Photosynthesis Research (v.135, #1-3)

Introduction: light harvesting for photosynthesis by Anjali Pandit; Ivo H. M. van Stokkum; Herbert van Amerongen; Roberta Croce (1-2).

Progress in measuring and understanding the mechanism of the elementary energy transfer steps in photosynthetic light harvesting from roughly 1949 to the present is sketched with a focus on the group of scientists born in 1949 ± 1. Improvements in structural knowledge, laser spectroscopic methods, and quantum dynamical theories have led to the ability to record and calculate with reasonable accuracy the timescales of elementary energy transfer steps. The significance of delocalized excited states and of near-field Coulombic coupling is noted. The microscopic understanding enables consistent coarse graining and should enable a much-improved understanding of the regulation of photosynthetic light harvesting.
Keywords: Femtosecond spectroscopy; Förster theory; Generalized Förster theory; Hierarchy equations of motion

Characterisation of a pucBA deletion mutant from Rhodopseudomonas palustris lacking all but the pucBAd genes by June Southall; Sarah L. Henry; Alastair T. Gardiner; Aleksander W. Roszak; William Mullen; Anne-Marie Carey; Sharon M. Kelly; Claire Ortmann de Percin Northumberland; Richard J. Cogdell (9-21).
Rhodopseudomonas palustris is a species of purple photosynthetic bacteria that has a multigene family of puc genes that encode the alpha and beta apoproteins, which form the LH2 complexes. A genetic dissection strategy has been adopted in order to try and understand which spectroscopic form of LH2 these different genes produce. This paper presents a characterisation of one of the deletion mutants generated in this program, the pucBAd only mutant. This mutant produces an unusual spectroscopic form of LH2 that only has a single large NIR absorption band at 800 nm. Spectroscopic and pigment analyses on this complex suggest that it has basically a similar overall structure as that of the wild-type HL LH2 complex. The mutant has the unique phenotype where the mutant LH2 complex is only produced when cells are grown at LL. At HL the mutant only produces the LH1-RC core complex.
Keywords: Photosynthesis; Purple bacteria; Puc genes; Light-harvesting; LH2

C-terminal cleavage of the LH1 α-polypeptide in the Sr2+-cultured Thermochromatium tepidum by Yukihiro Kimura; Tomoaki Kawakami; Teruhisa Arikawa; Yong Li; Long-Jiang Yu; Takashi Ohno; Michael T. Madigan; Zheng-Yu Wang-Otomo (23-31).
The light-harvesting 1 reaction center (LH1-RC) complex in the thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum binds Ca ions as cofactors, and Ca-binding is largely involved in its characteristic Q y absorption at 915 nm and enhanced thermostability. Ca2+ can be biosynthetically replaced by Sr2+ in growing cultures of Tch. tepidum. However, the resulting Sr2+-substituted LH1-RC complexes in such cells do not display the absorption maximum and thermostability of those from Ca2+-grown cells, signaling that inherent structural differences exist in the LH1 complexes between the Ca2+- and Sr2+-cultured cells. In this study, we examined the effects of the biosynthetic Sr2+-substitution and limited proteolysis on the spectral properties and thermostability of the Tch. tepidum LH1-RC complex. Preferential truncation of two consecutive, positively charged Lys residues at the C-terminus of the LH1 α-polypeptide was observed for the Sr2+-cultured cells. A proportion of the truncated LH1 α-polypeptide increased during repeated subculturing in the Sr2+-substituted medium. This result suggests that the truncation is a biochemical adaptation to reduce the electrostatic interactions and/or steric repulsion at the C-terminus when Sr2+ substitutes for Ca2+ in the LH1 complex. Limited proteolysis of the native Ca2+-LH1 complex with lysyl protease revealed selective truncations at the Lys residues in both C- and N-terminal extensions of the α- and β-polypeptides. The spectral properties and thermostability of the partially digested native LH1-RC complexes were similar to those of the biosynthetically Sr2+-substituted LH1-RC complexes in their Ca2+-bound forms. Based on these findings, we propose that the C-terminal domain of the LH1 α-polypeptide plays important roles in retaining proper structure and function of the LH1-RC complex in Tch. tepidum.
Keywords: Thermochromatium tepidum ; Thermophilic purple bacteria; Light-harvesting 1 reaction center; Calcium; Strontium; C-terminal truncation

