Multiple retinal isomerizations during the early phase of the bestrhodopsin photoreaction.

Bestrhodopsins constitute a class of light-regulated pentameric ion channels that consist of one or two rhodopsins in tandem fused with bestrophin ion channel domains. Here, we report on the isomerization dynamics in the rhodopsin tandem domains of Phaeocystis antarctica bestrhodopsin, which binds all-trans retinal Schiff-base (RSB) absorbing at 661 nm and, upon illumination, converts to the meta-stable P540 state with an unusual 11-cis RSB. The primary photoproduct P682 corresponds to a mixture of highly distorted 11-cis and 13-cis RSB directly formed from the excited state in 1.4 ps. P673 evolves from P682 in 500 ps and contains highly distorted 13-cis RSB, indicating that the 11-cis fraction in P682 converts to 13-cis. Next, P673 establishes an equilibrium with P595 in 1.2 µs, during which RSB converts to 11-cis and then further proceeds to P560 in 48 µs and P540 in 1.0 ms while remaining 11-cis. Hence, extensive isomeric switching occurs on the early ground state potential energy surface (PES) on the hundreds of ps to µs timescale before finally settling on a metastable 11-cis photoproduct. We propose that P682 and P673 are trapped high up on the ground-state PES after passing through either of two closely located conical intersections that result in 11-cis and 13-cis RSB. Co-rotation of C11=C12 and C13=C14 bonds results in a constricted conformational landscape that allows thermal switching between 11-cis and 13-cis species of highly strained RSB chromophores. Protein relaxation may release RSB strain, allowing it to evolve to a stable 11-cis isomeric configuration in microseconds.

The Orange Carotenoid Protein Triggers Cyanobacterial Photoprotection by Quenching Bilins via a Structural Switch of Its Carotenoid.

Cyanobacteria were the first microorganisms that released oxygen into the atmosphere billions of years ago. To do it safely under intense sunlight, they developed strategies that prevent photooxidation in the photosynthetic membrane, by regulating the light-harvesting activity of their antenna complexes-the phycobilisomes-via the orange-carotenoid protein (OCP). This water-soluble protein interacts with the phycobilisomes and triggers nonphotochemical quenching (NPQ), a mechanism that safely dissipates overexcitation in the membrane. To date, the mechanism of action of OCP in performing NPQ is unknown. In this work, we performed ultrafast spectroscopy on a minimal NPQ system composed of the active domain of OCP bound to the phycobilisome core. The use of this system allowed us to disentangle the signal of the carotenoid from that of the bilins. Our results demonstrate that the binding to the phycobilisomes modifies the structure of the ketocarotenoid associated with OCP. We show that this molecular switch activates NPQ, by enabling excitation-energy transfer from the antenna pigments to the ketocarotenoid.

Energy Transfer and Radical-Pair Dynamics in Photosystem I with Different Red Chlorophyll a Pigments.

We establish a general kinetic scheme for the energy transfer and radical-pair dynamics in photosystem I (PSI) of Chlamydomonas reinhardtii, Synechocystis PCC6803, Thermosynechococcus elongatus and Spirulina platensis grown under white-light conditions. With the help of simultaneous target analysis of transient-absorption data sets measured with two selective excitations, we resolved the spectral and kinetic properties of the different species present in PSI. WL-PSI can be described as a Bulk Chl a in equilibrium with a higher-energy Chl a, one or two Red Chl a and a reaction-center compartment (WL-RC). Three radical pairs (RPs) have been resolved with very similar properties in the four model organisms. The charge separation is virtually irreversible with a rate of ≈900 ns-1. The second rate, of RP1 → RP2, ranges from 70-90 ns-1 and the third rate, of RP2 → RP3, is ≈30 ns-1. Since RP1 and the Red Chl a are simultaneously present, resolving the RP1 properties is challenging. In Chlamydomonas reinhardtii, the excited WL-RC and Bulk Chl a compartments equilibrate with a lifetime of ≈0.28 ps, whereas the Red and the Bulk Chl a compartments equilibrate with a lifetime of ≈2.65 ps. We present a description of the thermodynamic properties of the model organisms at room temperature.

A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Pyglotaran: a lego-like Python framework for global and target analysis of time-resolved spectra.

