Harvesting far-red light:Lessons from Photosystem I

Oxygenic photosynthesis is the fundamental process by which sunlight energy is stored as chemical energy in organic compounds and oxygen is released in the atmosphere. It starts with the capture of a photon by one of the pigments embedded within one of the two photosystems, Photosystem I (PSI) or II (PSII). These photosystems are large assemblies of many pigments held together by the protein scaffold. The absorption of the photon brings the pigment to an electronic excited state. The excitation energy is then transferred from pigment-to-pigment to the reaction center (RC) of the photosystem, where it is used to perform charge separation (CS). The pigment-to-pigment energy transfer within photosynthetic complexes occurs on a very fast, femtosecond (fs, 10^(-15) second) to picosecond (ps, 10^(-12) second) timescale, which ensures that the photosystems are extremely efficient in using the energy for charge separation. In this thesis, aspects of the light-harvesting of photosynthetic pigment-protein complexes were investigated. The spectroscopic properties (absorption, emission) and energy-transfer processes were studied with a variety of different techniques, including advanced ultrafast time-resolved spectroscopic methods (two-dimensional electronic spectroscopy (2DES) and time-resolved fluorescence spectroscopy). In these time-resolved experiments, the complexes are excited with ultrashort (fs temporal width) pulses of light, after which the optical response (photon-echo, fluorescence) is monitored in time. By measuring these signals, excitation energy transfer (EET) and energy trapping within these complexes can be determined. Oxygenic photosynthesis is mainly powered by visible light in the 400–700 nm range. Expanding the absorption range to 750 nm would result in 19% more photons available for photosynthesis [Chen, M. & Blankenship, R. E. (2011) Trends Plant Sci., 16, 427–431]. Moreover, improved far-red light-harvesting can be advantageous in shaded environments. For these reasons extention of the absorption beyond the 400–700 nm range is an important approach in the global aim to improve crop productivity to meet the increasing global demands for food production. This thesis focuses on the far-red light-harvesting properties of PSI from higher plants and cyanobacteria. The aim is to understand underlying aspects that are important for far-red light (FRL, 700–800 nm) absorption and EET within PSI. These aspects can be useful to enhance the far-red light-harvesting in photosynthesis of other organism, such as plants. The chapters of this thesis contain several insights that can be generally important in the goal to enhance the far-red light-harvesting abilities of photosynthetic complexes. The integration of new low-energy states, such as from long-wavelength Chlorophylls (Chls) or new Chl a red form states, is a viable strategy to enhance the absorption of far-red light of these complexes. However, additional alterations to the light-harvesting mechanism may be required to obtain a highly efficient complex with optimally enhanced far-red light absorption. Notably, as shown by the investigated natural (light-harvesting complex I and FRL-specific PSI complex) and artificial (Chl f-containing hybrid PSI) complexes the protein scaffold is a determining factor in optimization of the far-red light-harvesting properties of photosynthetic complexes. Conclusively, in this thesis we provide several important lessons for the aim to effectively enhance the far-red light-harvesting capacity of other photosynthetic organisms

Complete mapping of energy transfer pathways in the plant light-harvesting complex Lhca4

The Lhca4 antenna complex of plant Photosystem I (PSI) is characterized by extremely red-shifted and broadened absorption and emission bands from its low-energy chlorophylls (Chls). The mixing of a charge-transfer (CT) state with the excited state manifold causing these so-called red forms results in highly complicated multi-component excited energy transfer (EET) kinetics within the complex. The two-dimensional electronic spectroscopy experiments presented here reveal that EET towards the CT state occurs on three timescales: fast from the red Chls (within 1 ps), slower (5-7 ps) from the stromal side Chls, and very slow (100-200 ps) from a newly discovered 690 nm luminal trap. The excellent agreement between the experimental data with the previously presented Redfield-Förster exciton model of Lhca4 strongly supports the equilibration scheme of the bulk excitations with the dynamically localized CT on the stromal side. Thus, a complete picture of the energy transfer pathways leading to the population of the CT final trap within the whole Lhca4 complex is presented. In view of the environmental sensitivity of the CT contribution to the Lhca4 energy landscape, we speculate that one role of the CT states is to regulate the EET from the peripheral antenna to the PSI core

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals. However due to the low fluorescence intensity, these constructs require use of much higher light intensity than other optogenetic tools. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity

Harvesting far-red light: Lessons from Photosystem I

Oxygenic photosynthesis is the fundamental process by which sunlight energy is stored as chemical energy in organic compounds and oxygen is released in the atmosphere. It starts with the capture of a photon by one of the pigments embedded within one of the two photosystems, Photosystem I (PSI) or II (PSII). These photosystems are large assemblies of many pigments held together by the protein scaffold. The absorption of the photon brings the pigment to an electronic excited state. The excitation energy is then transferred from pigment-to-pigment to the reaction center (RC) of the photosystem, where it is used to perform charge separation (CS). The pigment-to-pigment energy transfer within photosynthetic complexes occurs on a very fast, femtosecond (fs, 10^(-15) second) to picosecond (ps, 10^(-12) second) timescale, which ensures that the photosystems are extremely efficient in using the energy for charge separation. In this thesis, aspects of the light-harvesting of photosynthetic pigment-protein complexes were investigated. The spectroscopic properties (absorption, emission) and energy-transfer processes were studied with a variety of different techniques, including advanced ultrafast time-resolved spectroscopic methods (two-dimensional electronic spectroscopy (2DES) and time-resolved fluorescence spectroscopy). In these time-resolved experiments, the complexes are excited with ultrashort (fs temporal width) pulses of light, after which the optical response (photon-echo, fluorescence) is monitored in time. By measuring these signals, excitation energy transfer (EET) and energy trapping within these complexes can be determined. Oxygenic photosynthesis is mainly powered by visible light in the 400–700 nm range. Expanding the absorption range to 750 nm would result in 19% more photons available for photosynthesis [Chen, M. & Blankenship, R. E. (2011) Trends Plant Sci., 16, 427–431]. Moreover, improved far-red light-harvesting can be advantageous in shaded environments. For these reasons extention of the absorption beyond the 400–700 nm range is an important approach in the global aim to improve crop productivity to meet the increasing global demands for food production. This thesis focuses on the far-red light-harvesting properties of PSI from higher plants and cyanobacteria. The aim is to understand underlying aspects that are important for far-red light (FRL, 700–800 nm) absorption and EET within PSI. These aspects can be useful to enhance the far-red light-harvesting in photosynthesis of other organism, such as plants. The chapters of this thesis contain several insights that can be generally important in the goal to enhance the far-red light-harvesting abilities of photosynthetic complexes. The integration of new low-energy states, such as from long-wavelength Chlorophylls (Chls) or new Chl a red form states, is a viable strategy to enhance the absorption of far-red light of these complexes. However, additional alterations to the light-harvesting mechanism may be required to obtain a highly efficient complex with optimally enhanced far-red light absorption. Notably, as shown by the investigated natural (light-harvesting complex I and FRL-specific PSI complex) and artificial (Chl f-containing hybrid PSI) complexes the protein scaffold is a determining factor in optimization of the far-red light-harvesting properties of photosynthetic complexes. Conclusively, in this thesis we provide several important lessons for the aim to effectively enhance the far-red light-harvesting capacity of other photosynthetic organisms

Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis–trans and trans–cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright “on” and dark “off” states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete

Complete mapping of energy transfer pathways in the plant light-harvesting complex Lhca4

The Lhca4 antenna complex of plant Photosystem I (PSI) is characterized by extremely red-shifted and broadened absorption and emission bands from its low-energy chlorophylls (Chls). The mixing of a charge-transfer (CT) state with the excited state manifold causing these so-called red forms results in highly complicated multi-component excited energy transfer (EET) kinetics within the complex. The two-dimensional electronic spectroscopy experiments presented here reveal that EET towards the CT state occurs on three timescales: fast from the red Chls (within 1 ps), slower (5–7 ps) from the stromal side Chls, and very slow (100–200 ps) from a newly discovered 690 nm luminal trap. The excellent agreement between the experimental data with the previously presented Redfield–Fo¨rster exciton model of Lhca4 strongly supports the equilibration scheme of the bulk excitations with the dynamically localized CT on the stromal side. Thus, a complete picture of the energy transfer pathways leading to the population of the CT final trap within the whole Lhca4 complex is presented. In view of the environmental sensitivity of the CT contribution to the Lhca4 energy landscape, we speculate that one role of the CT states is to regulate the EET from the peripheral antenna to the PSI core

Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein

The fast-switching M159T mutant of the reversibly photoswitchable fluorescent protein Dronpa has an enhanced yield for the on-to-off reaction. The forward and reverse photoreactions proceed via cis–trans and trans–cis photoisomerization, yet protonation and deprotonation of the hydroxyphenyl oxygen of the chromophore is responsible for the majority of the resulting spectroscopic contrast. Ultrafast visible-pump, infrared-probe spectroscopy was used to detect the picosecond, nanosecond, as well as metastable millisecond intermediates. Additionally, static FTIR difference measurements of the Dronpa-M159T mutant correspond very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone chromophore in the cis anion and trans neutral forms in the bright “on” and dark “off” states, respectively. Green excitation of the on state is followed by dominant radiative decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1 ns, and additionally reveals spectral changes belonging to the species decaying with a 1.1 ns time constant, associated with both protein and chromophore modes. A 1 ms measurement of the on state identifies bleach features that correspond to those seen in the static off-minus-on Fourier transform infrared (FTIR) difference spectrum, indicating that thermal protonation of the hydroxyphenyl oxygen proceeds within this time window. Blue excitation of the off state directly resolves the formation of the primary photoproduct with 0.6 and 14 ps time constants, which is stable on the nanosecond time scale. Assignment of the primary photoproduct to the cis neutral chromophore in the electronic ground state is supported by the frequency positions expected relative to those for the nonplanar distorted geometry for the off state. A 1 ms measurement of the off state corresponds closely with the on-minus-off FTIR difference spectrum, indicating thermal deprotonation and rearrangement of the Arg66 side chain to be complete

Maternal hypothyroxinaemia in early pregnancy and problem behavior in 5-year-old offspring

Introduction There is evidence, though not consistent, that offspring born to mothers with subtle decreases in thyroid function early in their pregnancies may be at risk of cognitive impairments and attention problems. However, other types of problem behavior have not been addressed thus far. We tested whether maternal thyroid function in early pregnancy is associated with several types of problem behavior in offspring at age 5–6 years. Methods This was a longitudinal study that included the data of 2000 mother-child pairs from the Amsterdam Born Children and their Development study. At a median gestational age of 12.9 (interquartile range: 11.9–14.1) weeks, maternal blood was sampled for assessment of free T4 and TSH. Overall problem behavior, hyperactivity/inattention, conduct problems, emotional problems, peer relationship problems and prosocial behavior were measured at age 5–6 years using the Strengths and Difficulties Questionnaire, which was filled out by both parents and teachers. Results Maternal hypothyroxinaemia <5th percentile was associated with a 1.70 (95% confidence interval (CI): 1.01–2.86) increased odds of teacher-reported hyperactivity/inattention after adjustment for confounders. By increasing the cut-off level to <10th percentile, the odds ratio became 1.47 (95% CI: 0.99–2.20). There were no associations between maternal thyroid function parameters and hyperactivity/inattention as reported by parents, nor with teacher or parent reports of other types of problem behavior. Conclusions Our results partially confirm previous observations, showing that early disruptions in the maternal thyroid hormone supply may be associated with ADHD symptoms in offspring. Our study adds that there is no evidence for an effect on other types of problem behavior

Harvesting far-red light:Functional integration of chlorophyll f into Photosystem I complexes of Synechococcus sp. PCC 7002

The heterologous expression of the far-red absorbing chlorophyll (Chl) f in organisms that do not synthesize this pigment has been suggested as a viable solution to expand the solar spectrum that drives oxygenic photosynthesis. In this study, we investigate the functional binding of Chl f to the Photosystem I (PSI) of the cyanobacterium Synechococcus 7002, which has been engineered to express the Chl f synthase gene. By optimizing growth light conditions, one-to-four Chl f pigments were found in the complexes. By using a range of spectroscopic techniques, isolated PSI trimeric complexes were investigated to determine how the insertion of Chl f affects excitation energy transfer and trapping efficiency. The results show that the Chls f are functionally connected to the reaction center of the PSI complex and their presence does not change the overall pigment organization of the complex. Chl f substitutes Chl a (but not the Chl a red forms) while maintaining efficient energy transfer within the PSI complex. At the same time, the introduction of Chl f extends the photosynthetically active radiation of the new hybrid PSI complexes up to 750 nm, which is advantageous in far-red light enriched environments. These conclusions provide insights to engineer the photosynthetic machinery of crops to include Chl f and therefore increase the light-harvesting capability of photosynthesis