Folie 1

1
Structure of Cyanobacterial Photosystem II
B. Loll, J. Kern, W. Saenger, A. Zouni, J.
Biesiadka
Erice, June 16th 2006
2
Photosynthesis
incident light energy on earth 0.5-1x1022
kJ/year energy used by photosynthesis 2-4x 1018
kJ/year
3
Oxygenic photosynthesis
T. elongatus, Dimer, 3.8 Å, Zouni et al, Nature
409, 739-43 (2001), 1FE1 3.5 Å, Ferreira et al,
Science 303, 1831-8 (2004),1S5L 3.0 Å, Loll et
al, Nature 438, 1040-44 (2005), 2AXT
T. elongatus Trimer,2.5 Å, Jordan et al, Nature
411, 909-17 (2001), 1JBO Pea, Monomer, 4.4 Å,
Ben-Shem, et al. Nature 426, 630-5 (2003), 1QZV
P. laminosus Dimer, 3.0 Å, Kurisu et al, Science
302, 1009-14 (2003), 1VF5 C. reinhardtii Dimer,
3.1 Å, Stroebel et al, Nature 426, 413-8 (2003),
1Q90
4
Photosystem II
Dimer 750 kDa 5500 amino acids ca 20
subunits/monomer
QA
QB
Fe
e-
CP43
CP47
light driven transmembrane electron transfer
Pheo
Pheo
P680
D1
D2
reduces plastoquinone to plastoquinol
oxidises water to molecular oxygen
e-
YZ
YD
hn
Cyt c550
12kDa
2H2O
33kDa
O2 4H
oxygen evolving complex
5
Water oxidation
Forbush, B. et al. (1974) Photochem.
Photobiol., 14, 309.

• Energetically demanding reaction
• 4 light flashes per reaction cycle
• Mn4Ca cluster couples 1 photon ET reaction
  to 4 electron water oxidation

Kok-cycle
6
Questions

• generation of high oxidation potential of P680
• structure of the water oxidising site
  (Mn-cluster)
• coupling of antenna to the electron transfer
  system
• role of the two cytochromes
• function of the small subunits
• regulation of substrate access
• location and role of the carotenoids
• ...

7
T. elongatus
x 10k
x 20k
PBR Photobioreactor
Thermosynechococcus elongatus BP1 unicellular
thermophilic cyanobacterium
8
Protein preparation
cultivation at 56C
cell disruption
solubilization
1st chromatography
2nd chromatography
mild non-ionic detergen (bDM) mild chromatography
(weak anion exchanger, low salt concentration)
precipitation
9
Protein characterization
cofactors
activity
10
Crystallization

• Crystallization according to the batch method
• PSII concentration 12 mg/mL
• 6 8 PEG 2000
• 100 mM PIPES pH 7.0
• 5 mM CaCl2

11
Diffuse scattering

• Background variations problems for accurate
  integration of intensities
• Symmetry in diffuse scattering could contain
  usefull information (resembles fiber diffraction
  images)

12
Anisotropic diffraction
13
Data collection

• complete dataset 60 70 degree, around the
  good orientation
• translation of the crystal
• exact calculation of the total x-ray dose for
  each segment
• adjusting of Df according to mosaicity

14
3.0 Å resolution dataset
Space group P212121 a127.7 b225.4 c306.1

• X-ray source ESRF (Grenoble) ID14-2
• Wavelength Å 0.933
• Crystals 2
• Resolution Å 20 - 3.0 (3.05-3.00)
• Mosaicity 0.3 - 0.5
• Unique reflections 155,380
• Rsym 0.103 (0.466)
• Completness 88.2 (73.3)
• ltI/sig(I)gt 14.8 (2.2)
• Redundancy 5.6
• Refinement
• R/Rfree 0.238 / 0.290
• Rmsd (bonds/angles) 0.010/1.449
• Number of atoms 48272
• Number of non-protein atoms 7284

