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«PROBLEM OF ORIGIN OF LIFE» International Conference in Honor of 1 2 0 th 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:

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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 firstPresident 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

OriginandEvolution 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 Gyrsago 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 excessabsorbed energy protects against photodestruction.

  • Carotenoid-less non-photochemical quenching by Photosystem I

    PSI complex exists in cyanobacteria as atrimer, in plants asa monomer.

3.4Å structure of PSI monomer of P. sativum

Amunts et al., Nature (2007)

2.5Åstructure of PSI trimer of Th. elongatus

Jordan et al., Nature (2001)

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 coreantenna of cyanobacteria and

plants (Gobets…Karapetyan et al., Biophys. J. 2001)

6 K

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)


290 K





Spectral characteristics of LWC in PSI trimers and monomers of

A. platensis andTh. elongatus; amount of Chl molecules - in parenthesis (Karapetyan et al., FEBS Lett. 1999)

Fluorescence DAS

(decay associated spectra)

LWC delaythe 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)




P700+efficientlyquenches F760 of PSI trimers of A. platensisand F735of PSI trimers of Th. elongatus(Schlodder… Karapetyan, BBA 2011)

PSI trimers

PSI monomers


A. platensis



Th. elongatus

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

P700is involved in charge separation

P700+or3P700 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 ofA. platensis(Karapetyan et al., Photosynth. Res. 1999)

Time-course of F760 quenching and P700+ formation in PSI trimers of A. platensis at 77K

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

PSI trimer of Th. elongatus(Jordan et al., 2001)

Localization of LWC in PSI antenna of Th. elongatus:

trimer 719 (F741) - 4Chls; 708 (F732) - 4Chls

monomer 719 (F730) - 2Chls;708 (F728) - 4Chls

Chl719 (F741) might beB7/A32/A31

Chl719 is notB31/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 orA26/A27





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 sideclose to trimerization point,

time of energy transfer to P700+ is 110 ps, dipol is oriented parallel to membrane

Chl708(F727)=B38/B37, A38/A39B18/B19or A25/A16/A17

Distance between Chl740 and Chl708:

Chl740 Chl708

A32/A31/B7to B38/B37= 22Å

A32/A31/B7 to A25/A16/A17=48Å

A32/A31/B7to А38/A39 = 57Å

A32/A31/B7to B18/B19 = 52Å




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,….KarapetyanBBA 2012)

Scheme of energy migration in antenna of PSI trimers of A. platensis

No interaction of some LWC708 and LWC740at 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











1. Conclusions: PSI-induced energy dissipation in cyanobacteria

1. LWCdelay the energy equilibration and trapping in PSI core antenna. LWC function as terminal acceptors of excitationlike P700 and transfer uphill energy to P700.

2.P700+ quenches the LWC fluorescence of PSI trimers and monomers ofA. platensis and Th. elongatus but with different efficiency.

3. LWC740 (F760) in PSI ofA. platensismay correspond to peripherally localized A31/A32/B7trimericaggregate. Localization of LWC719 in PSI of Th. elongatusmay 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




In 2004 we have found that illumination by blue-green light of Synechocystis cellsquenches the fluoresence of PBSat660 nm; quenching is reversible in dark (Rakhimberdieva et al., FEBS Lett. 2004).


dark (non-quenched)

after BL (quenched)

Action spectrum of quenching

Quenching decreases PBSfluorescence at 660 nm (exc. 580 nm)

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 ∆pHindependent (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 Synechoystismutant cells innon-quenched andquenched states.

PSI activity was measured for PSII-less mutant, PSII activity - for PSI-less mutant.

Orange Carotenoid-binding protein (OCP)


OCP(35 kDa) from A. maxima - two-domain homodimer containing 3’-hydroxiechinenone(Kerfeld et al., 2003)


down regulation of photosynthesis

Quantum efficiency of PBS absorption in Synechocystis cellsin quenched state drops by about 40% (P700 photooxidation and PSII fluorescence induction). OCP-triggered energy dissipation in PBS of Synechocystisdivertsexcitation away from both RC (Rakhimberdieva et al., BBA 2010).


NPQ norm. max

















Wavelength, nm

BL-induced quenchingtakes place even at the absence of PSI and PSII

(Rakhimberdieva et al., FEBS Lett. 2011)

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

77K fluorescencespectra (exc. 570 nm) ofWT andPSI/PSII-less mutant


288 К

77 К

288 К



Light saturation curves ofquenching centre formation


Kuzminov….. Karapetyan BBA 2012

2. Conclusions on OCP-induced NPQ

1.Carotenoidis photosensitizer of PBS quenching, APC is a target of OCP-induced fluorescence quenching in Synechocystis cells.

2. OCP-induced quenchingof APC fluorescence in Synechocystiscellsdiverts excitation energy from PBS to PSI and PSII reaction centresdecreasingtheenergyflowfromPBS.

3. Excitation of carotenoid in Synechocystisinduces 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. Biology Faculty, Genetics Dep., MSU

Kuzminov F.I. Physics Faculty, Dep. of Non-linear Fluorimetry, MSU

Schlodder E. Max-Volmer Laboratorium, Technical University Berlin,


Rögner M. Plant Biochemistry Dep., Ruhr-University-Bochum, Germany

Vermaas W.F.J. School of Life Sciences, Arizona State University, Tempe, USA

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