and DNA sequencing to show that Acaryochloris-like cyanobacteria thrive underneath crustose coralline algae in a widespread endolithic habitat on coral reefs. This finding suggests an important role of Chl d-containing cyanobacteria in a range of hitherto unexplored endolithic habitats, where NIR light-driven oxygenic photosynthesis may be significant.     

Triplet-triplet energy transfer in Peridinin-Chlorophyll a-protein reconstituted with Chl a and Chl d as revealed by optically detected magnetic resonance and pulse EPR: Comparison with the native PCP complex from Amphidinium carterae

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at ∼ 620 and ∼ 640 nm in the Triplet-minus-Singlet (T − S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.