Photoprotective Energy Dissipation in the Light-Harvesting Antenna Petar H. Lambrev Biological Research Centre, Szeged, Hungary

Thanks to Max-Planck Institute for Chemical Energy Conversion Biological Research Centre Max-Planck Institute for Chemical Energy Conversion Dr. Győző Garab Dr. László Kovács Dr. Yuliya Miloslavina Prof. Dr. Alfred Holzwarth Michael Reus Prof. Dr. Leszek Fiedor Dr. Johanna Fiedor Dr. Jedrzej Tworzydło Prof. Dr. Peter Jahns Manuela Nilkens Prof. Dr. Rienk van Grondelle Prof. Dr. Marloes Groot Dr. Andreas Stahl Dr. Ivo van Stokkum

Outline Intro – photoprotection in plants Non-photochemical quenching in plants Far-red fluorescence of LHCII The two sites of NPQ in plants NPQ mechanisms in time Relationship between NPQ and photoprotection Energy dissipation in bacterial light-harvesting antennas containing Ni-BChl

Photoprotection in plants Excess light can damage and kill the organism Aerobic photosynthetic organisms must protect against excess light Light Intensity Energy Absorbed Energy Utilized Energy Excess light This is why all plants and most all other photosynthetic organisms have mechanisms to dissipate the excess light energy safely as heat. This process is termed non-photochemical quenching. NPQ is a major photoprotective feature in plants.

Photoprotection strategies in plants Organism level Heliotropic movements Photonastic movements Leaf rolling Tissue level Optical screening Phenolic Flavonoids Anthoc. Cars Cellular level Chloroplast movements Chloroplast level Antenna size NPQ Water-water cycle Cyclic ET ROS scavenging Repair

Photoprotection strategies in plants Organism level Heliotropic movements Photonastic movements Leaf rolling Tissue level Optical screening Phenolic Flavonoids Anthoc. Cars Cellular level Chloroplast movements Chloroplast level Antenna size NPQ Water-water cycle Cyclic ET ROS scavenging Repair

Photoprotection strategies in plants Organism level Heliotropic movements Photonastic movements Leaf rolling Tissue level Optical screening Phenolic Flavonoids Anthoc. Cars Cellular level Chloroplast movements Chloroplast level Antenna size NPQ Water-water cycle Cyclic ET ROS scavenging Repair Decrease of absorption

Photoprotection strategies in plants Organism level Heliotropic movements Photonastic movements Leaf rolling Tissue level Optical screening Phenolic Flavonoids Anthoc. Cars Cellular level Chloroplast movements Chloroplast level Antenna size NPQ Water-water cycle Cyclic ET ROS scavenging Repair Decrease of absorption Prevention of damage

Photoprotection strategies in plants Organism level Heliotropic movements Photonastic movements Leaf rolling Tissue level Optical screening Phenolic Flavonoids Anthoc. Cars Cellular level Chloroplast movements Chloroplast level Antenna size NPQ Water-water cycle Cyclic ET ROS scavenging Repair Decrease of absorption Prevention of damage Damage repair

Non-photochemical quenching NADPH ADP+P ATP PSII PSI LHCII PSII Cyt b6f e− e− Where does the dissipation take place? This is a rough scheme of the photosynthetic membrane – light energy is absorbed by the antenna of PSII and transferred to the PSII core where charge separation occurs, the electrons are transferred to PSI when with a second photon the electrons finally reduce NADP. H2O 2H++½O2 The primary site of NPQ is the antenna of Photosystem II

NPQ components NPQ relaxation phases: qE – rapidly relaxing qI – slowly relaxing dark light Fm qI NPQ Chlorophyll fluorescence qE 500 s Time

Xanthophyll conversion Factors governing NPQ ΔpH Xanthophyll conversion PsbS Violaxanthin (Vx) VDE Antheraxanthin H+ H+ VDE Zeaxanthin (Zx)

A model of NPQ based on time-resolved spectroscopy of leaves PSII in dark-adapted state High-light-adapted state light + ΔpH, Zx, PsbS Q1 Q2 Holzwarth et al. (2009) Chem. Phys. Lett. 483:262-267

Far-red fluorescence as a probe for NPQ 77 K RT DAS 77 K fluorescence emission spectra and RT decay-associated spectra of LHCII trimers, aggregates and LHCII in leaves Miloslavina et al. (2008) FEBS Lett.