The dynamics of molecular systems can be studied with time-resolved spectroscopy combined with model-based analysis. A Python framework for global and target analysis of time-resolved spectra is introduced with the help of three case studies. The first study, concerning broadband absorption of intersystem crossing in 4-thiothymidine, demonstrates the framework's ability to resolve vibrational wavepackets with a time resolution of ≈10 fs using damped oscillations and their associated spectra and phases. Thereby, a parametric description of the "coherent artifact" is crucial. The second study addresses multichromophoric systems composed of two perylene bisimide chromophores. Here, pyglotaran's guidance spectra and lego-like model composition enable the integration of spectral and kinetic properties of the parent chromophores, revealing a loss process, the undesired production of a radical pair, that reduces the light harvesting efficiency. In the third, time-resolved emission case study of whole photosynthetic cells, a megacomplex containing ≈500 chromophores of five different types is described by a combination of the kinetic models for its elements. As direct fitting of the data by theoretical simulation is unfeasible, our global and target analysis methodology provides a useful 'middle ground' where the theoretical description and the fit of the experimental data can meet. The pyglotaran framework enables the lego-like creation of kinetic models through its modular design and seamless integration with the rich Python ecosystem, particularly Jupyter notebooks. With extensive documentation and a robust validation framework, pyglotaran ensures accessibility and reliability for researchers, serving as an invaluable tool for understanding complex molecular systems.

Coherent vibrational modes promote the ultrafast internal conversion and intersystem crossing in thiobases.

Thionated nucleobases are obtained by replacing oxygen with sulphur atoms in the canonical nucleobases. They absorb light efficiently in the near-ultraviolet, populating singlet states which undergo intersystem crossing to the triplet manifold on an ultrashort time scale with a high quantum yield. Nonetheless there are still important open questions about the primary mechanisms responsible for this ultrafast transition. Here we track both the electronic and the vibrational ultrafast excited-state dynamics towards the triplet state for solvated 4-thiothymidine (4TT) and 4-thiouracil (4TU) with sub-30 fs broadband transient absorption spectroscopy in the ultraviolet. A global and target analysis allows us to simultaneously resolve the contributions of the different electronically and vibrationally excited states to the whole data set. Our experimental results, combined with state-of-the-art quantum mechanics/molecular mechanics simulations and Damped Oscillation Associated Spectra (DOAS) and target analysis, support that the relaxation to the triplet state is mediated by conical intersections promoted by vibrational coherences through the population of an intermediate singlet state. In addition, the analysis of the coherent vibrational dynamics reveals that, despite sharing the same relaxation mechanism and similar chemical structures, 4TT and 4TU exhibit rather different geometrical deformations, characterized by the conservation of planarity in 4TU and its partial rupture in 4TT.

pH dependence, kinetics and light-harvesting regulation of nonphotochemical quenching in Chlamydomonas.

Sunlight drives photosynthesis but can also cause photodamage. To protect themselves, photosynthetic organisms dissipate the excess absorbed energy as heat, in a process known as nonphotochemical quenching (NPQ). In green algae, diatoms, and mosses, NPQ depends on the light-harvesting complex stress-related (LHCSR) proteins. Here we investigated NPQ in Chlamydomonas reinhardtii using an approach that maintains the cells in a stable quenched state. We show that in the presence of LHCSR3, all of the photosystem (PS) II complexes are quenched and the LHCs are the site of quenching, which occurs at a rate of ∼150 ps-1 and is not induced by LHCII aggregation. The effective light-harvesting capacity of PSII decreases upon NPQ, and the NPQ rate is independent of the redox state of the reaction center. Finally, we could measure the pH dependence of NPQ, showing that the luminal pH is always above 5.5 in vivo and highlighting the role of LHCSR3 as an ultrasensitive pH sensor.

Vibronic dynamics resolved by global and target analysis of ultrafast transient absorption spectra.

