An unusual triplet population pathway in the Reaction Centre of the Chlorophyll-d binding Photosystem I of A. marina, as revealed by a combination of TR-EPR and ODMR spectroscopies.

Photo-induced Chlorophyll (Chl) triplet states in the isolated Photosystem I (PSI) of Acaryochloris marina, that harbours Chl d as its main pigment, were investigated by Optically Detected Magnetic Resonance (ODMR) and Time-Resolved Electron Paramagnetic Resonance (TR-EPR), and as a function of pre-illumination of the sample under reducing redox poising. Fluorescence Detected Magnetic Resonance (FDMR) allowed resolving four Chl d triplet (3Chl d) populations (T1-T4) both in untreated and illuminated samples in the presence of ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The FDMR signals increased following the pre-illumination treatment, particularly for the T3 and T4 populations, which are therefore sensitive to the redox state of PSI cofactors. Microwave-induced Triplet minus Singlet (TmS) spectra were detected in the |D|-|E| resonance window of the T3 and T4 triplets. These showed a broad singlet bleaching centred at 740 nm and also displayed complex spectral structure with several derivative-like features, indicating that both the T3 and T43Chl d populations are associated with the PSI reaction centre (RC) triplet, P3740. Parallel measurements by TR-EPR demonstrated that triplet signals observed under all conditions investigated are dominated by an electron spin polarisation (esp), which is typical of intersystem crossing, differently from what expected for recombination triplet states formed from a radical pair precursor. Moreover, stronger reductant conditions obtained by pre-illumination of the samples in the presence of dithionite and 5-methylphenazinium methyl sulfate (PMS) did not lead to a recombination triplet state esp, but rather to a decrease of the whole signal intensity. The energetics of A. marina PSI and the possible occurrence of distributions of cofactors redox properties are discussed in order to address the unexpected P3740 esp.

Ecological diversification of a cyanobacterium through divergence of its novel chlorophyll d-based light-harvesting system.

The evolution of novel traits can have important consequences for biological diversification. Novelties such as new structures are associated with changes in both genotype and phenotype that often lead to changes in ecological function.1,2 New ecological opportunities provided by a novel trait can trigger subsequent trait modification or niche partitioning3; however, the underlying mechanisms of novel trait diversification are still poorly understood. Here, we report that the innovation of a new chlorophyll (Chl) pigment, Chl d, by the cyanobacterium Acaryochloris marina was followed by the functional divergence of its light-harvesting complex. We identified three major photosynthetic spectral types based on Chl fluorescence properties for a collection of A. marina laboratory strains for which genome sequence data are available,4,5 with shorter- and longer-wavelength types more recently derived from an ancestral intermediate phenotype. Members of the different spectral types exhibited extensive variation in the Chl-binding proteins as well as the Chl energy levels of their photosynthetic complexes. This spectral-type divergence is associated with differences in the wavelength dependence of both growth rate and photosynthetic oxygen evolution. We conclude that the divergence of the light-harvesting apparatus has consequently impacted A. marina ecological diversification through specialization on different far-red photons for photosynthesis.

Chlorophyll triplet states in thylakoid membranes of Acaryochloris marina. Evidence for a triplet state sitting on the photosystem I primary donor populated by intersystem crossing.