Carotenoid to bacteriochlorophyll energy transfer in the RC–LH1–PufX complex from Rhodobacter sphaeroides containing the extended conjugation keto-carotenoid diketospirilloxanthin by Václav Šlouf; Gürkan Keşan; Radek Litvín; David J. K. Swainsbury; Elizabeth C. Martin; C. Neil Hunter; Tomáš Polívka (33-43).
RC–LH1–PufX complexes from a genetically modified strain of Rhodobacter sphaeroides that accumulates carotenoids with very long conjugation were studied by ultrafast transient absorption spectroscopy. The complexes predominantly bind the carotenoid diketospirilloxanthin, constituting about 75% of the total carotenoids, which has 13 conjugated C=C bonds, and the conjugation is further extended to two terminal keto groups. Excitation of diketospirilloxanthin in the RC–LH1–PufX complex demonstrates fully functional energy transfer from diketospirilloxanthin to BChl a in the LH1 antenna. As for other purple bacterial LH complexes having carotenoids with long conjugation, the main energy transfer route is via the S2–Qx pathway. However, in contrast to LH2 complexes binding diketospirilloxanthin, in RC–LH1–PufX we observe an additional, minor energy transfer pathway associated with the S1 state of diketospirilloxanthin. By comparing the spectral properties of the S1 state of diketospirilloxanthin in solution, in LH2, and in RC–LH1–PufX, we propose that the carotenoid-binding site in RC–LH1–PufX activates the ICT state of diketospirilloxanthin, resulting in the opening of a minor S1/ICT-mediated energy transfer channel.
Keywords: Light-harvesting; Carotenoids; Ultrafast spectroscopy; Purple bacteria; Energy transfer; Intramolecular charge transfer state

Carotenoid-to-bacteriochlorophyll energy transfer through vibronic coupling in LH2 from Phaeosprillum molischianum by Erling Thyrhaug; Craig N. Lincoln; Federico Branchi; Giulio Cerullo; Václav Perlík; František Šanda; Heiko Lokstein; Jürgen Hauer (45-54).
The peripheral light-harvesting antenna complex (LH2) of purple photosynthetic bacteria is an ideal testing ground for models of structure–function relationships due to its well-determined molecular structure and ultrafast energy deactivation. It has been the target for numerous studies in both theory and ultrafast spectroscopy; nevertheless, certain aspects of the convoluted relaxation network of LH2 lack a satisfactory explanation by conventional theories. For example, the initial carotenoid-to-bacteriochlorophyll energy transfer step necessary on visible light excitation was long considered to follow the Förster mechanism, even though transfer times as short as 40 femtoseconds (fs) have been observed. Such transfer times are hard to accommodate by Förster theory, as the moderate coupling strengths found in LH2 suggest much slower transfer within this framework. In this study, we investigate LH2 from Phaeospirillum (Ph.) molischianum in two types of transient absorption experiments—with narrowband pump and white-light probe resulting in 100 fs time resolution, and with degenerate broadband 10 fs pump and probe pulses. With regard to the split Qx band in this system, we show that vibronically mediated transfer explains both the ultrafast carotenoid-to-B850 transfer, and the almost complete lack of transfer to B800. These results are beyond Förster theory, which predicts an almost equal partition between the two channels.
Keywords: LH2; Ultrafast spectroscopy; Excitation energy transfer; Excitons; Photosynthesis

Effects of tunable excitation in carotenoids explained by the vibrational energy relaxation approach by Vytautas Balevičius Jr; Craig N. Lincoln; Daniele Viola; Giulio Cerullo; Jürgen Hauer; Darius Abramavicius (55-64).
Carotenoids are fundamental building blocks of natural light harvesters with convoluted and ultrafast energy deactivation networks. In order to disentangle such complex relaxation dynamics, several studies focused on transient absorption measurements and their dependence on the pump wavelength. However, such findings are inconclusive and sometimes contradictory. In this study, we compare internal conversion dynamics in $$eta$$ β -carotene, pumped at the first, second, and third vibronic progression peak. Instead of employing data fitting algorithms based on global analysis of the transient absorption spectra, we apply a fully quantum mechanical model to treat the high-frequency symmetric carbon–carbon (C=C and C–C) stretching modes explicitly. This model successfully describes observed population dynamics as well as spectral line shapes in their time-dependence and allows us to reach two conclusions: Firstly, the broadening of the induced absorption upon excess excitation is an effect of vibrational cooling in the first excited state ( $$S_{1}$$ S 1 ). Secondly, the internal conversion rate between the second excited state ( $$S_{2}$$ S 2 ) and $$S_{1}$$ S 1 crucially depends on the relative curve displacement. The latter point serves as a new perspective on solvent- and excitation wavelength-dependent experiments and lifts contradictions between several studies found in literature.
Keywords: Internal conversion; $$eta$$ β -carotene; Transient absorption spectroscopy; Vibrational cooling

