Energetic Landscape and Terminal Emitters of Phycobilisome Cores from Quantum Chemical Modeling.

Phycobilisomes (PBs) are giant antenna supercomplexes of cyanobacteria that use phycobilin pigments to capture sunlight and transfer the collected energy to membrane-bound photosystems. In the PB core, phycobilins are bound to particular allophycocyanin (APC) proteins. Some phycobilins are thought to be terminal emitters (TEs) with red-shifted fluorescence. However, the precise identification of TEs is still under debate. In this work, we employ multiscale quantum-mechanical calculations to disentangle the excitation energy landscape of PB cores. Using the recent atomistic PB structures from Synechoccoccus PCC 7002 and Synechocystis PCC 6803, we compute the spectral properties of different APC trimers and assign the low-energy pigments. We show that the excitation energy of APC phycobilins is determined by geometric and electrostatic factors and is tuned by the specific protein-protein interactions within the core. Our findings challenge the simple picture of a few red-shifted bilins in the PB core and instead suggest that the red-shifts are established by the entire TE-containing APC trimers. Our work provides a theoretical microscopic basis for the interpretation of energy migration and time-resolved spectroscopy in phycobilisomes.

ApcE plays an important role in light-induced excitation energy dissipation in the Synechocystis PCC6803 phycobilisomes.

Phycobilisomes (PBs) play an important role in cyanobacterial photosynthesis. They capture light and transfer excitation energy to the photosynthetic reaction centres. PBs are also central to some photoprotective and photoregulatory mechanisms that help sustain photosynthesis under non-optimal conditions. Amongst the mechanisms involved in excitation energy dissipation that are activated in response to excessive illumination is a recently discovered light-induced mechanism that is intrinsic to PBs and has been the least studied. Here, we used single-molecule spectroscopy and developed robust data analysis methods to explore the role of a terminal emitter subunit, ApcE, in this intrinsic, light-induced mechanism. We isolated the PBs from WT Synechocystis PCC 6803 as well as from the ApcE-C190S mutant of this strain and compared the dynamics of their fluorescence emission. PBs isolated from the mutant (i.e., ApcE-C190S-PBs), despite not binding some of the red-shifted pigments in the complex, showed similar global emission dynamics to WT-PBs. However, a detailed analysis of dynamics in the core revealed that the ApcE-C190S-PBs are less likely than WT-PBs to enter quenched states under illumination but still fully capable of doing so. This result points to an important but not exclusive role of the ApcE pigments in the light-induced intrinsic excitation energy dissipation mechanism in PBs.

Sub-lithic photosynthesis in hot desert habitats.

In hyper-arid soil environments, photosynthetic microorganisms are largely restricted to hypolithic (sub-lithic) habitats: i.e., on the ventral surfaces of translucent pebbles in desert pavements. Here, we combined fluorometric, spectroscopic, biochemical and metagenomic approaches to investigate in situ the light transmission properties of quartz stones in the Namib Desert, and assess the photosynthetic activity of the underlying hypolithic cyanobacterial biofilms. Quartz pebbles greatly reduced the total photon flux to the ventral surface biofilms and filtered out primarily the short wavelength portion of the solar spectrum. Chlorophylls d and f were not detected in biofilm pigment extracts; however, hypolithic cyanobacterial communities showed some evidence of adaptation to sub-lithic conditions, including the prevalence of genes encoding Helical Carotenoid Proteins, which are associated with desiccation stress. Under water-saturated conditions, hypolithic communities showed no evidence of light stress, even when the quartz stones were exposed to full midday sunlight. This initial study creates a foundation for future in-situ and laboratory exploration of various adaptation mechanisms employed by photosynthetic organisms forming hypolithic microbial communities.

Charge transfer states in phycobilisomes.

Phycobilisomes (PBs) absorb light and supply downstream photosynthetic processes with excitation energy in many cyanobacteria and algae. In response to a sudden increase in light intensity, excess excitation energy is photoprotectively dissipated in PBs by means of the orange carotenoid protein (OCP)-related mechanism or via a light-activated intrinsic decay channel. Recently, we have identified that both mechanisms are associated with far-red emission states. Here, we investigate the far-red states involved with the light-induced intrinsic mechanism by exploring the energy landscape and electro-optical properties of the pigments in PBs. While Stark spectroscopy showed that the far-red states in PBs exhibit a strong charge-transfer (CT) character at cryogenic temperatures, single molecule spectroscopy revealed that CT states should also be present at room temperature. Owing to the strong environmental sensitivity of CT states, the knowledge gained from this study may contribute to the design of a new generation of fluorescence markers.

