«PROBLEM OF ORIGIN OF LIFE»
International Conference in Honor of 120th Birth Anniversary of
acad. A.I. Oparin
Karapetyan N.V.
A.N. Bach Institute of Biochemistry RAS, Moscow
How cyanobactria managed to survive under
intense solar radiation billions years ago:
Photoprotection mechanisms
September 26, 2014
Acad. A.I. Oparin was elected as the first President of ISSOL
(photo was taken in Pont- á-Mousson, France 1970)
.
«You are our Pope, we are your monks!»
«Вы наш пастырь, мы Ваши иноки!»
Acad. A.I. Oparin was the Director of A.N. Bach Institute of
Biochemistry for 1946-1980
Many laboratories of our
. Institute have been involved in study of
Origin and Evolution of Life. My contribution:
“Photoprotecton mechanisms against photodestruction by excess
absorbed energy in cyanobacteria.”
We have found two mechanisms of photoprotection in cyanobacteria:
1.Carotenoid-less non-photochemical quenching by Photosystem I
2.Carotenoid-induced non-photochemical quenching of Phycobilisomes
Cyanobacteria, the first photosynthetic organisms, have originated about 2.5-3
Gyrs ago in conditions of intense UV and VIS light at the absence of ozone
layer.
Irradiance conditions on the Earth surface NOW on the width of equator:
UV-C (190-280 nm) - does not penetrate the ozone layer
UV-B (280-320 nm) - 7-8 W m-2
UV-A (320-400 nm) - 45-50 W m-2 (generates singlet oxygen)
VIS light (400-700 nm) - 1100 W m-2
To be protected against intense solar light and UV, cyanobacteria were habituated
in deep ocean waters or in hydrothermal sources.
Oxygenic photosynthesis
Photosynthesis is optimal under the balance of the activity and stability of the
photosynthetic apparatus. Over-excitation of antenna Chls generates reactive
oxygen species that destroy the photosynthetic apparatus.
Dissipation (or quenching) of excess absorbed energy protects against photodestruction.
1.
Carotenoid-less non-photochemical quenching by Photosystem I
PSI complex exists in cyanobacteria as a trimer, in plants as a monomer.
2.5Å structure of PSI trimer of Th. elongatus
Jordan et al., Nature (2001)
3.4Å structure of PSI monomer of P. sativum
Amunts et al., Nature (2007)
Organization of Chlorophyll (Chl) antenna in cyanobacteria
Chls in cyanobacteria are located only in core antenna of PSI and PSII
since cyanobacteria are deficient in Chl-containing Lhca.
Cyanobacteria are highly enriched with PSI: PSI/PSII ratio is 3-5.
Thus main part of Chls (~90%) in cyanobacteria is located in PSI.
About 90% of antenna Chls in PSI of cyanobacteria belong to bulk while
10% of antenna Chls belong to long-wavelength Chls (LWC).
The origin of LWC and the role in PSI was not clear.
We have studied the role of the red-most LWC in energy balance and in
energy dissipation in the cyanobacterium Arthrospira platensis
Some information about LWC of PSI in cyanobacteria.
LWC in PSI core antenna of cyanobacteria and
plants (Gobets…Karapetyan et al., Biophys. J. 2001)
1,0
0,8
absorbance
0,6
6K
0,4
708 (7)
0,2
740 (3)
0,0
550
600
650
700
750
wavelength / nm
Gaussian deconvolution of 5 K absorption spectrum
of PSI trimers of A. platensis:
LWC740 (F760) = 3; LWC708 (F730) = 7
(Schlodder,….Karapetyan et al., BBA 2005)
730 nm
290 K
trimer
monomer
740
708
800
Spectral characteristics of LWC in PSI trimers and monomers of
A. platensis and Th. elongatus; amount of Chl molecules - in parenthesis
(Karapetyan et al., FEBS Lett. 1999)
Cyanobacteria
Absorbance
bands
Fluorescence
λmax with
P700 red.
Fluorescence
λmax with
P700 ox.
A. platensis
trimers
708 (7)
740 (3)
727
760
726
A. platensis
monomers
708 (7)
727
726
Th. elongatus
trimers
708 (4)
719 (4)
730
741
732
Th. elongatus
monomers
708 (4)
719 (2)
730
728
trimer
monomer
Fluorescence DAS
(decay associated spectra)
LWC delay the energy equilibration in core
antenna and trapping by P700; it is
dependent on spectral properties of LWC:
35 ps in PSI trimers of Th. elongatus - C
37 ps in PSI monomers of A. platensis - D
50 ps in PSI trimers of A. platensis - E.