Time-resolved NPQ measurements Picosecond fluorescence kinetics measured by TCSPC from intact leaves in two states: Dark-adapted (Fm) Light-adapted (Fnpq)

Kinetic model scheme PS II PS I kD Red1* (Ant/RC)* RP1 RP2 (Ant/RC)* This kinetic model allows us to calculate all decay lifetimes associated with the two photosystems and their fluorescence spectra.

Decay-associated spectra Fm FNPQ

Q2 (quenching of PSII antenna) The role of Zx and PsbS Wild type npq4 L17 npq1 Q1 (detached antenna) 30% 0% 56% Q2 (quenching of PSII antenna) 4.2 4.3 5.3 1.6 Q1 is PsbS-dependent Q2 is Zx-dependent

Dual-LED spectrofluorimeter Lambrev et al. (2010) Plant Physiol. 152:1611-1624

NPQ vs. time and wavelength wild type npq4 Lambrev et al. (2010) Plant Physiol. 152:1611-1624

Emission spectra during light adaptation and re-darkening Light On Light Off Lambrev et al. (2010) Plant Physiol. 152:1611-1624

Emission spectra during light adaptation and re-darkening Light On Light Off Lambrev et al. (2010) Plant Physiol. 152:1611-1624

Fluorescence kinetics at different wavelengths Holzwarth et al. (2009) Chem. Phys. Lett. 483:262-267

Emission spectra of npq4 (PsbS-deficient) mutant Light On Light Off Lambrev et al. (2010) Plant Physiol. 152:1611-1624

Emission spectra of the npq4 mutant Light On Light Off Lambrev et al. (2010) Plant Physiol. 152:1611-1624

Fluorescence kinetics in the npq4 mutant Lambrev et al. (2010) Plant Physiol. 152:1611-1624

L17 (PsbS overexpressor) NPQ parameter* Fluorescence ratios** ΔF720 is not proportional to NPQ ΔF720 has different kinetics ΔF720 is PsbS-dependent * NPQ = Fm / F − 1 ** F720 multiplied by 4

The spectral change ΔF720 and qE

Theoretical modeling Of PSII Photoprotection

Questions How do different photoprotective processes affect the fluorescencmechanisms are most/least efficient with resp. to photoprotection? What is the quantitative relationship between fluorescence and photoprotection? e yield? What is the real photoprotection gain? Which NPQ

Photoprotection (definition) Main mechanism of damage of PSII: 3Chl in the RC (charge recombination of 3RP) Damage from antenna 3Chl is neglected (quenched by Car’s) Photoprotection is any process preventing the formation of 3Chl in the PSII RC

Photoprotection mechanisms Detachment of the PSII antenna Quenching of the (attached) PSII antenna Charge recombination in PSII* Simulated separately and in combination * Vass & Cser (2009) Trends Plant Sci. 42:200-205

ERPE model scheme Ant/RC* kCS RP1 k1 RP2 krec k-1 kD kT 3Chl Lambrev et al. (2012) BBA 1817:760-769

Population kinetics Lambrev et al. (2012) BBA 1817:760-769

2. Simulate photoprotection Simulation workflow Set initial conditions Calculate φF Calculate φT 1. Initial state Change initial conditions 2. Simulate photoprotection Relative φF change Relative φT change 3. Estimate effects