Two-Step Structural Changes in Orange Carotenoid Protein Photoactivation Revealed by Time-Resolved Fourier Transform Infrared Spectroscopy.

The orange carotenoid protein (OCP), which is essential in cyanobacterial photoprotection, is the first photoactive protein containing a carotenoid as an active chromophore. Static and time-resolved Fourier transform infrared (FTIR) difference spectroscopy under continuous illumination at different temperatures was applied to investigate its photoactivation mechanism. Here, we demonstrate that in the OCP, the photo-induced conformational change involves at least two different steps, both in the second timescale at 277 K. Each step involves partial reorganization of α-helix domains. At early illumination times, the disappearance of a nonsolvent-exposed α-helix (negative 1651 cm-1 band) is observed. At longer times, a 1644 cm-1 negative band starts to bleach, showing the disappearance of a solvent-exposed α-helix, either the N-terminal extension and/or the C-terminal tail. A kinetic analysis clearly shows that these two events are asynchronous. Minor modifications in the overall FTIR difference spectra confirm that the global protein conformational change consists of-at least-two asynchronous contributions. Comparison of spectra recorded in H2O and D2O suggests that internal water molecules may contribute to the photoactivation mechanism.

The role of far-red spectral states in the energy regulation of phycobilisomes.

The main light-harvesting pigment-protein complex of cyanobacteria and certain algae is the phycobilisome, which harvests sunlight and regulates the flow of absorbed energy to provide the photochemical reaction centres with a constant energy throughput. At least two light-driven mechanisms of excited energy quenching in phycobilisomes have been identified: the dominant mechanism in many strains of cyanobacteria depends on the orange carotenoid protein (OCP), while the second mechanism is intrinsically available to a phycobilisome and is possibly activated faster than the former. Recent single molecule spectroscopy studies have shown that far-red (FR) emission states are related to the OCP-dependent mechanism and it was proposed that the second mechanism may involve similar states. In this study, we examined the dynamics of simultaneously measured emission spectra and intensities from a large set of individual phycobilisome complexes from Synechocystis PCC 6803. Our results suggest a direct relationship between FR spectral states and thermal energy dissipating states and can be explained by a single phycobilin pigment in the phycobilisome core acting as the site of both quenching and FR emission likely due to the presence of a charge-transfer state. Our experimental method provides a means to accurately resolve the fluorescence lifetimes and spectra of the FR states, which enabled us to quantify a kinetic model that reproduces most of the experimentally determined properties of the FR states.

Switching an Individual Phycobilisome Off and On.

Photosynthetic organisms have found various smart ways to cope with unexpected changes in light conditions. In many cyanobacteria, the lethal effects of a sudden increase in light intensity are mitigated mainly by the interaction between phycobilisomes (PBs) and the orange carotenoid protein (OCP). The latter senses high light intensities by means of photoactivation and triggers thermal energy dissipation from the PBs. Due to the brightness of their emission, PBs can be characterized at the level of individual complexes. Here, energy dissipation from individual PBs was reversibly switched on and off using only light and OCP. We reveal the presence of quasistable intermediate states during the binding and unbinding of OCP to PB, with a spectroscopic signature indicative of transient decoupling of some of the PB rods during docking of OCP. Real-time control of emission from individual PBs has the potential to contribute to the development of new super-resolution imaging techniques.

Phycocyanin: One Complex, Two States, Two Functions.

Solar energy captured by pigments embedded in light-harvesting complexes can be transferred to neighboring pigments, dissipated, or emitted as fluorescence. Only when it reaches a reaction center is the excitation energy stabilized in the form of a charge separation and converted into chemical energy. Well-directed and regulated energy transfer within the network of pigments is therefore of crucial importance for the success of the photosynthetic processes. Using single-molecule spectroscopy, we show that phycocyanin can dynamically switch between two spectrally distinct states originating from two different conformations. Unexpectedly, one of the two states has a red-shifted emission spectrum. This state is not involved in energy dissipation; instead, we propose that it is involved in direct energy transfer to photosystem I. Finally, our findings suggest that the function of linker proteins in phycobilisomes is to stabilize one state or the other, thus controlling the light-harvesting functions of phycocyanin.

