Femtosecond energy transfer in LHCII of green plants

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In vivo regulation of photocurrents
Herbert van Amerongen
Herbert van Amerongen Laboratory of
Biophysics Wageningen
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Photosynthesis occurs in the chloroplasts of
green plants
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The complexes of the thylakoid membrane
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Absorption properties of the photosynthetic
pigments
S2
Qy
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Absorption
Vibronic excited states
Electronic excited state
Absorption 6 fs
Electronic ground state
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Decay of the excited state
Vibronic excited states
Electronic excited state
Internal conversion to the lowest excited
state ( 100 fs) Energy lost as heat
Electronic ground state
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Decay of the excited state
Vibronic excited states
Electronic excited state
Internal conversion to the electronic ground
state (IC) Energy lost as heat
Electronic ground state
kIC 0.02 ns-1 tIC 50 ns
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Decay of the excited state
Vibronic excited states
Electronic excited state
Decay by emission of a photon Energy lost as
fluorescence
Electronic ground state
kF 0.06 ns-1 tF 17 ns
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Decay of the excited state
Electronic excited state
Decay to the triplet state (T) (Intersystem
crossing)
1 ms
Electronic ground state
kT 0.125 ns-1 tIT 8 ns
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Lifetime of the excited state
t (k)-1 (kF kIC kT)-1
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Decay of the excited state 2e
Vibronic excited states
Electronic excited state
Electron transfer to nearby molecule
Electronic ground state
Molecule 1
Molecule 2
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carotenoids in plant photosynthesis
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Carotenoid Absorption Spectra
S2 ? S0
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Femtoseconde pump-probe
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Femtoseconde pump-probe
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Femtoseconde pump-probe
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Femtoseconde pump-probe
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LHCII energy transfer from carotenoids to Chl
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• Energy transfer from Chl b and Car to Chl a is
  ultrafast
• Car and Chl need physical contact for energy
  transfer
• Almost all the excitations are localized on Chl a
• Energy transfer to the reaction center occurs via
  Chls a

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Photosystems I and II are separated in the
thylakoid membrane
PSII and LHCII.
PSI.
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Photosystems I and II can be studied separately
PSII and LHCII.
PSI.
We used BBY particles, no PSI present
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Photosystem 2 (PSII)
PS2-LHC2 supercomplex
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Picosecond fluorescence reveals excited-state
dynamics in PSII membranes
Excitation at 420 nm and 483 nm lead to different
extent of excitation of the outer antenna 68
and 86
Dt
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Charge separation in PSII in the membrane
35 ps
5.3 ps
e-
5.5 ps
-
Antenna
DG 826 cm-1
e-
137 ps
e-
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• Excited-state lifetime of PSII in thylakoid
  membrane is around 300 ps

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The destiny of the excited states
lt1
4
2
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Triplet quenching in the antenna complexes LHCII
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Triplet quenching in the antenna
10ms
1ns
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Measure triplets with slow absorption
difference spectroscopy

• Fast detector
• Relatively more probe light than in a fs-ps
  experiment,

Ns laserpulse
?OD
Monochromator
Lamp
sample
Photomultiplieror photodiode
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Triplets in LHCII
Peterman et al. 1995 Croce et al. 2007
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525
507
510
490
L2
L1
Neoxanthin quenches singlet oxygen Violaxanthin
is available for xanthophyll cycle
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3Chla
3Car
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The other antenna complexes are protected in a
similar way

• CP24, CP26 and CP29 act in the same way as LHCII
  (lutein and neoxanthin)
• CP47 and CP43 use b-carotene

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Two stages of photosynthesis
H2O
Chloroplast
ATP and NADPH
Water oxydation
The light is absorbed by chlorophylls
O2
Light reactions
Transformation of light energy into chemical
energy (ATP and NADPH)
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If the photosynthetic apparatus gets saturated
somewhere (high light), this leads to blocking of
electron transfer in the RC ? Chl triplet
formation in the RC
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There is no carotenoid in contact with Chl in
the RC
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How to avoid photodamage in the RC?