(Gobets,.. Karapetyan et al., Biophys. J.
2001)
trimer
P700+ efficiently quenches F760 of PSI trimers of A. platensis and
F735 of PSI trimers of Th. elongatus (Schlodder… Karapetyan, BBA 2011)
PSI trimers
P700 reduced
P700 oxidized
P700AoA1-FxFA-FB-
0,8
PS I trimer
A. platensis
77 K
0,7
0,5
ex = 500 nm
727
1,0
fluorescence
A. platensis
fluorescence
1,0
PSI monomers
0,8
PS I monomer
A. platensis
77 K
ex = 500 nm
726
P700 reduced
P700 oxidized
red - ox
0,6
0,4
0,3
0,2
0,2
0,0
660
760
680
P700+AoA1FXFAFB
700
720
740
760
wavelength / nm
Th. elongatus
780
800
820
840
731
0,0
660
680
700
720
740
760
wavelength / nm
780
800
820
840
Energy transfer in PSI antenna depends on redox state of the cofactors of the
PSI Rection Center (RC):
open RC – charge separation
Chl → P700A0A1FX → P700+Ao- A1FX
closed RC – dissipation of absorbed energy
Chl → P700+A0A1FX or Chl → 3P700A0A1-FX
P700 is involved in charge separation
P700+ or 3P700 are involved in energy dissipation
Origin of LWC: interaction of Chl molecules on the surface of various PSI
monomers is forming the red-most LWC (F760) in PSI trimers of A. platensis
(Karapetyan et al., Photosynth. Res. 1999)
1,0
F760
0,8
0,6
0,4
0,2
0,0
0,0
0,2
0,4
0,6
0,8
+
1,0
P700/(P700 + P700 )
Time-course of F760
quenching and P700+
formation in PSI trimers
of A. platensis at 77K
PSI trimer of Th. elongatus
(Jordan et al., 2001)
Non-linear dependence of F760
on P700+ amount in PSI trimers
of A. platensis indicates on
energy exchange between PSI
monomers within trimer
Localization of LWC in PSI antenna of Th. elongatus:
trimer 719 (F741) - 4 Chls; 708 (F732) - 4 Chls
monomer 719 (F730) - 2 Chls; 708 (F728) - 4 Chls
Chl719 (F741) might be B7/A32/A31
F741
Chl719 is not B31/B32/B33 – 3
Chls, big distance to P700 (50Å)
Candidates for Chl708 (F732) are
B38/B37, А38/A39, B18/B19 or
A16/A17/A25 (strong coupling
between Chls, dig distance to
P700).
Chl715 (F734) = B24/B25 or A26/A27
F734
F734
F732
Schlodder…Karapetyan, BBA (2012)
Localization of LWC in PSI complexes of A. platensis
PSI trimer: 740 (F760) - 3 Chl; 708 (F727) - 7 Chl
PSI monomer: 708 (F726) – 7 Chl (three different aggregates).
Chl740 (F760 ) might be A31/A32/B7 on lumenal side close to trimerization point,
time of energy transfer to P700+ is 110 ps, dipol is oriented parallel to membrane
Chl708(F727)=B38/B37, A38/A39
B18/B19 or A25/A16/A17
F760
Distance between Chl740 and Chl708:
Chl740
F727
Chl708
A32/A31/B7 to B38/B37 = 22Å
A32/A31/B7 to A25/A16/A17= 48Å
A32/A31/B7 to А38/A39 = 57Å
A32/A31/B7 to B18/B19 = 52Å
or F727
Schlodder…Karapetyan, BBA (2012)
Different orientation
of Chls in various LWC730 of
PSI antenna in A. platensis
SMS data
Fluorescence spectra of a single
PSI trimer of as a function of
the orientation of polarizer in
front of the spectrograph
Chls in F730 polarized differently
since 2-3 different emitters
form this LWC. Chls in F760
are polarized equally.
(Brecht,….Karapetyan BBA 2012)
Scheme of energy migration in antenna of PSI trimers of A. platensis
No interaction of some LWC708 and LWC740 at cryogenic temperatures:
- big distance between F760 (А31-A32-B7) and LWC726 (different complexes)
- different orientation of the transient dipole moments in LWC708
(Karapetyan et al., Biochemistry-Moscow 2014)
Bulk Chl
LWC708
F726
P700
LWC708
~F726
LWC740
F760
P700+
heat
А31-A32-B7
1. Conclusions: PSI-induced energy dissipation in cyanobacteria
1. LWC delay the energy equilibration and trapping in PSI core antenna. LWC function as
terminal acceptors of excitation like P700 and transfer uphill energy to P700.
