Johannes Messinger - szbk.u-szeged.hu · Time of flight (TOF) Vacuum. Membrane-inlet mass...
Transcript of Johannes Messinger - szbk.u-szeged.hu · Time of flight (TOF) Vacuum. Membrane-inlet mass...
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July 5, 2010
Szeged
Light-driven water-splitting in
photosystem II
Johannes Messinger
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Photosynthetic electron transport chain
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Function of the Water-Splitting Complex
• concerted 4-electron or 2+2 electron abstraction from two water molecules during S3→S4→S0transition
• Decoupling of proton and oxygen release (1:0:1:2 vs. 0:0:0:1 pattern) to avoid high charge in WOC
• Coupling of electron and proton transport
1: Coupling of fast 1-electron photo-reactions (3 ps; 10-12 s)
with the slow 4-electron chemical-reaction (1 ms; 10-3 s)
2: Redox-levelling
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H2O-binding Geometric Structure
IV
IV
IV
III
-180
-55
-5
-50
S2 state
Electronic Structure
Cofactors
Ca2+,Cl- and HCO3-
Protein
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Problem: crystallography models are different
1) Limited resolution (≥ 3.0 Å)
2) Radiation damage
Mn EXAFS
Intact PS II
Dose
Mn XANES of PS II crystals
Yano et al.,PNAS 2005
A: 25% Mn(II)B: 45% Mn(II)C: 90% Mn(II)
Ferreira et al., 2004; Loll et al., 2005
2.7 Å
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Possible Mn4Ca structures and orientations
Yano et al., Science, 2006
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Problem: Water is substrate and ‚solvent‘!
When, where and how do the two substrate water molecules bind?
How study H2O-Binding to PSII?
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Solution 1: Studying water binding with substrate analogs
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Methanol increases the miss parameter
Effect fully reversible!
Nöring et al. (2008) Photosynth Res DOI: 10.1007/s11120-008-9364-4
Methanol appears tobind at a substrate site.
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Solution 2: Stable Isotopes
Oxygen: 16O, 17O and 18OHydrogen: 1H and 2H
16O 17O 18O 1H 2H technique
Mass, u 16 17 18 1 2 MS, FTIR
Nuclear spin
(I)
- 5/2 - ½ 1 EPR, NMR
Price, EUR/g - 2000 50 - 5? VR
Probe kinetic isotope effects
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• there are 15 options for the
sequence, in which two water
molecules can bind to five Si
states
• Problem: water is substrate
and solvent!
• Time-resolved isotope
studies are required:
H216O/H2
18O (Radmer and
Ollinger, 1986)
In which Si state(s) do the two substrate water molecules bind?
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Mass spectrometry
Ion source Mass analyzer Ion detection
Electron impact, EI Magnetic sector field Farraday cup
Electrospray, ESI Quadrupole, ion trap Electron multiplyer
Matrix assisted laser
desorption, MALDI
Time of flight (TOF)
Vacuum
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Membrane-inlet mass spectrometry (MIMS)A powerful tool to study natural photosynthetic water-splitting
MIMS
cell
ThermoFinnigan
DELTAPlus XP
TR-MIMS advantages:
Continuous on-line sampling
Analysis of the abundance and
the isotope ratio of gases
(such as H2, O2, N2, CO2 and
N2O)
Simultaneous measurements
up to 7 different masses (m/z)
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Time-resolved MIMS
(high vacuum)
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Time-resolved MIMS
kf = 40 s-1
ks = 2 s-1Messinger, Badger, Wydrzynski (1995) PNAS 92, 3209-3213
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Substrate water binding: When
Hillier and Messinger 2005 In:
Wydrzynski T and Satoh K (eds) Photosystem II.
Ws 620
Wf nd
Ws 1
Wf nd
Ws 70
Wf 6000Ws 70
Wf 2000
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2D pulse EPR: HYSCORE
results
2H
2
16O
1H
2
17O
(- +): |A/2|>nI(+ +): |A/2|<nI
(- +): |A/2|>nI(+ +): |A/2|<nI
n2H = 2.232 MHz
n17O = 2.05 MHz
Under reinvestigation !