An improved crystal structure of C-phycoerythrin from the marine cyanobacterium Phormidium sp. A09DM by Ravi R. Sonani; Aleksander W. Roszak; Claire Ortmann de Percin Northumberland; Datta Madamwar; Richard J. Cogdell (65-78).
C-Phycoerythrin (PE) from Phormidium sp. A09DM has been crystallized using different conditions and its structure determined to atomic resolution (1.14 Å). In order for the pigment present, phycoerythrobilin (PEB), to function as an efficient light-harvesting molecule it must be held rigidly (Kupka and Scheer in Biochim Biophys Acta 1777:94–103, 2008) and, moreover, the different PEB molecules in PE must be arranged, relative to each other, so as to promote efficient energy transfer between them. This improved structure has allowed us to define in great detail the structure of the PEBs and their binding sites. These precise structural details will facilitate theoretical calculations of each PEB’s spectroscopic properties. It was possible, however, to suggest a model for which chromophores contribute to the different regions of absorption spectrum and propose a tentative scheme for energy transfer. We show that some subtle differences in one of these PEB binding sites in two of the 12 subunits are caused by crystal contacts between neighboring hexamers in the crystal lattice. This explains some of the differences seen in previous lower resolution structures determined at two different pH values (Kumar et al. in Photosyn Res 129:17–28, 2016).
Keywords: Photosynthesis; Cyanobacteria; Phycobilisomes; Phycoerythrin; PEB chromophores; Atomic resolution crystal structure

Picosecond excitation energy transfer of allophycocyanin studied in solution and in crystals by Reza Ranjbar Choubeh; Ravi R. Sonani; Datta Madamwar; Paul C. Struik; Arjen N. Bader; Bruno Robert; Herbert van Amerongen (79-86).
Cyanobacteria perform photosynthesis with the use of large light-harvesting antennae called phycobilisomes (PBSs). These hemispherical PBSs contain hundreds of open-chain tetrapyrrole chromophores bound to different peptides, providing an arrangement in which excitation energy is funnelled towards the PBS core from where it can be transferred to photosystem I and/or photosystem II. In the PBS core, many allophycocyanin (APC) trimers are present, red-light-absorbing phycobiliproteins that covalently bind phycocyanobilin (PCB) chromophores. APC trimers were amongst the first light-harvesting complexes to be crystallized. APC trimers have two spectrally different PCBs per monomer, a high- and a low-energy pigment. The crystal structure of the APC trimer reveals the close distance (~21 Å) between those two chromophores (the distance within one monomer is ~51 Å) and this explains the ultrafast (~1 ps) excitation energy transfer (EET) between them. Both chromophores adopt a somewhat different structure, which is held responsible for their spectral difference. Here we used spectrally resolved picosecond fluorescence to study EET in these APC trimers both in crystallized and in solubilized form. We found that not all closely spaced pigment couples consist of a low- and a high-energy pigment. In ~10% of the cases, a couple consists of two high-energy pigments. EET to a low-energy pigment, which can spectrally be resolved, occurs on a time scale of tens of picoseconds. This transfer turns out to be three times faster in the crystal than in the solution. The spectral characteristics and the time scale of this transfer component are similar to what have been observed in the whole cells of Synechocystis sp. PCC 6803, for which it was ascribed to EET from C-phycocyanin to APC. The present results thus demonstrate that part of this transfer should probably also be ascribed to EET within APC trimers.
Keywords: Allophycocyanin crystals; Excitation energy transfer; Phycobilisome; Cyanobacteria; Time-resolved fluorescence spectroscopy

A functional compartmental model of the Synechocystis PCC 6803 phycobilisome by Ivo H. M. van Stokkum; Michal Gwizdala; Lijin Tian; Joris J. Snellenburg; Rienk van Grondelle; Herbert van Amerongen; Rudi Berera (87-102).
In the light-harvesting antenna of the Synechocystis PCC 6803 phycobilisome (PB), the core consists of three cylinders, each composed of four disks, whereas each of the six rods consists of up to three hexamers (Arteni et al., Biochim Biophys Acta 1787(4):272–279, 2009). The rods and core contain phycocyanin and allophycocyanin pigments, respectively. Together these pigments absorb light between 400 and 650 nm. Time-resolved difference absorption spectra from wild-type PB and rod mutants have been measured in different quenching and annihilation conditions. Based upon a global analysis of these data and of published time-resolved emission spectra, a functional compartmental model of the phycobilisome is proposed. The model describes all experiments with a common set of parameters. Three annihilation time constants are estimated, 3, 25, and 147 ps, which represent, respectively, intradisk, interdisk/intracylinder, and intercylinder annihilation. The species-associated difference absorption and emission spectra of two phycocyanin and two allophycocyanin pigments are consistently estimated, as well as all the excitation energy transfer rates. Thus, the wild-type PB containing 396 pigments can be described by a functional compartmental model of 22 compartments. When the interhexamer equilibration within a rod is not taken into account, this can be further simplified to ten compartments, which is the minimal model. In this model, the slowest excitation energy transfer rates are between the core cylinders (time constants 115–145 ps), and between the rods and the core (time constants 68–115 ps).
Keywords: Excitation energy transfer; Global analysis; Light harvesting; Orange carotenoid protein; Target analysis