A functional compartmental model of the Synechocystis PCC 6803 phycobilisome.

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).

Controlling Light Harvesting with Light.

When exposed to intense sunlight, all organisms performing oxygenic photosynthesis implement various photoprotective strategies to prevent potentially lethal photodamage. The rapidly responding photoprotective mechanisms, occurring in the light-harvesting pigment-protein antennae, take effect within tens of seconds, while the dramatic and potentially harmful light intensity fluctuations manifest also on shorter time scales. Here we show that, upon illumination, individual phycobilisomes from Synechocystis PCC 6803, which, in vivo under low-light conditions, harvest solar energy, and have the built-in capacity to switch rapidly and reversibly into light-activated energy-dissipating states. Simultaneously measured fluorescence intensity, lifetime, and spectra, compared with a multicompartmental kinetic model, revealed that essentially any subunit of a phycobilisome can be quenched, and that the core complexes were targeted most frequently. Our results provide the first evidence for fluorescence blinking from a biologically active system at physiological light intensities and suggest that the light-controlled switches to intrinsically available energy-dissipating states are responsible for a novel type of photoprotection in cyanobacteria. We anticipate other photosynthetic organisms to employ similar strategies to respond instantly to rapid solar light intensity fluctuations. A detailed understanding of the photophysics of photosynthetic antenna complexes is of great interest for bioinspired solar energy technologies.

A method to decompose spectral changes in Synechocystis PCC 6803 during light-induced state transitions.

Cyanobacteria have developed responses to maintain the balance between the energy absorbed and the energy used in different pigment-protein complexes. One of the relatively rapid (a few minutes) responses is activated when the cells are exposed to high light intensities. This mechanism thermally dissipates excitation energy at the level of the phycobilisome (PB) antenna before it reaches the reaction center. When exposed to low intensities of light that modify the redox state of the plastoquinone pool, the so-called state transitions redistribute energy between photosystem I and II. Experimental techniques to investigate the underlying mechanisms of these responses, such as pulse-amplitude modulated fluorometry, are based on spectrally integrated signals. Previously, a spectrally resolved fluorometry method has been introduced to preserve spectral information. The analysis method introduced in this work allows to interpret SRF data in terms of species-associated spectra of open/closed reaction centers (RCs), (un)quenched PB and state 1 versus state 2. Thus, spectral differences in the time-dependent fluorescence signature of photosynthetic organisms under varying light conditions can be traced and assigned to functional emitting species leading to a number of interpretations of their molecular origins. In particular, we present evidence that state 1 and state 2 correspond to different states of the PB-PSII-PSI megacomplex.

Resolving the contribution of the uncoupled phycobilisomes to cyanobacterial pulse-amplitude modulated (PAM) fluorometry signals.

Pulse-amplitude modulated (PAM) fluorometry is extensively used to characterize photosynthetic organisms on the slow time-scale (1-1000 s). The saturation pulse method allows determination of the quantum yields of maximal (F(M)) and minimal fluorescence (F(0)), parameters related to the activity of the photosynthetic apparatus. Also, when the sample undergoes a certain light treatment during the measurement, the fluorescence quantum yields of the unquenched and the quenched states can be determined. In the case of cyanobacteria, however, the recorded fluorescence does not exclusively stem from the chlorophyll a in photosystem II (PSII). The phycobilins, the pigments of the cyanobacterial light-harvesting complexes, the phycobilisomes (PB), also contribute to the PAM signal, and therefore, F(0) and F(M) are no longer related to PSII only. We present a functional model that takes into account the presence of several fluorescent species whose concentrations can be resolved provided their fluorescence quantum yields are known. Data analysis of PAM measurements on in vivo cells of our model organism Synechocystis PCC6803 is discussed. Three different components are found necessary to fit the data: uncoupled PB (PB(free)), PB-PSII complexes, and free PSI. The free PSII contribution was negligible. The PB(free) contribution substantially increased in the mutants that lack the core terminal emitter subunits allophycocyanin D or allophycocyanin F. A positive correlation was found between the amount of PB(free) and the rate constants describing the binding of the activated orange carotenoid protein to PB, responsible for non-photochemical quenching.