• The expression of light-harvesting complexes can
  be up- and down-regulated (hours)

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State transitions

• Only 15 of the LHC can migrate to PSI. Cyclic
  electron transfer around PSI contributes to
  proton gradient ATP formation can continue while
  PSII receives less photons.
• State transitions are much more important in
  green algae (Chlamydomonas reinhardtii) gt80 of
  the LHC can migrate to PSI.
• Phycobilisomes in cyanobacteria also participate
  in state transitions,

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Light stress
chloroplast
chloroplast
NPQ
Heat production
Photosynthesis
Heat production
Photosynthesis
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Nonphotochemical quenching (NPQ) acts on a
second-minute time scale throw away excess
energy as heat
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NPQ

• Required DpH across thylakoid membrane (result
  of extensive charge separation)
• PsbS can sense the low pH in the lumen via
  protonation (direct quencher or allosteric
  regulator)
• Low pH in the lumen creates zeaxanthin (Zea) via
  the xanthophyll cycle (direct quencher or
  allosteric regulator)

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PsbS belongs to lhc family, binds no Chls,
becomes protonated when lumen has low pH
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Xanthophyll cycle leads to Zea formation at low
pH in the lumen
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Is zeaxanthin a direct quencher of excitations?

• Carotenoids with 11 conjugated bonds can quench
  Chl excitation via excitation energy transfer,
  whereas carotenoids with 9 conjugated bonds
  cannot.
• R. Berera et al. (2006) Proc. Natl. Acad. Sci
  USA 103 5343

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THE SYSTEM
A zinc Phthalocyanine covalently attached to a
series of carotenoids with conjugation length of
9,10 and 11.
Absorption in THF
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THE EXPERIMENT
dyad 1, dyad 2 and dyad 3
Solvents
THF(e07.58), Acetone(e020.6), DMSO(e040.5)
We probe the visible (450-730 nm) and n-IR
regions (850-1000 nm) at different time delays (0
4.8 ns)
We pump the system at 680 nm (Qy
state of Pc)
excitation frequency 1 Khz width 100
fs energy 100 nJ/pulse
Objectives
Can a carotenoid directly quench the singlet
exited state of a Pc-based tetrapyrrole?
How does the process depend on the conjugation
length?
What is the underlying quenching mechanism?
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KINETIC TRACES AT 680 nm IN THF
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QUENCHING MODEL
Qy
slow
S1
ICT
fast
S0
S0
phtalocyanine
carotenoid
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But zeaxanthin can also quench via electron
transfer

• N.E. Holt et al. (2005) Science 307 433.

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NPQ consists of rapid (seconds) qE (no Zea
required) and slower (minutes) qI (Zea required)
Picture from Peter Horton
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LHCII can switch between 2 states
light-harvesting
Heat producing
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Fluorescence quenching by pressure (4 kbar)
?Vqn 510-6 m3/mol 0.006 ? small
conformational change ?G0qn 7 kJ/mol 3
kT/LHCII
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LHCII aggregation as a model system
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FLIM of LHCII
Raman shows that crystal is similar to quenched
aggregates
LHCII in crystal is quenched as compared to LHCII
in detergent
Pascal et al. 2005, Nature 436, 134-137
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Switching behavior in vivo
Nature (next week) A.V. Ruban et al. (2007)
Nature 450 575.
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Upon aggregation, excitations decay via a Car S1
state (femtosecond pump-probe spectroscopy)
A.V. Ruban et al. (2007) Nature 450 575
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Target analysis reveals the spectrum of the
quencher
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proposed molecular model (LHCII)
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Interactions Chl612/Lut620 are also present in
CP24, CP26, CP29 (Croce et al.)
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conclusions

• Luteins 1 and 2 quench triplets in
  light-harvesting complexes in PSII
• Neoxanthin quenches singlet oxygen
• Violaxanthin is source for zeaxanthin formation
• DpH leads to zeaxanthin formation (NPQ, qI)
• PsbS senses DpH and is needed for NPQ (qE and qI)
• Zeaxanthin can in principle also quench in other
  ways
• LHCII is responsible for qE via lutein 1 and Chl
  612
• CP24, CP26 and CP29 have an identical quenching
  cluster and probably also participate in qE

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Other systems

• Diatoms have NPQ like higher plants but stronger
• Cyanobacteria less NPQ (OCP and state
  transitions)
• Green algae less NPQ, more state-transitions
• Green bacteria Chlorosomes triplet excitons

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Coworkers and collaborators
Wageningen Bart van Oort Koen Broess
Sashka Krumova Arie van Hoek Cor Dijkema
Jan Willem Borst Vilnius Gediminas
Trinkunas Leonas Valkunas Amsterdam Jan
Dekker Rienk van Grondelle Rudi
Berera Groningen Roberta Croce Szeged
Gyozo Garab
Sheffield Peter Horton Marseille Stefano
Caffarri London Jim Barber Alexander
Ruban Tel Aviv Nathan Nelson Verona
Roberto Bassi Saclay/Paris Bruno Robert
Andy Pascal Frankfurt Werner Kuhlbrandt
.A Ground state, B Excited state, C, D E
States reached after charge separation and
charge stabilization