2. P700+ quenches the LWC fluorescence of PSI trimers and monomers of A. platensis and
Th. elongatus but with different efficiency.
3. LWC740 (F760) in PSI of A. platensis may correspond to peripherally localized
A31/A32/B7 trimeric aggregate. Localization of LWC719 in PSI of Th. elongatus may
differ since aggregate contains 4 Chls.
2. Caroteboid-induced NPQ of Phycobilisomes (PBS) fluorescence in
cyanobacteria; PBS are the main light-harvesting complex in cyanobacteria
Structure of Phycobilisomes, interaction with Photosystems
PBS
PSI
PSII
In 2004 we have found that illumination by blue-green light of Synechocystis
cells quenches the fluoresence of PBS at 660 nm; quenching is reversible in
dark (Rakhimberdieva et al., FEBS Lett. 2004).
=APC
dark (non-quenched)
after BL
(quenched)
Quenching decreases PBS fluorescence at 660
nm (exc. 580 nm)
Action spectrum of quenching
Photoprotective dissipation of energy in cyanobacteria.
1. PBS is the quenching target, carotenoid is photosensitizer (Rakhimberdieva et al., 2004)
2. Quenching - only at physiological temperatures (Rakhimberdieva et al., 2004, 2007)
3. Quenching is ∆pH independent (Rakhimberdieva et al., 2006; Wilson et al., 2006)
4.OCP-red (=OCP*) may be fluorescence quencher (Wilson et al., 2006, 2008).
Main strategy to reveal the mechanism of quenching - comparison of the activity of PSI
and PSII in Synechoystis mutant cells in non-quenched and quenched states.
PSI activity was measured for PSII-less mutant, PSII activity - for PSI-less mutant.
Orange Carotenoidbinding protein (OCP)
non-quenched
OCP (35 kDa) from
A. maxima - twodomain homodimer
containing 3’hydroxiechinenone
(Kerfeld et al., 2003)
quenched
down regulation of photosynthesis
Quantum efficiency of PBS absorption in Synechocystis cells in quenched state drops by
about 40% (P700 photooxidation and PSII fluorescence induction). OCP-triggered energy
dissipation in PBS of Synechocystis diverts excitation away from both RC
(Rakhimberdieva et al., BBA 2010).
PS2-less strain
PS1-less strain
100
Relative P700 signal amplitude
0,8
0,6
80
60
+RL
+BL
+
Relative FPAM signal amplitude
1,0
+RL
+BL
0,4
0,2
40
20
0
0,0
0
20
40
60
0,5s flash intensity
80
100
0
2
4
6
8
0,3s flash intensity
10
12
BL-induced quenching takes place even at the absence of PSI and PSII
(Rakhimberdieva et al., FEBS Lett. 2011)
77K
PSI/PSII-less
Fluorescence quenching spectra at 77 K
and RT (top) and the second derivative
of quenching spectrum at RT (down).
NPQ norm. max
77K fluorescence spectra (exc. 570
nm) of WT and PSI/PSII-less
mutant
0
288 К
77 К
-1
WT
WT
0
+
660
680
288 К
620640660680700720740760780
Wavelength, nm
Light saturation curves of quenching centre formation
BL
Kuzminov….. Karapetyan BBA 2012
2. Conclusions on OCP-induced NPQ
1. Carotenoid is photosensitizer of PBS quenching, APC is a target of OCPinduced fluorescence quenching in Synechocystis cells.
2. OCP-induced quenching of APC fluorescence in Synechocystis cells diverts
excitation energy from PBS to PSI and PSII reaction centres decreasing the
energy flow from PBS.
3. Excitation of carotenoid in Synechocystis induces the multistep OCP
transformation as sensitizer and as quencher.
Thanks to colleagues
Rakhimberdieva M.G. A.N. Bach Institute of Biochemistry RAS, Moscow
Shubin V.V.
Bolychevtseva Y.V
Terekhova I.V.
Elanskaya I.V.
Kuzminov F.I.
Biology Faculty, Genetics Dep., MSU
Physics Faculty, Dep. of Non-linear Fluorimetry, MSU
Schlodder E.
Max-Volmer Laboratorium, Technical University Berlin,
Germany
Rögner M.
Plant Biochemistry Dep., Ruhr-University-Bochum, Germany
Vermaas W.F.J.
School of Life Sciences, Arizona State University, Tempe, USA