Small CAB-like proteins (SCPs) are single-helix light-harvesting-like proteins found in all organisms performing oxygenic photosynthesis. We investigated the effect of growth in moderate salt stress on these stress-induced proteins in the cyanobacterium Synechocystis sp. PCC 6803 depleted of Photosystem I (PSI), which expresses SCPs constitutively, and compared these cells with a PSI-less/ScpABCDE mutant. SCPs, by stabilizing chlorophyll-binding proteins and Photosystem II (PSII) assembly, protect PSII from photoinhibitory damages, and in their absence electrons accumulate and will lead to ROS formation. The presence of 0.2 M NaCl in the growth medium increased the respiratory activity and other PSII electron sinks in the PSI-less/ScpABCDE strain. We postulate that this salt-induced effect consumes the excess of PSII-generated electrons, reduces the pressure of the electron transport chain, and thereby prevents 1O2 production.
Keywords: Small CAB-like proteins (SCPs); Photosystem II photoinhibition; Singlet oxygen; Salt stress; Terminal oxidases

Energy transfer and trapping in Synechococcus WH 7803 by Alonso M. Acuña; Claire Lemaire; Rienk van Grondelle; Bruno Robert; Ivo H. M. van Stokkum (115-124).
Excitation energy transfer (EET) and trapping in Synechococcus WH 7803 whole cells and isolated photosystem I (PSI) complexes have been studied by time-resolved emission spectroscopy at room temperature (RT) and at 77 K. With the help of global and target analysis, the pathways of EET and the charge separation dynamics have been identified. Energy absorbed in the phycobilisome (PB) rods by the abundant phycoerythrin (PE) is funneled to phycocyanin (PC645) and from there to the core that contains allophycocyanin (APC660 and APC680). Intra-PB EET rates have been estimated to range from 11 to 68/ns. It was estimated that at RT, the terminal emitter of the phycobilisome, APC680, transfers its energy at a rate of 90/ns to PSI and at a rate of 50/ns to PSII. At 77 K, the redshifted Chl a states in the PSI core were heterogeneous, with maximum emission at 697 and 707 nm. In 72% of the PSI complexes, the bulk Chl a in equilibrium with F697 decayed with a main trapping lifetime of 39 ps.
Keywords: Excitation energy transfer; Global analysis; Light harvesting; Target analysis

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein by Marcus Moldenhauer; Nikolai N. Sluchanko; Neslihan N. Tavraz; Cornelia Junghans; David Buhrke; Mario Willoweit; Leonardo Chiappisi; Franz-Josef Schmitt; Vladana Vukojević; Evgeny A. Shirshin; Vladimir Y. Ponomarev; Vladimir Z. Paschenko; Michael Gradzielski; Eugene G. Maksimov; Thomas Friedrich (125-139).
In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1–11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.
Keywords: Orange carotenoid protein; Fluorescence recovery protein; Fluorescein-maleimide; Site-specific fluorescence labeling; Mass spectroscopy; Fluorescence correlation spectroscopy

Erratum to: Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein by Marcus Moldenhauer; Nikolai N. Sluchanko; Neslihan N. Tavraz; Cornelia Junghans; David Buhrke; Mario Willoweit; Leonardo Chiappisi; Franz‑Josef Schmitt; Vladana Vukojević; Evgeny A. Shirshin; Vladimir Y. Ponomarev; Vladimir Z. Paschenko; Michael Gradzielski; Eugene G. Maksimov; Thomas Friedrich (141-142).
In Fig. 1a in the original article, the amino acid side chains were incorrectly labeled in the structure representation of the orange carotenoid protein (OCP). The corrected figure is printed in this erratum.

Photoactivation and relaxation studies on the cyanobacterial orange carotenoid protein in the presence of copper ion by Haijun Liu; Yue Lu; Benjamin Wolf; Rafael Saer; Jeremy D. King; Robert E. Blankenship (143-147).
Photosynthesis starts with absorption of light energy by light-harvesting antenna complexes with subsequent production of energy-rich organic compounds. However, all photosynthetic organisms face the challenge of excess photochemical conversion capacity. In cyanobacteria, non-photochemical quenching (NPQ) performed by the orange carotenoid protein (OCP) is one of the most important mechanisms to regulate the light energy captured by light-harvesting antennas. This regulation permits the cell to meet its cellular energy requirements and at the same time protects the photosynthetic apparatus under fluctuating light conditions. Several reports have revealed that thermal dissipation increases under excess copper in plants. To explore the effects and mechanisms of copper on cyanobacteria NPQ, photoactivation and relaxation of OCP in the presence of copper were examined in this communication. When OCPo (OCP at orange state) is converted into OCPr(OCP at red state), copper ion has no effect on the photoactivation kinetics. Relaxation of OCPr to OCPo, however, is largely delayed—almost completely blocked, in the presence of copper. Even the addition of the fluorescence recovery protein (FRP) cannot activate the relaxation process. Native polyacrylamide gel electrophoresis (PAGE) analysis result indicates the heterogeneous population of Cu2+-locked OCPr. The Cu2+-OCP binding constant was estimated using a hyperbolic binding curve. Functional roles of copper-binding OCP in vivo are discussed.
Keywords: Copper; Orange carotenoid protein; Photosynthesis; Photoprotection

Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded β-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active α1β·α2β-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.
Keywords: Cryptophyta; Phycobilin; Phycobiliprotein; Translation; TAT-pathway; Proteomics

The identification of IsiA proteins binding chlorophyll d in the cyanobacterium Acaryochloris marina by Zheng-Ke Li; Yan-Chao Yin; Lu-Dan Zhang; Zhong-Chun Zhang; Guo-Zheng Dai; Min Chen; Bao-Sheng Qiu (165-175).
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).
Keywords: Accessory chlorophyll-binding proteins (CBPs); Iron limitation; Iron-stress-induced protein A (IsiA); Photosynthesis; Six transmembrane helix family of chlorophyll-binding proteins/antenna; Acaryochloris marina

Characterization of a newly isolated freshwater Eustigmatophyte alga capable of utilizing far-red light as its sole light source by Benjamin M. Wolf; Dariusz M. Niedzwiedzki; Nikki Cecil M. Magdaong; Robyn Roth; Ursula Goodenough; Robert E. Blankenship (177-189).
Oxygenic phototrophs typically utilize visible light (400–700 nm) to drive photosynthesis. However, a large fraction of the energy in sunlight is contained in the far-red region, which encompasses light beyond 700 nm. In nature, certain niche environments contain high levels of this far-red light due to filtering by other phototrophs, and in these environments, organisms with photosynthetic antenna systems adapted to absorbing far-red light are able to thrive. We used selective far-red light conditions to isolate such organisms in environmental samples. One cultured organism, the Eustigmatophyte alga Forest Park Isolate 5 (FP5), is able to absorb far-red light using a chlorophyll (Chl) a-containing antenna complex, and is able to grow under solely far-red light. Here we characterize the antenna system from this organism, which is able to shift the absorption of Chl a to >705 nm.
Keywords: Light harvesting complex; Stramenopila; Eustigmatophyte; Far-red light; Antenna

Light-harvesting complexes of Botryococcus braunii by Tomas E. van den Berg; Bart van Oort; Roberta Croce (191-201).
The colonial green alga Botryococcus braunii (BB) is a potential source of biofuel due to its natural high hydrocarbon content. Unfortunately, its slow growth limits its biotechnological potential. Understanding its photosynthetic machinery could help to identify possible growth limitations. Here, we present the first study on BB light-harvesting complexes (LHCs). We purified two LHC fractions containing the complexes in monomeric and trimeric form. Both fractions contained at least two proteins with molecular weight (MW) around 25 kDa. The chlorophyll composition is similar to that of the LHCII of plants; in contrast, the main xanthophyll is loroxanthin, which substitutes lutein in most binding sites. Circular dichroism and 77 K absorption spectra lack typical differences between monomeric and trimeric complexes, suggesting that intermonomer interactions do not play a role in BB LHCs. This is in agreement with the low stability of the BB LHCII trimers as compared to the complexes of plants, which could be related to loroxanthin binding in the central (L1 and L2) binding sites. The properties of BB LHCII are similar to those of plant LHCII, indicating a similar pigment organization. Differences are a higher content of red chlorophyll a, similar to plant Lhcb3. These differences and the different Xan composition had no effect on excitation energy transfer or fluorescence lifetimes, which were similar to plant LHCII.
Keywords: Botryococcus braunii ; LHC; Light-harvesting; Loroxanthin; Pigment-protein complexes

The structure of FCPb, a light-harvesting complex in the diatom Cyclotella meneghiniana by Anja Röding; Egbert Boekema; Claudia Büchel (203-211).
Diatoms possess fucoxanthin chlorophyll proteins (FCP) as light-harvesting systems. These membrane intrinsic proteins bind fucoxanthin as major carotenoid and Chl c as accessory chlorophyll. The relatively high sequence homology to higher plant light-harvesting complex II gave rise to the assumption of a similar overall structure. From centric diatoms like Cyclotella meneghiniana, however, two major FCP complexes can be isolated. FCPa, composed of Fcp2 and Fcp6 subunits, was demonstrated to be trimeric, whereas FCPb, known to contain Fcp5 polypeptides, is of higher oligomeric state. No molecular structure of either complex is available so far. Here we used electron microscopy and single particle analysis to elucidate the overall architecture of FCPb. The complexes are built from trimers as basic unit, assembling into nonameric moieties. The trimer itself is smaller, i.e. more compact than LHCII, but the main structural features are conserved.
Keywords: Electron microscopy; Fucoxanthin chlorophyll protein; Oligomeric state; Single particle analysis