15
PSII dimer top view
CP47 (red)
D1 (yellow) D2 (orange)
Cyt b-559 (green/cyan)
CP43 (magenta)
16
PSII dimer side view
Number of a-helices 2 x 80 (2 x 36 TMH) Number
of metal cations 2 x 7 Number of organic
cofactors 2 x 70 n
17
Antenna system
29 Chla 16 at CP47 and 13 at CP43 11
Carotenoids 2 in reaction centre 8 in the antenna
subunits (5 at CP47 and 3 at CP43) 1 within low
molecular weight subunits
CP43
CP47
CP47
CP43
D1/D2
D1/D2
18
Light harvesting
CP47
D1/D2
?
CP43
D1/D2
CP43
?
CP47
19
Light harvesting - photoprotection
CP47
D1/D2
?
CP43
CP43
?
D1/D2
CP47
20
The electron transfer chain
21
The electron transfer chain
22
Quinone binding pockets and non-haem Fe2
D2
MGDG
QA
QB
D1
SQDG
23
Lipid molecules
14 different lipid and 3 detergent molecules
within PSII - 4 digalactosyldiacylglycerol
(DGDG) - 6 monogalactosyldiacylglycerol (MGDG) -
3 sulfoquinovosyldiacylglycerol (SQDG) - 1
phosphatidyldiacylglycerol (PG) - 3 ß-DM
(n-dodecyl-ß-D-maltoside)
digalactosyldiacylglycerol
sulphoquinovosyldiacylglycerol
24
Lipid molecules

• non-annular lipids
• belt around D1/D2 core
• monomer-monomer interface
• between subunits

QB
QA
25
Lipid molecules

• Possible functions of lipids
• monomer-monomer interaction
• promoting subunit exchange (D1 turnover)
• assembly of the multi- subunit complex
• influence on redox- potential of other
  cofactors

26
QB diffusion pathway
PsbJ
cyt b-559
QB
PsbK
27
QB diffusion pathway
QBpocket
28
Protein surrounding of Mn-cluster
PsbU
PsbO
CP43
PsbV
Mn4Ca-cluster
CP47
29
Mn4Ca cluster
30
Mn4Ca cluster
31
Mn4Ca cluster Protein ligands
Asp 170
TyrZ
Glu 354
Ala 344
Glu 333
His190
Glu 189
His 332
Asp 342
32
Mn4Ca cluster
2.7 Å 3.4 Å 3.3 Å
33
Mn4Ca cluster radiation damage
Solution 13,3 keV, 100 K
Solution6,6 keV, 100 K

• Before X-ray measurement
• O2 activity 40 Chla/active center
• AAS 3.7 0.5 Mn/36 Chla in crystals
• EPR (on solution samples) lt1 free Mn2

Crystals13,3 keV, 100 K
Mn(II)-content ()
Measuring Mn-edge-position after various
radiation doses
Crystals13,3 keV, 10 K
Mn(II) is formed during diffraction data
collection
X-ray dose 1014 photons/(100µm)2
Measurement J. Yano, V. Yachandra
34
Setup for single crystal XAS
Energy Resolving 30 element Ge detector for XANES
and EXAFS
X-ray Diffraction Detector
Liquid He Cryostat
Incident X-rays
35
Mn EXAFS of PSII single crystals in S1
36
Proposed Models
Ca position not shown
37
Model from single crystal EXAFS spectra
Simulated spectra
2.7 Å
3.3 Å
Experimental spectra
2.7 Å
2.7 Å
best fitting model
38
Summary

• Detailed biochemical characterization of protein
  samples and crystals
• Complete model of the PSII super-complex at 3.0 Å
• 14 novel lipid molecules
• Possible diffusion pathway for QB
• Full antenna system (ß-carotene and Chla)
• Mn-cluster geometry and surrounding
• Combination with single crystal X-ray
  spectroscopy to improve information on Mn-cluster

