photosystem ii – engine of life fikret mamedov

Molecular Biomimetics / Fotomol / Ångström laboratory

Fikret Mamedov

Department of Photochemistry and

Molecular Science, Uppsala University

The Dynamics of

Thylakoid Membranes

from Higher Plants

Photosynthesis – a global

perspective

Takes place almost everywhere on Earth – where

green plants, algae and photosynthetic bacteria

can be found


perspective

• Energy for photosynthesis comes from sun light

• Two sets of reactions – light dependent and light

independnt

• Affected by temperature, light intensity/quality

and CO2 level


perspective

• Ultimate energy sourse for living organisms –all

food and oxygen in Earth’s biosphere arrive from

photosynthesis

• Sourse for all fossil fuel reserves – products of

photosynthesis were converted into fuels over

millions of years

• One tree makes 12 kg of

biomass and outputs 9400 L

of oxygen in 24 h – enough

for family of FIVE!

Photosynthesis – where it all

takes place

Tree Leaf Plant cell

Thylakoid membrane Chloroplasts

Outer and inner

memranes

Intermembrane

space

Stroma

Photosynthetic membranes

0.1 mm

0.5 mm

Photosynthetic bacteria Cyanobacteria Marine cyanobacteria

Rhodopseudomonas viridis Synechocystis sp. PCC 6803 Prochlorococcus marinum

1 mm

1 mm

Green alga

Chlamydomonas

reinhardtii

Chloroplast

Spinacia

oleracia L.

Domains of the thylakoid

membrane

from higher plants

Thylakoid membrane complexes

and electron/proton transfer reactions

Cytochrome b6f

Photosystem IPhotosystem II

Photosystem I

• Light driven plastocianine feredoxine oxidoreductase

• Electron transfer reactions

• Analogous to green sulphur and hellobacteria (iron sulfur type reaction center)

• ~ 300 kDa, about 15 protein subunits

• Trimer in cyanobacteria, monomer in higher plants

• Crystal structure is solved to 2.0 Å resolution

• Branched electron transfer is debated

Lumen

Stroma

Cytochrome b6f complex

• Plastoquinone plastocyanine oxidoreductase

• Electron and proton transfer reactions

• Q cycle to translocate proton through the membrane

• Found in the dimeric form

• Analogous to Cytochrome bc1 complex in photosynthetic bacteria and mitochondria

• Crystal structure is solved to the 3.0 Å resolution

• A single Chl molecule is found; function is unknown

StromaLumen

Stroma

Lumen

Photosystem II• Light driven water plastoquinone

oxidoreductase. Can split water

and O2 is released as a byproduct,

turnover rate is about 100 molecules per second

• Electron and proton transfer reactions

• Analogous to purple bacteria

(quinone type reaction center)

• ~ 900 kDa, more than 25 protein subunits, structurally highly heterogenic

• Operates at highly oxidizing potentials

• Crystal structure is solved to the medium 3.0 Å resolution

• Water oxidation mechanism is unknown

Lumen

Stroma

e-

2H204H+

02

The catalytic site of Photosystem II CaMn4 cluster and the S-state cycle

S1

S2

S3S4

TyrZ

S0e-

e- e-

e-

H+

H+

2H+

O2

H20

H20

Oxygen release pattern: the S state cycle

e-

2H204H+

02

PSI

LHCII

Cytb6f

ATP-synthase

Grana

Stroma lamellae

Distribution of Photosystems in the

thylakoid membrane from higher

plants

PSII dimer

PSII monomer

Methods to study photosynthetic

complexes : Biochemistry

• Separation of different parts of the thylakoid membrane

(different domains) without disturbing their native

composition

• Isolation and purification of the photosynthetic

membranes and complexes on the different levels –

chloroplasts, thylakoid membranes, PSI or PSII

membranes, PSI and PSII core complexes, reaction

centers etc. from plants, green algae and cyanobacteria

• Supramolecular and protein composition analysis of

different complexes in the thylakoid membrane


complexes : Biochemistry

Preparation Activity (i.e. PSII oxygen

evolution, mmol O2 / mg Chl h)EPR signal

Cells, chloroplasts

80

Thylakoid embranes

120

PSII membranes 500 - 700

PSII core reparations 5000

Reaction Center

preparations 0


complexes: Biophysics and

Spectroscopy

• Electron and proton transport

measurements

• Optical and fluorescence spectroscopy;

time resolved measurements

• EPR spectroscopy – conventional and

advansed (pulse) methods

• Application of the short (ns) laser flashes

to study different intermediates of the

catalytic meachanisms (i.e. S states)

flash 10 ms 2 s

QA- QB(QB

-): 200-600 ms

QA- QB rebinding: 2-8 ms

QA- donor side of PSII:

> 200 ms

Variable fluorescence

Temperature, 'C

-20 -10 0 10 20 30 40 50 60 70

S2QA- S2QB

-

Thermoluminescence

Electron Paramagnetic Resonance

(EPR) spectroscopy from PSII

e-

2H204H+

02

Electron Paramagnetic Resonance

Spectroscopy on the S-states

5 0 0 G

g = 2 . 0500 G

g=4.9

g= 2. 0

500 G

500 G

g=4.3

500 G

g =4.1

g = 4.1

500 G

g = 2.0

500 Gg = 10

500 G S0

S1S2

S4

TyrZ

S3e-

e-

e-e-

Photosystem II life cyclephotoinhibition / repair cycle

• Photosystem II is highly vulnerable to environmental stress

• Exhibit functional and structural heterogeneity and unevenly distributed in the thylakoid membrane

• Pocess several protective meachanisns such as energy dissipation in antenna, xanthophyll cycle, protein phosphorylation, state transition, etc

• Excess of light leads to inhibition of Photosystem II (photoinhibition). At the normal day light conditions every 30 minone Photosystem II is destroyed

• Reparation of Photosystem II is a complex process, which takes place in the different parts of the thylakoid membrane and requires the lateral movement of Photosystem II centers in the thylakoid membrane

Photosystem II life cyclePhotoinhibition

Grana

Stroma

lamellae

4Mn

P680

QA QB

YZYD

PheoD1D2P680

QA

YZYD

Pheo

4Mn

D1D2

QBH2

4Mn

P680

QA=

YZYD

PheoD1D2

QAH2

4Mn

P680

YZYD

PheoD1D2

P680

QA QB

YZYD

PheoD1D2

4Mn

P680

QA QB

YZYD

PheoD1D2

4Mn

P680

QA QB

YZYD

PheoD1D2

Chl+

Car+

D1P680

QA QB

YD

Pheo

D2

Chl+

Car+

YZ

D1

QB

Chl+

Car+

D1P680+

QA

YD

Pheo

D2

YZ

QB

Chl+

Car+

P680+

QA

YD

Pheo

YZ

D1D2

D1

4Mn

YZYD

Pheo-

D2 3P680

O2

D1D2

4Mn

YZYD

Pheo-

3P6801O2

acceptor

side

induced

donor

side

induced

How to study Repair process?

• Separation of the thylakoid domains and study of their biochemical and biophysical properties

• Application of the imaging technology – confocal fluorescence miscroscopy, EPR imaging, etc.

• Biogenesis of the photosynthetic complexes. In this case, the assembly and activation of the PSI, PSII or cyt b6f complexes can be studied during greening of the etiolated plants

• Photoactivation experiments (assembly of the CaMn4–cluster) (dark gron alga are an excellent model)

Understanding membrane dynamics

– study of the thylakoid membrane

domains

• Non-invasive, two phase

separation of the different

fractions of thylakoid

membrane

• Biochemical and biophysical

characterization of PSII, PSI

and Cyt b6f complex (antenna

properties, protein

composition, electron transfer

reactions)

Grana Core

Margins

Y100

Grana

Stroma lamellae

Isolation of the different membrane

fractions Two-phase separation technique

Stroma lamellae

Grana

Grana Margins

Grana Core

mixing

mixing

sonication sentrifugation

The end membrane (End of Grana) and the purified stroma lamellae

(Y100) also can be separated

Domain O2 evolution O2 evolving centers Fv/Fo Chl a/b

(mmol / mg of Chl h) (% of total PSII centers) (mol/mol)

Grana Core 250-300 91 0.87-1.30 1.8-2.0

Grana 200-250 84 0.81-1.10 2.2-2.4

Margins 102 66 0.45-0.50 3.0-3.3

Stroma 80 43 0.27 4.5-5.0

Y100 0 0 0.20 6.0-6.7

Thylakoids 120 80 0.70 2.9

Grana Core

Margins

Y100

Grana

Stroma lamellae

Characterization

of Photosystem II

in different fractions

Thylakoid membrane domains

Quantification of PSI and PSII

Fractions

PS

I/P

SII

0

2

4

6

8

10

12

Grana corevesicles

Grana vesicles

Margins Thylakoid Stromalamellavesicles

Y-100

0.25 0.430.97 1.13

2.58

11.8

Magnetic field, G

3440 3460 3480 3500 3520

EPR spectroscopy

3460 3500Magnetic field, G

Chemically or light

oxidized sample

Dark adapted sample

TyrD (1 spin/PSII center)