Quenching of chlorophyll triplet states by carotenoids in algal light-harvesting complexes related to fucoxanthin-chlorophyll protein by Petro Khoroshyy; David Bína; Zdenko Gardian; Radek Litvín; Jan Alster; Jakub Pšenčík (213-225).
We have used time-resolved absorption and fluorescence spectroscopy with nanosecond resolution to study triplet energy transfer from chlorophylls to carotenoids in a protective process that prevents the formation of reactive singlet oxygen. The light-harvesting complexes studied were isolated from Chromera velia, belonging to a group Alveolata, and Xanthonema debile and Nannochloropsis oceanica, both from Stramenopiles. All three light-harvesting complexes are related to fucoxanthin-chlorophyll protein, but contain only chlorophyll a and no chlorophyll c. In addition, they differ in the carotenoid content. This composition of the complexes allowed us to study the quenching of chlorophyll a triplet states by different carotenoids in a comparable environment. The triplet states of chlorophylls bound to the light-harvesting complexes were quenched by carotenoids with an efficiency close to 100%. Carotenoid triplet states were observed to rise with a ~5 ns lifetime and were spectrally and kinetically homogeneous. The triplet states were formed predominantly on the red-most chlorophylls and were quenched by carotenoids which were further identified or at least spectrally characterized.
Keywords: Algae; Energy transfer; Light harvesting; Photoprotection; Photosynthesis; Transient spectroscopy

In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells by Fatemeh Azadi-Chegeni; Christo Schiphorst; Anjali Pandit (227-237).
We report the application of NMR dynamic spectral editing for probing the structure and dynamics of molecular constituents in fresh, intact cells and in freshly prepared thylakoid membranes of Chlamydomonas reinhardtii (Cr.) green algae. For isotope labeling, wild-type Cr. cells were grown on 13C acetate-enriched minimal medium. 1D 13C J-coupling based and dipolar-based MAS NMR spectra were applied to distinguish 13C resonances of different molecular components. 1D spectra were recorded over a physiological temperature range, and whole-cell spectra were compared to those taken from thylakoid membranes, evaluating their composition and dynamics. A theoretical model for NMR polarization transfer was used to simulate the relative intensities of direct, J-coupling, and dipolar-based polarization from which the degree of lipid segmental order and rotational dynamics of the lipid acyl chains were estimated. We observe that thylakoid lipid signals dominate the lipid spectral profile of whole algae cells, demonstrating that with our novel method, thylakoid membrane characteristics can be detected with atomistic precision inside intact photosynthetic cells. The experimental procedure is rapid and applicable to fresh cell cultures, and could be used as an original approach for detecting chemical profiles, and molecular structure and dynamics of photosynthetic membranes in vivo in functional states.
Keywords: Solid-state NMR; Polarization transfer; Conformational dynamics; Thylakoid membrane

Excitation transfer and trapping kinetics in plant photosystem I probed by two-dimensional electronic spectroscopy by Parveen Akhtar; Cheng Zhang; Zhengtang Liu; Howe-Siang Tan; Petar H. Lambrev (239-250).
Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon’s energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet–singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5 ps. We further observed spectral equilibration component in the core complex with a 3–4 ps lifetime between the bulk Chl states and a state absorbing at 700 nm. Trapping in the core complex occurred with a 20 ps lifetime, which in the supercomplex split into two lifetimes, 16 ps and 67–75 ps. The experimental data could be modelled with two alternative models resulting in equally good fits—a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3–4 ps component is ascribed to equilibration with a “red” core antenna pool absorbing at 700 nm. Conversely, if these low-energy states are identified with the P700 reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model.
Keywords: Excitation energy transfer; Light harvesting; Light-harvesting complexes; Multidimensional spectroscopy; Ultrafast spectroscopy

LHCI, the peripheral antenna system of Photosystem I, includes four light-harvesting proteins (Lhca1-Lhca4) in higher plants, all of which are devoid in the Arabidopsis thaliana knock-out mutant ΔLhca. PSI absorption cross-section was reduced in the mutant, thus affecting the redox balance of the photosynthetic electron chain and resulting in a more reduced PQ with respect to the wild type. ΔLhca plants developed compensatory response by enhancing LHCII binding to PSI. However, the amplitude of state transitions, as measured from changes of chlorophyll fluorescence in vivo, was unexpectedly low than the high level of PSI–LHCII supercomplex established. In order to elucidate the reasons for discrepancy, we further analyzed state transition in ΔLhca plants. The STN7 kinase was fully active in the mutant as judged from up-regulation of LHCII phosphorylation in state II. Instead, the lateral heterogeneity of thylakoids was affected by lack of LHCI, with LHCII being enriched in stroma membranes with respect to the wild type. Re-distribution of this complex affected the overall fluorescence yield of thylakoids already in state I and minimized changes in RT fluorescence yield when LHCII did connect to PSI reaction center. We conclude that interpretation of chlorophyll fluorescence analysis of state transitions becomes problematic when applied to mutants whose thylakoid architecture is significantly modified with respect to the wild type.
Keywords: Chloroplast; Photosystem I; LHCI; LHCII; State transitions; Grana; Stroma lamellae

