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.M.G., T.P.J.K. and R.v.G. were supported by R.v.G.'s advanced investigator grant (267333, PHOTPROT) from the European Research Council and TOP grant (700.58.305) from the Foundation of Chemical Sciences part of NWO. T.P.J.K. was additionally supported by the University of Pretoria‘s Research Development Programme (A0W679). R.v.G. gratefully acknowledges his ‘Academy Professor’ grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). M.G. was additionally funded by EMBO, the Claude Leon Foundation and the University of Pretoria.http://www.elsevier.com/locate/bbamem2020-04-01hj2019Physic

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.http://pubs.acs.org/journal/jpclcdhj2024Forestry and Agricultural Biotechnology Institute (FABI)PhysicsNon

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

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

DATA AVAILABILITY : Experimental data is available upon request.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.The African Laser Centre, the National Research Foundation (NRF), South Africa, the Vrije Universiteit Amsterdam–NRF Desmond Tutu Programme, the Department of Science and Innovation, the Rental Pool Programme of the Council for Scientific and Industrial Research's Photonics Centre, South Africa, the Claude Leon Foundation and the University of Pretoria. Open access funding provided by University of Pretoria.https://www.springer.com/journal/11120hj2024Forestry and Agricultural Biotechnology Institute (FABI)PhysicsNon

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.This material is based on work supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Award Number DE-FG02-07ER15892 (to W.E.M) and by the Dutch organization for scientific research (NWO-ALW) via a Vici grant (to R.C.). R.v.G. and T.P.J.K. were supported by the Netherlands Organization for Sciences, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305). R.v.G. was further supported by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT) and by the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his Academy Professor grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). T.P.J.K. was further supported by University of Pretoria’s Research Development Programme (grant no. A0W679). The authors would like to acknowledge the following fellowships: a Postdoctoral Fellowship from the Center for Molecular Analysis and Design at Stanford University (to G.S.S.-C.); a Kenneth and Nina Tai Stanford Graduate Fellowship (to H.-Y.Y.); and a Long Term Fellowship from EMBO (to M.G.).http://pubs.acs.org/journal/jpclcdhb2017Physic

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.This material is based on work supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Award Number DE-FG02-07ER15892 (to W.E.M) and by the Dutch organization for scientific research (NWO-ALW) via a Vici grant (to R.C.). R.v.G. and T.P.J.K. were supported by the Netherlands Organization for Sciences, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305). R.v.G. was further supported by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT) and by the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his Academy Professor grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). T.P.J.K. was further supported by University of Pretoria’s Research Development Programme (grant no. A0W679). The authors would like to acknowledge the following fellowships: a Postdoctoral Fellowship from the Center for Molecular Analysis and Design at Stanford University (to G.S.S.-C.); a Kenneth and Nina Tai Stanford Graduate Fellowship (to H.-Y.Y.); and a Long Term Fellowship from EMBO (to M.G.).http://pubs.acs.org/journal/jpclcdhb2017Physic