Difference spectra

P700+ (1 spin/PSI center)


Antenna properties

77 K fluorescence spectra

State transition phenomenon

Thylakoid membrane domainsAntenna properties

77 K fluorescence spectra

State transition phenomenon


Supramolecular composition of Photosystem II

BN-PAGE and the second dimension SDS-PAGE

of different fractions from the thylakoid membrane

A – PSII supercomplex

B – PSI, PSII dimers

C – ATPase

D – PSII monomer

E – Cytb6f dimer

F – LHCII trimer

G – Cytb6f monomer

Thylakoids Y100Grana Core Margins

47.5 kDa

32 kDa

25 kDa

16 kDa

SD

S P

AG

E

BN PAGEA B CDEF GH A B CDE F GH A B CD E FGH A BC DE FGH

CP47

CP43

D1

D2

CP47

CP43D2

D1

CP47

CP43

D2

D1

CP47

CP43

Thylakoids Y100Grana Core Margins

47.5 kDa

32 kDa

25 kDa

16 kDa

SD

S P

AG

E

BN PAGEA B CDEF GH A B CDE F GH A B CD E FGH A BC DE FGH

CP47

CP43

D1

D2

CP47

CP43D2

D1

CP47

CP43

D2

D1

CP47

CP43

Grana Core

Margins

Y-100

Thylakoids

PSII

supercomplex

PSII dimer

PSII monomer

CP43-less

PSII

monomer

PSII reaction

center

Immunoblot detection

Margin

Grana CoreY100

PSII Monomer

PSII Dimer

PSII Monomer

43 kDa

PSII RC D1/D2

PSII RC D1/D2

PSII Monomer

43 kDa

PSII Monomer PSII Dimer

PSII Supercomplex

Dimer + LHCII

PSII Supercomplex

Dimer + LHCII

PSII Dimer

PSII Monomer

PSII Monomer

43 kDa

482912 11

284114 13

452525


Supramolecular composition of Photosystem II

Thylakoid membrane domainsElectron transport properties – EPR spectroscopy

EPR measurements

on different fractions

TyrDox

%

QA- Fe2+

%

100 100

82 94

59 39

35 31

15 13

Domain of the

thylakoid

Grana Core

Grana

Margin

Stroma

Y100

Thylakoid 66 70

S2 State

%

O2

evolution

%

100 100

92 81

40 37

33 29

0 0

81 43

Magnetic field, G

3600 3800 4000 4200

a

b

c

d

e

f

QA-Fe2+

signal

Grana Core

Margins

Y100

S2 state

multiline

Magnetic field, G

0 1000 2000 3000 4000 5000

a

b

c

d

e

f

Thylakoid membrane domainsElectron transport properties – Fluorescence

Margins

Grana Core

Y100

2 ms 1 s

Domain QB binding,

ms

Y100 29

Stroma 29

Margin 46

--- ---

--- ---

Grana 6.5

Grana Core 5.9

Photoacti-

vation, min

QB binding,

ms

Dark grown 32

2 27

5 18

10 14

30 12

60 10

Light grown 8

Domain Recombi-

nation

Y100 39

Stroma 44

Margin 90

--- ---

--- ---

Grana 170

Grana Core 280

Photoacti-

vation, min

Recombi-

nation

Dark grown 90

2 110

5 170

10 460

30 720

60 670

Light grown 930

Flash-induced fluorescence decay

in different fractions

+ DCMU

Thylakoid membrane domainsConclusions on the repair process

• Photosystem II migrates from the stroma lamellae to the grana during

reparation process. Concomitantly with this lateral migration:

• The number of the Photosystem II centers is gradually increases

(from 5 to 60% of the total amount)

• Supramolecular and protein composition is changing from the minimal

monomeric protein unit to the fully assembled PSII supercomplexes

• Electron transport on both acceptor and donor side is activated leading

to the fully competent centers

Margin

Grana CoreY100

Botanical Garden

View from Uppsala Castle

Special thanks to:Stenbjorn Styring Molecular Biomimetics, Uppsala University, Sweden

Per-Åke Albertsson Biochemistry, Lund University, Sweden

Eva-Mari Aro Biology, University of Turku, Finland

Marjaana Suorsa

Yagut Allakhverdiyeva

photosystem ii – engine of life fikret mamedov

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