Photo-induced triplet states in the thylakoid membranes isolated from the cyanobacterium Acaryocholoris marina, that harbours Chlorophyll (Chl) d as its main chromophore, have been investigated by Optically Detected Magnetic Resonance (ODMR) and time-resolved Electron Paramagnetic Resonance (TR-EPR). Thylakoids were subjected to treatments aimed at poising the redox state of the terminal electron transfer acceptors and donors of Photosystem II (PSII) and Photosystem I (PSI), respectively. Under ambient redox conditions, four Chl d triplet populations were detectable, identifiable by their characteristic zero field splitting parameters, after deconvolution of the Fluorescence Detected Magnetic Resonance (FDMR) spectra. Illumination in the presence of the redox mediator N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and sodium ascorbate at room temperature led to a redistribution of the triplet populations, with T3 (|D|= 0.0245 cm-1, |E|= 0.0042 cm-1) becoming dominant and increasing in intensity with respect to untreated samples. A second triplet population (T4, |D|= 0.0248 cm-1, |E|= 0.0040 cm-1) having an intensity ratio of about 1:4 with respect to T3 was also detectable after illumination in the presence of TMPD and ascorbate. The microwave-induced Triplet-minus-Singlet spectrum acquired at the maximum of the |D|-|E| transition (610 MHz) displays a broad minimum at 740 nm, accompanied by a set of complex spectral features that overall resemble, despite showing further fine spectral structure, the previously reported Triplet-minus-Singlet spectrum attributed to the recombination triplet of PSI reaction centre, 3 P 740 [Schenderlein M, Çetin M, Barber J, et al. Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium Acaryochloris marina. Biochim Biophys Acta 1777:1400-1408]. However, TR-EPR experiments indicate that this triplet displays an eaeaea electron spin polarisation pattern which is characteristic of triplet sublevels populated by intersystem crossing rather than recombination, for which an aeeaae polarisation pattern is expected instead. It is proposed that the observed triplet, which leads to the bleaching of the P740 singlet state, sits on the PSI reaction centre.

Isolation and characterization of trimeric and monomeric PSI cores from Acaryochloris marina MBIC11017.

Photosystem I (PSI) catalyzes light-induced electron-transfer reactions and has been observed to exhibit various oligomeric states and different energy levels of chlorophylls (Chls) in response to oligomerization. However, the biochemical and spectroscopic properties of a PSI monomer containing Chls d are not well understood. In this study, we successfully isolated and characterized PSI monomers from the cyanobacterium Acaryochloris marina MBIC11017, and compared their properties with those of the A. marina PSI trimer. The PSI trimers and monomers were prepared using trehalose density gradient centrifugation after anion-exchange and hydrophobic interaction chromatography. The polypeptide composition of the PSI monomer was found to be consistent with that of the PSI trimer. The absorption spectrum of the PSI monomer showed the Qy band of Chl d at 704 nm, which was blue-shifted from the peak at 707 nm observed in the PSI-trimer spectrum. The fluorescence-emission spectrum of the PSI monomer measured at 77 K exhibited a peak at 730 nm without a broad shoulder in the range of 745-780 nm, which was clearly observed in the PSI-trimer spectrum. These spectroscopic properties of the A. marina PSI trimer and monomer suggest different formations of low-energy Chls d between the two types of PSI cores. Based on these findings, we discuss the location of low-energy Chls d in A. marina PSIs.

Reacquisition of light-harvesting genes in a marine cyanobacterium confers a broader solar niche.

The evolution of phenotypic plasticity, i.e., the environmental induction of alternative phenotypes by the same genotype, can be an important mechanism of biological diversification.1,2 For example, an evolved increase in plasticity may promote ecological niche expansion as well as the innovation of novel traits;3 however, both the role of phenotypic plasticity in adaptive evolution and its underlying mechanisms are still poorly understood.4,5 Here, we report that the Chlorophyll d-producing marine cyanobacterium Acaryochloris marina strain MBIC11017 has evolved greater photosynthetic plasticity by reacquiring light-harvesting genes via horizontal gene transfer. The genes, which had been lost by the A. marina ancestor, are involved in the production and degradation of the light-harvesting phycobiliprotein phycocyanin. A. marina MBIC11017 exhibits a high degree of wavelength-dependence in phycocyanin production, and this ability enables it to grow with yellow and green light wavelengths that are inaccessible to other A. marina. Consequently, this strain has a broader solar niche than its close relatives. We discuss the role of horizontal gene transfer for regaining a lost phenotype in light of Dollo's Law6 that the loss of a complex trait is irreversible.

The identification of IsiA proteins binding chlorophyll d in the cyanobacterium Acaryochloris marina.

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (-F6) isolated from iron-deficient culture contained Chl d-bound PSI-IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).

Excitation energy transfer in phycobiliproteins of the cyanobacterium Acaryochloris marina investigated by spectral hole burning.