39
PSII Team
Free University BerlinB. Loll (now at MPI
Heidelberg),J. Biesiadka, W. Saenger
Technical University BerlinD. DiFiore, C.
Lüneberg, J. Kern, A. Zouni
Former coworkers P. Fromme, K.-D. Irrgang, N.
Krauss, P. Orth, H.-T. Witt
Collaborators J. Yano, J. Pushkar, K. Sauer, V.
Yachandra (all LBNL, Berkeley) J. Messinger (MPI
Mülheim) U. Bergmann, M. Latimer (SSRL) P.
Dörmann (MPI Golm) H. Lokstein (Uni Potsdam) F.
Müh, W. Knapp, T. Renger, P. Franke (FU Berlin)
Support at ESRF, SLS, SSRL, DESY and BESSY is
gratefully acknowledged. This work was supported
by the DFG (SFB 498), the NIH and the DOE
40
PSII dimer top view
CP47 (red)
D1 (yellow) D2 (orange)
Cyt b-559 (green/cyan)
CP43 (magenta)
41
Photosynthetic organisms
42
Protein surrounding of Mn-cluster
CP43 ef-loop (Glu354)
D1 C-terminus
D1 ab loop
D1 cd-helix
D1 C-terminal helix
43
Antenna system
7 Car close to antenna Chla (lt5Å)
44
PSII team
Freie Universität Technische Universität
B. Loll, J. Biesiadka, A. Zouni, J.
Kern, W. Saenger D. DiFiore, C.
Lüneberg
present address MPI Heidelberg
Former coworkers P. Fromme, K.-D. Irrgang, N.
Krauss, P. Orth, H.-T. Witt
Collaborators Junko Yano, Julia Pushkar, Kenneth
Sauer, Vittal Yachandra (all LBNL, Berkeley)
Support at ESRF, SLS, DESY and BESSY is
acknowledged. This work was supported by DFG,
SFB 498
45
Mn4Ca cluster radiation damage
Mn EXAFS of PS II
A 25 Mn(II) B 45 Mn(II) C 90 Mn(II)
I
III
Peak I 1.8 Å Mn-ligand interactions Peak
II 2.7 Å Mn-Mn (2-3) Peak III 3.3 Å Mn-Mn
(1) 3.4 Å Mn-Ca (1-2)
II
A
B
One 2.7 Å Mn-Mn bond probably remains 3.0 Å
Mn-Mn distance?
Increasing number of longer Mn-ligand
bonds (2Å)
C
Similar to the Mn(II) solution
46
Unassigned electron density
47
Mn4Ca cluster
48
Subunits of Photosystem II
SDS-PAGE of redissolved crystals
MALDI-TOF MS of small subunits
large intrinsic PsbA (D1), PsbB (CP47), PsbC
(CP43), PsbD (D2) small intrinsic PsbE (cyt
b559a), PsbF (cyt b559b), PsbH, PsbI, PsbJ,
PsbK, PsbL, PsbM, PsbT, PsbX, PsbY, PsbZ
extrinsic PsbO (33 kDa), PsbU(12 kDa), PsbV
(cyt c550)
19 subunits (16 intrinsic, 3 extrinsic)
49
ETC top view
Cyt c-550
Cyt b-559
CarD2
ChlzD1
TyrZ
QB
Mn4Ca cluster
PD1
ChlzD2
PD2
TyrD
QA
CarD1
50
Mn4Ca cluster radiation damage
51
Antenna system top view
52
Evolution
Structural similarities

• arrangement of Chla in antennae (PSI and PSII)
• topology of transmembrane ?-helices (PSI, PSII
  and BRC)
• non heme iron and quinone binding site (BRC and
  PSII)
• arrangement of central cofactors (BRC and PSII)

53
Water oxidizing complex
Composition of WOC 4 Mn-ions 1 Ca2 1 Cl- TyrZ
? Base/other amino acids ?
54
Photosystem II of higher plants
Spinach
Nield et al., Nature Structural Biology (2000)