July 5, 2010

Szeged

Light-driven water-splitting in

photosystem II

Johannes Messinger

Photosynthetic electron transport chain

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

H2O-binding Geometric Structure

IV

IV

IV

III

-180

-55

-5

-50

S2 state

Electronic Structure

Cofactors

Ca2+,Cl- and HCO3-

Protein

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

Possible Mn4Ca structures and orientations

Yano et al., Science, 2006

Problem: Water is substrate and ‚solvent‘!

When, where and how do the two substrate water molecules bind?

How study H2O-Binding to PSII?

Solution 1: Studying water binding with substrate analogs

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.

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

• 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?

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

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)

Time-resolved MIMS

(high vacuum)

Time-resolved MIMS

kf = 40 s-1

ks = 2 s-1Messinger, Badger, Wydrzynski (1995) PNAS 92, 3209-3213

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

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 !

Cofactors

• Cl- and HCO3-

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

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

MIMS cell

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

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 ↑?

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

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?

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!

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

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

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

Thank you

for attention!