Single-Molecule Identification of Quenched and Unquenched States of LHCII.

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.

Excited states of the inactive and active forms of the orange carotenoid protein.

The orange carotenoid protein (OCP) is a crucial player in the process of nonphotochemical quenching in a large number of cyanobacteria. This water-soluble protein binds one pigment only, the keto carotenoid 3'-hydroxyechinenone, and needs to be photoactivated by strong (blue-green) light in order to induce energy dissipation within or from the phycobilisome, the main light harvesting system of these organisms. We performed transient-absorption spectroscopy on OCP samples frozen in the inactive and active forms at 77 K. By making use of target analysis we determined the excited state properties of the active form. Our results show that OCP photoactivation modifies the carotenoid excited state energy landscape. More specifically the photoactivated OCP is characterized by one state with predominantly ICT character (ICT/S1) and a lifetime of 2.3 ps, and another state with mainly S1 character (S1/ICT) with a lifetime of 7.6 ps. We also show that the kinetic model is fully consistent with the RT data obtained earlier (Berera et al., J. Phys. Chem. B 2012, 116, 2568-2574). We propose that this ICT/S1 state acts as the quencher in the OCP mediated nonphotochemical quenching.

Characterization of the Synechocystis PCC 6803 Fluorescence Recovery Protein involved in photoprotection.

Under high irradiance, most cyanobacteria induce a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers to avoid the formation of dangerous species of oxygen. This mechanism which rapidly increases the heat dissipation of excess energy at the level of the cyanobacterial antenna, the phycobilisomes, is triggered by the photoactivation of the Orange Carotenoid Protein (OCP). Under low light conditions, the Fluorescence Recovery Protein (FRP) mediates the recovery of the full antenna capacity by accelerating the deactivation of the OCP. Several FRP Synechocystis mutants were constructed and characterized in terms of the OCP-related photoprotective mechanism. Our results demonstrate that Synechocystis FRP starts at Met26 and not at Met1 (according to notation in Cyanobase) as was previously suggested. Moreover, changes in the genomic region upstream the ATG encoding for Met26 influenced the concentration of OCP in cells. A long FRP (beginning at Met1) is synthesized in Synechocystis cells when the frp gene is under the control of the psbA2 promoter but it is less active than the shorter protein. Overexpression of the short frp gene in Synechocystis enabled short FRP isolation from the soluble fraction. However, the high concentration of FRP in this mutant inhibited the induction of the photoprotective mechanism by decreasing the concentration of the activated OCP. Therefore, the amplitude of photoprotection depends on not only OCP concentration but also on that of FRP. The synthesis of FRP and OCP must be strictly regulated to maintain a low FRP to OCP ratio to allow efficient photoprotection.

The essential role of the N-terminal domain of the orange carotenoid protein in cyanobacterial photoprotection: importance of a positive charge for phycobilisome binding.

Most cyanobacteria, under high light conditions, decrease the amount of energy arriving at the reaction centers by increasing thermal energy dissipation at the level of the phycobilisome, the extramembranous antenna. This mechanism is induced by photoactivation of the Orange Carotenoid Protein (OCP). To identify how the activated OCP interacts with phycobilisomes (PBs), several OCP mutants were constructed, and the influence of mutations on photoactivity, stability, and binding to PBs was characterized. The disruption of the salt bridge between Arg155 and Glu244, which stabilizes the interaction between the N- and C-terminal domains, increased the rate of photoactivity and the stability of the photoactivated OCP, suggesting that the activated OCP has an open structure with decreased interdomain interaction. Changing Glu244 to leucine had no effect on OCP binding to PBs. By contrast, substitution of Arg155 with a neutral or a negatively charged amino acid largely decreased OCP binding to the PBs, whereas substitution with a lysine slightly perturbed the interaction. These results strongly suggest that the surface of the N-terminal domain, containing the Arg155, interacts with the PB and that the positive charge of Arg155 plays a key role in photoprotection.