High light acclimation of Chromera velia points to photoprotective NPQ by Erica Belgio; Eliška Trsková; Eva Kotabová; Daniela Ewe; Ondřej Prášil; Radek Kaňa (263-274).
It has previously been shown that the long-term treatment of Arabidopsis thaliana with the chloroplast inhibitor lincomycin leads to photosynthetic membranes enriched in antennas, strongly reduced in photosystem II reaction centers (PSII) and with enhanced nonphotochemical quenching (NPQ) (Belgio et al. Biophys J 102:2761–2771, 2012). Here, a similar physiological response was found in the microalga Chromera velia grown under high light (HL). In comparison to cells acclimated to low light, HL cells displayed a severe re-organization of the photosynthetic membrane characterized by (1) a reduction of PSII but similar antenna content; (2) partial uncoupling of antennas from PSII; (3) enhanced NPQ. The decrease in the number of PSII represents a rather unusual acclimation response compared to other phototrophs, where a smaller PSII antenna size is more commonly found under high light. Despite the diminished PSII content, no net damage could be detected on the basis of the Photosynthesis versus irradiance curve and electron transport rates pointing at the excess capacity of PSII. We therefore concluded that the photoinhibition is minimized under high light by a lower PSII content and that cells are protected by NPQ in the antennas.
Keywords: Nonphotochemical quenching; Photoinhibition; Chromera velia alga; High light acclimation; Uncoupling of antennas from Photosystem II.

Can red-emitting state be responsible for fluorescence quenching in LHCII aggregates? by Andrius Gelzinis; Jevgenij Chmeliov; Alexander V. Ruban; Leonas Valkunas (275-284).
Non-photochemical quenching (NPQ) is responsible for protecting the light-harvesting apparatus of plants from damage at high light conditions. Although it is agreed that the major part of NPQ, an energy-dependent quenching (qE), originates in the light-harvesting antenna, its exact mechanism is still debated. In our earlier work (Chmeliov et al. in Nat Plants 2:16045, 2016), we have analyzed the time-resolved fluorescence (TRF) from the trimers and aggregates of the major light-harvesting complexes of plants (LHCII) over a broad temperature range and came to a conclusion that three distinct states are required to describe the experimental data: two of them correspond to the emission bands centered at ~680 and ~700 nm, and the third state is responsible for the excitation quenching. This was opposite to earlier suggestions of a two-state model, where the red-shifted fluorescence and excitation quenching were assumed to be related. To examine such possibility, in the current work we repeat our analysis of the TRF data in terms of the two-state model. We find that even though it can reasonably describe the aggregate fluorescence, it fails to do so for the LHCII trimers. We conclude that the red-emitting state cannot be responsible for fluorescence quenching in the LHCII aggregates and reaffirm that the three-state model is the simplest possible description of the experimental data.
Keywords: Light-harvesting complex; Fluorescence; Temperature; LHCII; NPQ

Some mosses stay green and survive long even under desiccation. Dissipation mechanisms of excess excitation energy were studied in two drought-tolerant moss species adapted to contrasting niches: shade-adapted Rhytidiadelphus squarrosus and sun-adapted Rhytidium rugosum in the same family. (1) Under wet conditions, a light-induced nonphotochemical quenching (NPQ) mechanism decreased the yield of photosystem II (PSII) fluorescence in both species. The NPQ extent saturated at a lower illumination intensity in R. squarrosus, suggesting a larger PSII antenna size. (2) Desiccation reduced the fluorescence intensities giving significantly lower F 0 levels and shortened the overall fluorescence lifetimes in both R. squarrosus and R. rugosum, at room temperature. (3) At 77 K, desiccation strongly reduced the PSII fluorescence intensity. This reduction was smaller in R. squarrosus than in R. rugosum. (4) Global and target analysis indicated two different mechanisms of energy dissipation in PSII under desiccation: the energy dissipation to a desiccation-formed strong fluorescence quencher in the PSII core in sun-adapted R. rugosum (type-A quenching) and (5) the moderate energy dissipation in the light-harvesting complex/PSII in shade-adapted R. squarrosus (type-B quenching). The two mechanisms are consistent with the different ecological niches of the two mosses.
Keywords: Chlorophyll fluorescence; Drought tolerance; Fluorescence lifetime; Photodamage; Moss photosynthesis; Global analysis