We present a methodology that provides a complete parametric description of the time evolution of the electronically and vibrationally excited states as detected by ultrafast transient absorption (TA). Differently from previous approaches, which started fitting the data after ≈100 fs, no data are left out in our methodology, and the "coherent artifact" and the instrument response function are fully taken into account. In case studies, the method is applied to solvents, the dye Nile blue, and all-trans ß-carotene in cyclohexane solution. The estimated Damped Oscillation Associated Spectra (DOAS) and phases express the most important vibrational frequencies present in the molecular system. By global fit alone of the experimental data, it is difficult to interpret in detail the underlying dynamics. Since it is unfeasible to directly fit the data by a theoretical simulation, our enhanced DOAS methodology thus provides a useful "middle ground" where the theoretical description and the fit of the experimental data can meet. ß-carotene in cyclohexane was complementarily studied with femtosecond stimulated Raman spectroscopy (FSRS). The fs-ps dynamics of ß-carotene in cyclohexane in TA and FSRS experiments can be described by a sequential scheme S2 → hot S1 → S1' → S1 → S0 with lifetimes of 167 fs (fixed), 0.35, 1.1, and 9.6 ps. The correspondence of DOAS decaying concomitantly with hot S1 and the Species Associated Difference Spectra of hot S1 in TA and FSRS suggest that we observe here features of the vibrational relaxation and nuclear reorganization responsible for the hot S1 to S1 transition.

Unraveling the Excited-State Dynamics and Light-Harvesting Functions of Xanthophylls in Light-Harvesting Complex II Using Femtosecond Stimulated Raman Spectroscopy.

Photosynthesis in plants starts with the capture of photons by light-harvesting complexes (LHCs). Structural biology and spectroscopy approaches have led to a map of the architecture and energy transfer pathways between LHC pigments. Still, controversies remain regarding the role of specific carotenoids in light-harvesting and photoprotection, obligating the need for high-resolution techniques capable of identifying excited-state signatures and molecular identities of the various pigments in photosynthetic systems. Here we demonstrate the successful application of femtosecond stimulated Raman spectroscopy (FSRS) to a multichromophoric biological complex, trimers of LHCII. We demonstrate the application of global and target analysis (GTA) to FSRS data and utilize it to quantify excitation migration in LHCII trimers. This powerful combination of techniques allows us to obtain valuable insights into structural, electronic, and dynamic information from the carotenoids of LHCII trimers. We report spectral and dynamical information on ground- and excited-state vibrational modes of the different pigments, resolving the vibrational relaxation of the carotenoids and the pathways of energy transfer to chlorophylls. The lifetimes and spectral characteristics obtained for the S1 state confirm that lutein 2 has a distorted conformation in LHCII and that the lutein 2 S1 state does not transfer to chlorophylls, while lutein 1 is the only carotenoid whose S1 state plays a significant energy-harvesting role. No appreciable energy transfer takes place from lutein 1 to lutein 2, contradicting recent proposals regarding the functions of the various carotenoids (Son et al. Chem. 2019, 5 (3), 575-584). Also, our results demonstrate that FSRS can be used in combination with GTA to simultaneously study the electronic and vibrational landscapes in LHCs and pave the way for in-depth studies of photoprotective conformations in photosynthetic systems.

Modelling excitation energy transfer and trapping in the filamentous cyanobacterium Anabaena variabilis PCC 7120.

The phycobilisome (PBS) serves as the major light-harvesting system, funnelling excitation energy to both photosystems (PS) in cyanobacteria and red algae. The picosecond kinetics involving the excitation energy transfer has been studied within the isolated systems and intact filaments of the cyanobacterium Anabaena variabilis PCC 7120. A target model is proposed which resolves the dynamics of the different chromophore groups. The energy transfer rate of 8.5 ± 1.0/ns from the rod to the core is the rate-limiting step, both in vivo and in vitro. The PBS-PSI-PSII supercomplex reveals efficient excitation energy migration from the low-energy allophycocyanin, which is the terminal emitter, in the PBS core to the chlorophyll a in the photosystems. The terminal emitter of the phycobilisome transfers energy to both PSI and PSII with a rate of 50 ± 10/ns, equally distributing the solar energy to both photosystems. Finally, the excitation energy is trapped by charge separation in the photosystems with trapping rates estimated to be 56 ± 6/ns in PSI and 14 ± 2/ns in PSII.

Time-resolved fluorescence study of excitation energy transfer in the cyanobacterium Anabaena PCC 7120.

Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been studied by picosecond time-resolved fluorescence spectroscopy at room temperature. Global analysis of the time-resolved fluorescence kinetics revealed two lifetimes of spectral equilibration in the isolated PBS, 30-35 ps and 110-130 ps, assigned primarily to energy transfer within the rods and between the rods and the allophycocyanin core, respectively. An additional intrinsic kinetic component with a lifetime of 500-700 ps was found, representing non-radiative decay or energy transfer in the core. Isolated tetrameric PSI complexes exhibited biexponential fluorescence decay kinetics with lifetimes of about 10 ps and 40 ps, representing equilibration between the bulk antenna chlorophylls with low-energy "red" states and trapping of the equilibrated excitations, respectively. The cascade of EET in the PBS and in PSI could be resolved in intact filaments as well. Virtually all energy absorbed by the PBS was transferred to the photosystems on a timescale of 180-190 ps.

A Unified Experimental/Theoretical Description of the Ultrafast Photophysics of Single and Double Thionated Uracils.

Photoinduced processes in thiouracil derivatives have lately attracted considerable attention due to their suitability for innovative biological and pharmacological applications. Here, sub-20 fs broadband transient absorption spectroscopy in the near-UV are combined with CASPT2/MM decay path calculations to unravel the excited-state decay channels of water solvated 2-thio and 2,4-dithiouracil. These molecules feature linear absorption spectra with overlapping ππ* bands, leading to parallel decay routes which we systematically track for the first time. The results reveal that different processes lead to the triplet states population, both directly from the ππ* absorbing state and via the intermediate nπ* dark state. Moreover, the 2,4-dithiouracil decay pathways is shown to be strongly correlated either to those of 2- or 4-thiouracil, depending on the sulfur atom on which the electronic transition localizes.

Vibronic and excitonic dynamics in perylenediimide dimers and tetramer.

Broad-band pump-probe spectroscopy combined with global and target analysis is employed to study the vibronic and excitonic dynamics of two dimers and a tetramer of perylenediimides. A simultaneous analysis is developed for two systems that have been measured in the same conditions. This enhances the resolvability of the vibronic and excitonic dynamics of the systems, and the solvent contributions that are common in the experiments. We resolve two oscillations of 1399 cm-1 or 311 cm-1 damped with ≈30/ps involved in vibrational relaxation and two more oscillations of 537 cm-1 or 136 cm-1 damped with ≈3/ps. A relaxation process with a rate of 2.1/ps-3.2/ps that is positively correlated with the excitonic coupling was discovered in all three model systems, attributed to annihilation of the one but lowest exciton state.

Photoactivation Mechanism, Timing of Protein Secondary Structure Dynamics and Carotenoid Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV-visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid ß1-ring in picoseconds occurs at a low yield of <1%, whereby the ß1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 µs, which allow the carotenoid to translocate into the N-terminal domain in 10 µs. We identified infrared signatures of helical elements that dock on the C-terminal domain ß-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.

Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from Halorhodospira halophila.

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.

Spectrally decomposed dark-to-light transitions in a PSI-deficient mutant of Synechocystis sp. PCC 6803.

Cyanobacterial thylakoid membranes are known to host photosynthetic and respiratory complexes. This hampers a straight forward interpretation of the highly dynamic fluorescence originating from photosynthetic units. The present study focuses on dark-to-light transitions in whole cells of a PSI-deficient mutant of the cyanobacterium Synechocystis sp. PCC 6803. The time-dependent cellular fluorescence spectrum has been measured, while having previously exposed the cells to different conditions that affect respiratory activity. The analysis method used allows the detected signal to be decomposed in a few components that are then assigned to functional emitting species. Additionally, we have worked out a minimal mathematical model consisting of sensible postulated species to interpret the recorded data. We conclude that the following two functional complexes play a major role: a phycobilisome antenna complex coupled to a PSII dimer with either two or no closed reaction centers. Crucially, we present evidence for an additional species capable of strongly quenching fluorescence, whose formation requires the presence of oxygen.

Mechanisms of drought-induced dissipation of excitation energy in sun- and shade-adapted drought-tolerant mosses studied by fluorescence yield change and global and target analysis of fluorescence decay kinetics.

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.

Energy transfer and trapping in Synechococcus WH 7803.

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.

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).

Spectrally decomposed dark-to-light transitions in Synechocystis sp. PCC 6803.

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB-PSII complexes with closed (saturated) RCs, a quenched or open PB-PSII complex, and a PB-PSII 'not fully closed.' For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB-PSII-PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.