Michal Gwizdala

In Vitro Reconstitution of the Cyanobacterial Photoprotective Mechanism Mediated by the Orange Carotenoid Protein in Synechocystis PCC 6803

In the cyanobacterial photoprotective mechanism, one red-activated Orange Carotenoid Protein binds to the core of the phycobilisome and quenches all its fluorescence. This binding stabilizes the red form. Addition of the Fluorescence Recovery Protein accelerates fluorescence recovery in darkness by interacting with the red protein and destabilizing its binding to the phycobilisome. 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.

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.

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 Essential Role of the N-Terminal Domain of the Orange Carotenoid Protein in Cyanobacterial Photoprotection: Importance of a Positive Charge for Phycobilisome Binding

This article provides information about the interaction between the phycobilisomes and the Orange Carotenoid Protein needed for photoprotection. Its red light–activated form has an open structure that allows the interaction of its N-terminal domain, containing the Arg155, with the phycobilisome, permitting a closer interaction between the carotenoid and the phycobilisome chromophores. 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.

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.

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.

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.

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.

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.