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
Photosynthesis – a global
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
Photosynthesis – a global
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
Methods to study photosynthetic
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
Methods to study photosynthetic
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)
Thylakoid membrane domains
Antenna properties
77 K fluorescence spectra
State transition phenomenon
Thylakoid membrane domainsAntenna properties
77 K fluorescence spectra
State transition phenomenon
Thylakoid membrane domains
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
Thylakoid membrane domains
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