1. Initial state – calculate φF and φT Ant/RC* 3 8.5 0.4 2 0.5 0.9 RP1 RP2 𝜑 𝐹 = 𝜏 𝑎𝑣𝑔 ∗ 3Chl 𝜑 𝑇 = [ 3 Chl] 𝑡=∞ Lambrev et al. (2012) BBA 1817:760-769

2. Photoprotection mechanisms 1. Antenna detachment Ant/RC* kCS RP1 k1 RP2 krec k-1 kD 3. Recombination kT 2. Antenna quenching 3Chl Lambrev et al. (2012) BBA 1817:760-769

3. Calculation of effects Fluorescence quenching factor 𝑄 = 𝜑 𝐹 0 𝜑 𝐹 Photoprotection factor 𝑃 = 𝜑 𝑇 0 𝜑 𝑇 Lambrev et al. (2012) BBA 1817:760-769

Antenna quenching P and Q are equal P and Q are linearly dependent on kd Lambrev et al. (2012) BBA 1817:760-769

Antenna detachment P and Q are not equal (Q > P) Q depends on the yield of the detached antenna Lambrev et al. (2012) BBA 1817:760-769

Charge recombination P is linearly dependent on krec Q decreases with krec! Lambrev et al. (2012) BBA 1817:760-769

Detachment and quenching Lambrev et al. (2012) BBA 1817:760-769

Recombination and quenching Lambrev et al. (2012) BBA 1817:760-769

Modeling of experimental cases Ant/RC* RP1 RP2 Arabidopsis (wild type) dark-adapted leaves 3 8.5 0.4 2 0.5 0.9 70% Ant/RC* RP1 RP2 High-light-adapted leaves 30% Ant* 2.1 22 1.7 4 2 0.9 2.3 Lambrev et al. (2012) BBA 1817:760-769

Q and P in wild type Arabidopsis Lambrev et al. (2012) BBA 1817:760-769

Conclusions Quenching ≠ Photoprotection Different mechanisms operating in parallel enhance photoprotection Redundancy Robustness Responsiveness on different time scales from seconds to hours

Energy dissipation in LH1 antenna mediated by Ni-bacteriochlorophyll This project is run in collaboration with the Jagiellonian University in Krakow and VU Amsterdam.

LH1 in photosynthetic bacteria Closed ring or open oval Surrounding the RC Strong excitonic interaction between Bchl’s has two major consequences: Transition energy shifts to the infrared (880 nm) Extremely rapid energy transfer

Ni-substituted bacteriochlorophyll ~ 3 ns 100 fs Ni-BChl: ultrafast excitation deactivation (60 fs) Spectra similar to BChl – allow for efficient energy transfer A powerful quencher of BChl excitations In Chls and Bchls the central atom of the porphyrin ring is Mg. When the central atom is exchanged to another metal, the excited state properties drastically change. Substituting the Mg atom by Ni, which has vacant d-orbitals in the outer electron shell, creates ultrafast excitation deactivation channels. In normal BChl the excited state lifetime is several ns but in Ni-substituted BChl it becomes extremely short – less than 60 fs. However energy levels and therefore absorption spectra do not change significantly, so energy transfer is possible between BChl and Ni-BChl. Because of this Ni-Bchl is a powerful excitation quencher. In our work we created LH1 complexes in which BChl is partially exchanged with Ni-BChl. The aim of this was two-fold.

AFM images of reconstituted LH1 For reconstitution of LH1 without carotenoids, we used the carotenoidless G9 mutant of Rsp. rubrum. Without cars LH1 dissociates reversibly, depending on the detergent concentration, into alfa-beta heterodimers or to monomeric subunits. To reconstitute LH1 with Ni, the isolated chromatophores are first dissolved with nonionic detergent β-OG to obtain heterodimers, then incubated with Ni-bchl which replaces the bound Bchl. The excess pigment is then removed and the Lh1 subunits purified by ion exchange chromatography. Finally the oligomeric Lh1 is reconstituted by lowering the detergent concentration. Rings of 20-30 nm diameter are clearly visible on the AFM images. The complexes are partially aggregated.

