«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

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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!»

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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 layerUV-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. elongatusJordan et al., Nature (2001)

3.4Å structure of PSI monomer of P. sativumAmunts 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)

550 600 650 700 750 800

0,0

0,2

0,4

0,6

0,8

1,0

740 (3)708 (7)

ab

sorb

an

ce

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)

740 708

730 nm

trimer

monomer

290 K

6 K

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. elongatustrimers

708 (4) 719 (4)

730 741

732

Th. elongatusmonomers

708 (4) 719 (2)

730 728

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

trimer

monomer

P700+ efficiently quenches F760 of PSI trimers of A. platensis and F735 of PSI trimers of Th. elongatus (Schlodder… Karapetyan, BBA 2011)

760

PSI trimers PSI monomers

660 680 700 720 740 760 780 800 820 8400,0

0,2

0,3

0,5

0,7

0,8

1,0

fluor

esce

nce

wavelength / nm

P700 reduced P700 oxidized

PS I trimerA. platensis77 K

ex = 500 nm

660 680 700 720 740 760 780 800 820 840

0,0

0,2

0,4

0,6

0,8

1,0

731

fluor

esce

nce

wavelength / nm

P700 reduced P700 oxidized red - ox

PS I monomer A. platensis77 K

ex = 500 nm

726

727

A. platensis

Th. elongatus

P700AoA1-FxFA

-FB-

P700+AoA1FXFAFB

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)

0,0 0,2 0,4 0,6 0,8 1,0

0,0

0,2

0,4

0,6

0,8

1,0

F76

0

P700/(P700 + P700+)

Non-linear dependence of F760 on P700+ amount in PSI trimers of A. platensis indicates on energy exchange between PSI monomers within trimer

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)

F741

F734

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

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 F732

F734

Schlodder…Karapetyan, BBA (2012)

F760

F727

Localization of LWC in PSI complexes of A. platensis PSI trimer: 740 (F760) - 3 Chl; 708 (F727) - 7 ChlPSI monomer: 708 (F726) – 7 Chl (three different aggregates).

Schlodder…Karapetyan, BBA (2012)

Chl708(F727)=B38/B37, A38/A39 B18/B19 or A25/A16/A17

Distance between Chl740 and Chl708:

Chl740 Chl708

A32/A31/B7 to B38/B37 = 22Å

A32/A31/B7 to A25/A16/A17= 48Å

A32/A31/B7 to А38/A39 = 57ÅA32/A31/B7 to B18/B19 = 52Å

or F727

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

Different orientationof Chls in various LWC730 of PSI antenna in A. platensis

SMS dataFluorescence 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

P700LWC708

F726

LWC740

F760

P700+

heat

А31-A32-B7

LWC708

~F726

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

PSI PSII

PBS

Structure of Phycobilisomes, interaction with Photosystems

Action spectrum of quenching

=APC

dark (non-quenched)

after BL (quenched)

Quenching decreases PBS fluorescence at 660 nm (exc. 580 nm)

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).

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.

quenched

non-quenched

Orange Carotenoid-binding protein (OCP)

OCP (35 kDa) from A. maxima - two-

domain homodimer containing 3’-

hydroxiechinenone(Kerfeld et al., 2003)

Quantum efficiency of uantum efficiency of PBSPBS absorption in absorption in Synechocystis cellsSynechocystis cells in quenched state drops by in quenched state drops by about 40% (P700 photooxidation and PSII fluorescence induction). 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)..

down regulation of photosynthesis down regulation of photosynthesis

0 2 4 6 8 10 120

20

40

60

80

100

PS2-less strain

Rel

ativ

e P

700+ s

ign

al a

mp

litu

de

0,3s flash intensity

+RL +BL

0 20 40 60 80 1000,0

0,2

0,4

0,6

0,8

1,0

PS1-less strain

Rel

ativ

e F

PA

M s

ign

al a

mp

litu

de

0,5s flash intensity

+RL +BL

77K fluorescence spectra (exc. 570 nm) of WT and PSI/PSII-less mutant

WT

PSI/PSII-less

WT

77K

BL-induced quenching takes place even at the absence of PSI and PSII (Rakhimberdieva et al., FEBS Lett. 2011)

-1

0

620640660680700720740760780

NP

Q n

orm

. max

-0+

Wavelength, nm

288 К 77 К

660 680

Fluorescence quenching spectra at 77 K and RT (top) and the second derivative of quenching spectrum at RT (down).

288 К

Light saturation curves of quenching centre formation

Kuzminov….. Karapetyan BBA 2012

BL

2. Conclusions on OCP-induced NPQ

1. Carotenoid is photosensitizer of PBS quenching, APC APC is a target of is a target of OCPOCP-induced fluorescence -induced fluorescence

quenching in quenching in SynechocystisSynechocystis cells. cells.

2. 2. OCP-induced quenching of APC fluorescence in SynechocystisSynechocystis cellscells diverts excitation energy from PBS

to PSI and PSII reaction centres reaction centres decreasing the energy flow from PBS.

3. Excitation of carotenoid in SynechocystisSynechocystis induces the multistep OCPOCP 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. Biology Faculty, Genetics Dep., MSU

Kuzminov F.I. 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