The regulatory nucleotides, guanosine 5′-triphosphate 3′-diphosphate (pppGpp) and guanosine 5′-diphosphate 3′-diphosphate (ppGpp), were originally identified in Escherichia coli, and control a large set of gene expression and enzyme activities. The (p)ppGpp-dependent control of cell activities is referred to as the stringent response. A growing number of (p)ppGpp synthase/hydrolase homologs have been identified in plants, which are localized in plastids in Arabidopsis thaliana. We recently reported that the Arabidopsis mutant overproducing ppGpp in plastids showed dwarf chloroplasts, and transcript levels in the mutant plastids were significantly suppressed. Furthermore, the mutant showed more robust growth than the wild type (WT), especially under nutrient-deficient conditions, although the mechanisms are unclear. To better understand the impact of the ppGpp accumulation on plant responses to nutrient deficiency, photosynthetic activities and metabolic changes in the ppGpp-overproducing mutant were characterized here. Upon transition to the nitrogen-deficient conditions, the mutant showed reduction of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) contents, and effective and maximum quantum yield of photosystem II compared with WT. The mutant also showed more obvious changes in key metabolite levels including some amino acid contents than WT; similar metabolic change is known to be critical for plants to maintain carbon–nitrogen balance in their cells. These results suggest that artificially overproducing ppGpp modulates the organelle functions that play an important role in controlling photosynthetic performance and metabolite balance during nitrogen starvation.
Keywords: Photosynthesis; ppGpp; Stringent response; Arabidopsis ; Chloroplast

Zinc 3-hydroxymethyl-131-oxo-chlorins bearing a variety of primary alkyl groups at the 20-position were prepared as models of bacteriochlorophyll-c by chemical modification of naturally occurring chlorophyll-a. The synthetic chlorophyll-a derivatives self-aggregated in an aqueous Triton X-100 solution to afford large oligomers whose Soret and Qy bands were red-shifted and broadened, compared with the bands of their monomers in tetrahydrofuran. The oligomeric bands are similar to those of bacteriochlorophyll-c self-aggregates in chlorosomes, the main light-harvesting antennae of photosynthetic green bacteria. The 20-alkylation led to bathochromic shifts of the visible Soret maxima in J-type self-aggregates of the synthetic models, while elongation of the 20-alkyl group decreased the chlorosomal Qy maxima due to an increase in steric hindrance. Considering the light-harvesting and energy-transferring processes in a chlorosome, the 20-methylation in bacteriochlorophyll-c would be more suitable for efficient culturing of green bacteria than the 20-ethylation and propylation as well as the 20-unsubstitution in bacteriochlorophyll-d.
Keywords: Bacteriochlorophyll; Chlorosome; Self-aggregation; Photosynthetic green bacterium; Visible absorption spectroscopy

A chlorosome is a large and efficient light-harvesting antenna system found in some photosynthetic bacteria. This system comprises self-aggregates of bacteriochlorophyll (BChl) c, d, or e possessing a chiral 1-hydroxyethyl group at the 3-position, which plays a key role in the formation of the supramolecule. Biosynthesis of chlorosomal pigments involves stereoselective conversion of 3-vinyl group to 3-(1-hydroxyethyl) group facilitated by a 3-vinyl hydratase. This 3-vinyl hydration also occurs in BChl a biosynthesis, followed by oxidation that introduces an acetyl group at the 3-position. Herein, we present in vitro enzymatic assays of paralogous 3-vinyl hydratases derived from green sulfur bacteria, Chlorobaculum tepidum and Chlorobaculum limnaeum, the filamentous anoxygenic phototroph Chloroflexus aurantiacus, and the chloracidobacterium Chloracidobacterium thermophilum. All the hydratases showed hydration activities. The biosynthetic pathway of BChl a and other chlorosomal pigments is discussed considering the substrate specificity and stereoselectivity of the present hydratases.
Keywords: Bacteriochlorophyll; Chloracidobacterium; Chlorosome; Filamentous anoxygenic phototroph; Green sulfur bacterium; Hydratase

Spectrally selective fluorescence imaging of Chlorobaculum tepidum reaction centers conjugated to chelator-modified silver nanowires by Dorota Kowalska; Marcin Szalkowski; Khuram Ashraf; Justyna Grzelak; Heiko Lokstein; Joanna Niedziolka-Jonsson; Richard Cogdell; Sebastian Mackowski (329-336).
A polyhistidine tag (His-tag) present on Chlorobaculum tepidum reaction centers (RCs) was used to immobilize photosynthetic complexes on a silver nanowire (AgNW) modified with nickel-chelating nitrilo-triacetic acid (Ni-NTA). The optical properties of conjugated nanostructures were studied using wide-field and confocal fluorescence microscopy. Plasmonic enhancement of RCs conjugated to AgNWs was observed as their fluorescence intensity dependence on the excitation wavelength does not follow the excitation spectrum of RC complexes in solution. The strongest effect of plasmonic interactions on the emission intensity of RCs coincides with the absorption spectrum of AgNWs and is observed for excitation into the carotenoid absorption. From the absence of fluorescence decay shortening, we attribute the emission enhancement to increase of absorption in RC complexes.
Keywords: Plasmonic enhancement; Bacteriochlorophyll; Fluorescence; Silver nanowires; Conjugation