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Cofactors
• Cl- and HCO3-
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ACCEPTOR SIDE
BINDING
HCO3-
Ferreira et al., Science 2004
Loll et al., Nature 2005
HCO3- is a ligand of the non-heme iron
Photosystem II
(PSII)Wydrzynski & Govindjee, Biochim. Biophys. Acta 1975
Ferreira et al, Science 2004
HCO3- binding to photosystem II
An overview of the main historical statements
Water
Oxidizing
Complex
(WOC) O2↑ + 4 H+ + 4 e-2 H2O
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HCO3-
HCO3- has been tentatively included to
bind as a ligand to the Mn4OxCa cluster
DONOR SIDE
BINDING
Warburg, Annu. Rev. Biochem. 1964
Stemler & Govindjee, Plant Physiol. 1973
Stemler et al., Proc. Natl. Acad. Sci. USA 1974
HCO3- ions are required for both
maximal activity and stability of the
water-oxidizing complex
Klimov & Baranov, Biochim. Biophys. Acta 2001
Ferreira et al, Science 2004
HCO3- binding to photosystem II
An overview of the main historical statements
O2↑ + 4 H+ + 4 e-2 H2O
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MIMS cell
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CO2↑
Kd(MnII-HC complexes) is
≈ 10 orders higher the
Kd(MnIII-HC complexes)
Kozlov et al.,
Phys. Chem. Chem. Phys. 2004
C16O16O(m/z = 44)
Detection by MIMS
with 15N-labelled
NH2OH
15N216O
(m/z = 46)
+ NH2OHNH2OH specifically
destroys the Mn4OxCa
cluster by releasing Mn2+
Cheniae & Martin,
Plant Physiol. 1971
MIMS measurements
Probing HCO3- binding to the WOC by NH2OH
HCO3-
Mn2+
Mn2+
Mn2+solutionMn2+
Mn3+/Mn4+
cluster
HCO3-
N2O↑
15N2O was observed as
a product of 15NH2OH
oxidation by Mn4OxCa
cluster of PSII
N216O
(m/z = 44)
Kretschmann & Witt,
Biochim. Biophys. Acta 1993
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14N2O↑
15N2O↑
m/z
= 4
4 a
nd
m/z
= 4
6si
gn
als
, mV
2 NH2OH + 2 Mn3+/Mn4+(cluster) → N2O↑ + H2O + 4 Mn2+
(free) + 4 H+
Time
MIMS measurements
Probing HCO3- binding to the WOC by NH2OH
14N2O↑?
CO2 ↑?
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However, HCO3− has ‚indirect‘ effects
on the water oxidation in PSII:
HCO3− is not a structural part
of the Mn4OxCa cluster!
participation in the assembly of the WOCKlimov et al., FEBS Lett. 1997
Baranov et al., Biochemistry 2004
part of a proton relay networkShutova et al., EMBO J. 2008
Conclusions
Shevela et al., Photosynth. Res. 2007
Shevela et al., Biochim. Biophys. Acta 2008
stabilization of the WOCKlimov & Baranov, Biochim. Biophsy. Acta 2008
Pobeguts et al., Biochim. Biophys. Acta 2007
Ulas et al., Biochemistry 2008
Aoyama et al., Biochemistry 2008
Guskov et al., Nat. Struct. Mol. Biol. 2009
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Clausen & Junge, 2004
It was suggested that O2 evolution is half-inhibited by 2.3 bar external O2 pressure This
conclusion was based on UV absorption changes ascribed to the catalytic Mn4OxCa cluster.
Thermodynamic considerations
Does O2 pressure inhibit oxygen evolution in PSII?
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Does O2 pressure inhibit oxygen evolution in PSII? Special pressure cell constructed for MIMS measurements
~30%
20 bar
200 Xe
flashes
(2 Hz)
16O2
16O2
18O2
16O18O
[O2] increased
≈ 35 times over ambient
N2!
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Does O2 pressure inhibit oxygen evolution in PSII? MIMS measurements
16O2(20 bar)
14N2(20 bar)
• The above observations hold for
all conditions tested so far: sample
preparation, flashing and continious
light, two pH values
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Does O2 pressure inhibit oxygen evolution in PSII? Conclusions
• O2 pressure does not inhibit oxygen evolution in
photosystem II
• The results of this MIMS study appear to be in conflict
with the previous UV-study
• We have to try to understant the reproducibly different
outcomes of these two separate experimental
approaches
We must take things as we find them, and not as we would wish them to be
Napoleon Bonapart
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Thanks to
Dr. Dmitriy Shevela Dr. Katrin Beckmann
Prof. V. V. Klimov
Prof. Wolfgang Junge
Dr. Jürgen Clausen
Wallenberg’s
Foundation
Deutsche
Forchungsgemeinschaft
(DFG 1629/2-4)
Max Planck
Society
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Thank you
for attention!