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

3. 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

4. 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

5. 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.

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

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

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

14. 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:

15. 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.

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

17. 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.

18. Decay-associated spectraFm
FNPQ

19. 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

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

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

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

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

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

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

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

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

28. 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

29. The spectral change ΔF720 and qE

30. Theoretical modeling Of PSII Photoprotection

31. 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

32. 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

33. 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:

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

35. Population kinetics
Lambrev et al. (2012) BBA 1817:

36. 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

37. 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:

38. 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:

39. 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:

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

41. 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:

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

43. Detachment and quenching
Lambrev et al. (2012) BBA 1817:

44. Recombination and quenching
Lambrev et al. (2012) BBA 1817:

45. 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:

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

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

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

49. 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

50. 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.

51. 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 nm diameter are clearly visible on the AFM images.
The complexes are partially aggregated.

52. 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%.

53. 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.

54. 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.

55. 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 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.

56. 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