The cyanobacterium Acaryochloris marina developed two types of antenna complexes, which contain chlorophyll-d (Chl d) and phycocyanobilin (PCB) as light-harvesting pigment molecules, respectively. The latter membrane-extrinsic complexes are denoted as phycobiliproteins (PBPs). Spectral hole burning was employed to study excitation energy transfer and electron-phonon coupling in PBPs. The data reveal a rich spectral substructure with a total of four low-energy electronic states whose absorption bands peak at 633, 644, 654, and at about 673 nm. The electronic states at ~633 and 644 nm can be tentatively attributed to phycocyanin (PC) and allophycocyanin (APC), respectively. The remaining low-energy electronic states including the terminal emitter at 673 nm may be associated with different isoforms of PC, APC, or the linker protein. Furthermore, the hole burning data reveal a large number of excited state vibrational frequencies, which are characteristic for the chromophore PCB. In summary, the results are in good agreement with the low-energy level structure of PBPs and electron-phonon coupling parameters reported by Gryliuk et al. (BBA 1837:1490-1499, 2014) based on difference fluorescence line-narrowing experiments.

Energy Transfer Kinetics in Photosynthesis as an Inspiration for Improving Organic Solar Cells.

Clues to designing highly efficient organic solar cells may lie in understanding the architecture of light-harvesting systems and exciton energy transfer (EET) processes in very efficient photosynthetic organisms. Here, we compare the kinetics of excitation energy tunnelling from the intact phycobilisome (PBS) light-harvesting antenna system to the reaction center in photosystem II in intact cells of the cyanobacterium Acaryochloris marina with the charge transfer after conversion of photons into photocurrent in vertically aligned carbon nanotube (va-CNT) organic solar cells with poly(3-hexyl)thiophene (P3HT) as the pigment. We find that the kinetics in electron hole creation following excitation at 600 nm in both PBS and va-CNT solar cells to be 450 and 500 fs, respectively. The EET process has a 3 and 14 ps pathway in the PBS, while in va-CNT solar cell devices, the charge trapping in the CNT takes 11 and 258 ps. We show that the main hindrance to efficiency of va-CNT organic solar cells is the slow migration of the charges after exciton formation.

Solution structure and excitation energy transfer in phycobiliproteins of Acaryochloris marina investigated by small angle scattering.

The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

Excitation energy transfer and electron-vibrational coupling in phycobiliproteins of the cyanobacterium Acaryochloris marina investigated by site-selective spectroscopy.

In adaption to its specific environmental conditions, the cyanobacterium Acaryochloris marina developed two different types of light-harvesting complexes: chlorophyll-d-containing membrane-intrinsic complexes and phycocyanobilin (PCB) - containing phycobiliprotein (PBP) complexes. The latter complexes are believed to form a rod-shaped structure comprising three homo-hexamers of phycocyanin (PC), one hetero-hexamer of phycocyanin and allophycocyanin (APC) and probably a linker protein connecting the PBPs to the reaction centre. Excitation energy transfer and electron-vibrational coupling in PBPs have been investigated by selectively excited fluorescence spectra. The data reveal a rich spectral substructure with a total of five low-energy electronic states with fluorescence bands at 635nm, 645nm, 654nm, 659nm and a terminal emitter at about 673 nm. The electronic states at ~635 and 645 nm are tentatively attributed to PC and APC, respectively, while an apparent heterogeneity among PC subunits may also play a role. The other fluorescence bands may be associated with three different isoforms of the linker protein. Furthermore, a large number of vibrational features can be identified for each electronic state with intense phonon sidebands peaking at about 31 to 37cm⁻¹, which are among the highest phonon frequencies observed for photosynthetic antenna complexes. The corresponding Huang-Rhys factors S fall in the range between 0.98 (terminal emitter), 1.15 (APC), and 1.42 (PC). Two characteristic vibronic lines at about 1580 and 1634cm⁻¹ appear to reflect CNH⁺ and CC stretching modes of the PCB chromophore, respectively. The exact phonon and vibrational frequencies vary with electronic state implying that the respective PCB chromophores are bound to different protein environments. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.