Picosecond kinetics of light harvesting and photoprotective quenching in wild-type and mutant phycobilisomes isolated from the cyanobacterium Synechocystis PCC 6803.

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.

The photophysics of the orange carotenoid protein, a light-powered molecular switch.

To cope with the deleterious effects of excess illumination, photosynthetic organisms have developed photoprotective mechanisms that dissipate the absorbed excess energy as heat from the antenna system. In cyanobacteria, a crucial step in the process is the activation, by blue-green light, of a soluble protein, known as orange carotenoid protein (OCP), which binds the carotenoid 3'-hydroxyechinenone as its only pigment. While the spectroscopic properties of the inactive form of OCP have been described, the nature of the excited states in the active form still awaits elucidation. We applied transient absorption spectroscopy to the dark and the light activated forms of OCP to study and compare the excited state dynamics of both forms. We show that excitation of the photoactivated OCP leads to the population of new carotenoid excited states. One of these states populated shortly after excitation is characterized by a very pronounced charge transfer character and a lifetime of about 0.6 ps. When the illuminated sample is exposed to a dark relaxation period, it responds to excitation as the original dark sample, showing that photoactivation and decay of the photoactivated state are fully reversible. Thus OCP functions as a light-powered molecular switch that modulates its spectroscopic properties as a response to specific changes in light environment. We discuss the importance of this switch in cyanobacteria photoprotection and propose a mechanism wherein the red state of OCP echinenone acts as an energy dissipator via its charge transfer state.

ApcD, ApcF and ApcE are not required for the Orange Carotenoid Protein related phycobilisome fluorescence quenching in the cyanobacterium Synechocystis PCC 6803.

In cyanobacteria, strong blue-green light induces a photoprotective mechanism involving an increase of energy thermal dissipation at the level of phycobilisome (PB), the cyanobacterial antenna. This leads to a decrease of the energy arriving to the reaction centers. The photoactive Orange Carotenoid Protein (OCP) has an essential role in this mechanism. The binding of the red photoactivated OCP to the core of the PB triggers energy and PB fluorescence quenching. The core of PBs is constituted of allophycocyanin trimers emitting at 660 or 680nm. ApcD, ApcF and ApcE are the responsible of the 680nm emission. In this work, the role of these terminal emitters in the photoprotective mechanism was studied. Single and double Synechocystis PCC 6803 mutants, in which the apcD or/and apcF genes were absent, were constructed. The Cys190 of ApcE which binds the phycocyanobilin was replaced by a Ser. The mutated ApcE attached an unusual chromophore emitting at 710nm. The activated OCP was able to induce the photoprotective mechanism in all the mutants. Moreover, in vitro reconstitution experiments showed similar amplitude and rates of fluorescence quenching. Our results demonstrated that ApcD, ApcF and ApcE are not required for the OCP-related fluorescence quenching and they strongly suggested that the site of quenching is one of the APC trimers emitting at 660nm. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

In vitro reconstitution of the cyanobacterial photoprotective mechanism mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803.

In conditions of fluctuating light, cyanobacteria thermally dissipate excess absorbed energy at the level of the phycobilisome, the light-collecting antenna. The photoactive Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) have essential roles in this mechanism. Absorption of blue-green light converts the stable orange (inactive) OCP form found in darkness into a metastable red (active) form. Using an in vitro reconstituted system, we studied the interactions between OCP, FRP, and phycobilisomes and demonstrated that they are the only elements required for the photoprotective mechanism. In the process, we developed protocols to overcome the effect of high phosphate concentrations, which are needed to maintain the integrity of phycobilisomes, on the photoactivation of the OCP, and on protein interactions. Our experiments demonstrated that, whereas the dark-orange OCP does not bind to phycobilisomes, the binding of only one red photoactivated OCP to the core of the phycobilisome is sufficient to quench all its fluorescence. This binding, which is light independent, stabilizes the red form of OCP. Addition of FRP accelerated fluorescence recovery in darkness by interacting with the red OCP and destabilizing its binding to the phycobilisome. The presence of phycobilisome rods renders the OCP binding stronger and allows the isolation of quenched OCP-phycobilisome complexes. Using the in vitro system we developed, it will now be possible to elucidate the quenching process and the chemical nature of the quencher.