LH1 without carotenoids Absorption spectra LH1 without carotenoids LH1 with spheroidene The oligomeric LH1 has Qy absorption maximum at 880 nm and is easily distinguished from dimeric and monomeric subunits and free pigments. The abs spectra have well separated Car and BChl Qx and Qy peaks. That allowed us in the measurements to selectively excite Cars or BChls by tuning the excitation wavelength. We used three excitation wavelengths: 509, 590 and 860 nm to excite Sph, BChl Qx and BChl Qy. The successful incorporation of Ni-bchl is confirmed by measuring the fluorescence yield of the purified complexes, which was decreased by up to 97%.

TA results – LH1 with Sph control LH1 Ni-LH1 The result of measurement is the differential absorption (with and without pump pulse) as a function of wl and time. The main signal at around 890 nm is negative, because it reflects the depopulation of the ground state. I will present the results for Lh1 reconstituted with spheroidene. The figures depict the first 100 ps after excitation of the control and Ni-substituted Lh1. the ground state bleach shows very little decay in 100 ps in the control Lh1 but in the Ni-containing sample the signal quickly decays.

Time traces at 890 nm control LH1 Ni-LH1 The kinetics of ground state recovery is followed by the time traces at 890 nm. In these figures the different traces correspond to excitation wavelengths. Only the initial 10 ps after excitation are shown. In the control during this time there is no ground state recovery, except when exciting at 860 nm. On the contrary, when Spheroidene was excited (green), we could see the slow process of exciting BChl due to EET from Spheroidene. The TA kinetics of Ni-LH1 show fast decay of the ground state bleach. When exciting Bchl directly, 50% of the ground state was recovered in 4 ps. So the kinetics of excitation trapping by Ni-BChl is clearly visible. However, we did not expect to find a much slower relaxation when Sph was excited by the pump pulse. No significant relaxation in 10 ps Slow EET from Sph to BChl 50% GS recovery in 4 ps Slower kinetics with Sph exc.

Global analysis – control LH1, 590 nm excitation 60 fs 270 fs 11 ps 750 ps Qx Qy(2) Qy(1) Qy(0) Assignment of spectral bands: GSB at 880-890 nm Qx ESA at >900 nm Qy ESA at <860 nm SE at ~900 nm Next we analyse the kinetics of the same sample but after excitation of Spheroidene. The most important difference is that the lifetimes associated with Qx-Qy conversion are much slower, by a factor of 2-3. The decay lifetime remained at 700 ps. This result indicates the presence of slow (ps to 10’s of ps) energy transfer from Spheroidene to BChl. Interestingly though, the initially populated state has strong ESA that can be attributed to Qx, which means that a large portion of the energy is transferred so fast that it cannot be resolved. In conclusion there seem to be several EET pathways from Sph to BChl, some are ultrafast and some very slow.

Global analysis – Ni-LH1 Qx-Qy conversion as in control Main excited-state decay 2 ps Slower decay components indicate heterogeneity Excited state equilibration limits the quenching kinetics Still, the energy is effectively dissipated by Ni-BChl in few ps We then performed the same analyses to LH1 samples containing Ni-BChl as an excitation trap. These are the EADS spectra in the NIR region for BChl and Car excitation. Again it was possible to resolve the Qx to Qy conversion, which occured with lifetimes of 50 fs and 400 fs – similar to samples without Ni. Most importantly, we do not resolve any quenching components in this time range but only excitonic relaxation. The main excitation quenching component with BChl excitation was about 2 ps. Slower components are also present which may indicate heterogeneity due to the number of Ni-Bchl molecules per LH1 complex. Most likely though these components originate from non-intact LH1’s because they are blue shifted. As with control LH1, the excitation of Spheroidene slows down the kinetics significantly. We can conclude that carotenoid