Water splitting in natural and

artificial photosynthetic systems

Sergey Koroidov

Department of Chemistry
Umeå 2014
Responsible publisher under swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7459-800-1.
Elektronisk version tillgänglig på http://umu.diva-portal.org/
Tryck/Printed by: Service Center KBC
Umeå, Sweden 2014
This thesis is dedicated to the memory of my father
Table of Contents
Abstract iii
Abbreviations v
List of Publications vii
1. Introduction 1
1.1. Solar energy 1
1.2. Oxygenic photosynthesis 2
1.2.1. Photosynthetic energy storage 2
1.2.2. Photosynthetic water oxidation 7
1.2.3. Structure of the Mn4CaO5 cluster. 12
1.2.4. Oxidation of the Mn4CaO5 cluster. 13
1.2.5. Substrate water binding to the WOC. 15
1.3. Hydrogen carbonate as a cofactor of the WOC 16
1.4. Water splitting in artificial photosynthetic systems. 17
1.4.1. Concept of artificial photosynthetic devices. 19
1.4.1.1. Semiconductor particles. 19
1.4.1.2. Electrolyzers. 20
1.4.1.3. The artificial leaf 20
1.4.1.4. Dye-sensitized solar cells 21
1.4.2. Molecular catalysts for artificial water splitting 21
1.4.3. Cobalt and manganese oxides as a catalysts for artificial
water splitting 22
1.5. Relevance 24
2. Materials and Methods 26
2.1. Determination of the chlorophyll concentration 26
2.2. Membrane Inlet Mass Spectrometry 26
2.3. H218O – labeling 29
2.4. Joliot type electrode and flash induced oxygen evolution
patterns 29
2.4.1. Data evaluation 30
2.5. Clark electrode measurements of oxygen evolution 31
2.6. X-ray spectroscopy 32
2.7. Illumination setup 32
3. Femto-second serial X-ray crystallography and
spectroscopy: new tools for studying the Mn4CaO5 cluster in
different oxidation states. 37
3.1. Optimization of parameters for the light-induced Si state turnover
of PSII samples in a flow-illumination set-up. 40

i
4. Role of hydrogen carbonate in PSII 47
5. Water splitting in artificial photosynthetic systems. 52
5.1. Manganese oxides 54
5.2. Cobalt oxides 55
6. Summary 57
Acknowledgements 59
References 60

ii
Abstract
Photosynthesis is the unique biological process that converts
carbon dioxide into organic compounds, for example sugars, using the
energy of sunlight. Thereby solar energy is converted into chemical
energy. Nearly all life depends on this reaction, either directly, or
indirectly as the ultimate source of their food. Oxygenic photosynthesis
occurs in plants, algae and cyanobacteria. This process created the
present level of oxygen in the atmosphere, which allowed the formation
of higher life, since respiration allows extracting up to 15-times more
energy from organic matter than anaerobic fermentation. Oxygenic
photosynthesis uses as substrate for the ubiquitous water. The light-
induced oxidation of water to molecular oxygen (O2), catalyzed by the
Mn4CaO5 cluster associated with the photosystem II (PS II) complex, is
thus one of the most important and wide spread chemical processes
occurring in the biosphere. Understanding the mechanism of water-
oxidation by the Mn4CaO5 cluster is one of today’s great challenges in
science. It is believed that one can extract basic principles of catalyst
design from the natural system that than can be applied to artificial
systems. Such systems can be used in the future for the generation of fuel
from sunlight.

In this thesis the light-induced production of molecular oxygen

and carbon dioxide (CO2) by PSII was observed by membrane-inlet mass
spectrometry. By analyzing this observation is shown that CO2 not only is
the substrate in photosynthesis for the production of sugars, but that it
also regulates the efficiency of the initial steps of the electron transport
chain of oxygenic photosynthesis by acting, in form of HCO3-, as acceptor
for protons produced during water-splitting. This finding concludes the
50-years old search for the function of CO2/HCO3− in photosynthetic
water oxidation.

For understanding the mechanism of water oxidation it is crucial

to resolve the structures of all oxidation states, including transient once,
of the Mn4CaO5 cluster. With this application in mind a new illumination
setup was developed and characterized that allowed to bring the
Mn4CaO5 cluster of PSII microcrystals into known oxidation states while
they flow through a narrow capillary. The optimized illumination
conditions were employed at the X-ray free electron laser at the Linac

iii
Coherent Light Source (LCLS) to obtain simultaneous x-ray diffraction
(XRD) and x-ray emission spectroscopy (XES) at room temperature. This
two methods probe the overall protein structure and the electronic
structure of the Mn4CaO5 cluster, respectively. Data are presented from
both the dark state (S1) and the first illuminated state (S2) of PS II. This
approach opens new directions for studying structural changes during
the catalytic cycle of the Mn4CaO5 cluster, and for resolving the
mechanism of O-O bond formation.

In two other projects the mechanism of molecular oxygen

formation by artificial water oxidation catalysts containing inexpensive
and abundant elements were studied. Oxygen evolution catalyzed by
calcium manganese and manganese only oxides was studied in 18O-
enriched water. It was concluded that molecular oxygen is formed by
entirely different pathways depending on what chemical oxidant was
used. Only strong non-oxygen donating oxidants were found to support
‘true’ water-oxidation. For cobalt oxides a study was designed to
understand the mechanistic details of how the O-O bond forms. The data
demonstrate that O-O bond formation occurs by direct coupling between
two terminal water-derived ligands. Moreover, by detailed theoretical
modelling of the data the number of cobalt atoms per catalytic site was
derived.

iv
Abbreviations
ATP Adenosine triphosphate

Chl Chlorophyll

Ci Inorganic carbon (CO2, HCO3-, CO32-)

FIOP Flash-induced oxygen evolution pattern

MIMS Membrane-inlet mass spectrometry

NADPH Nicotinamide adenine dinucleotide phosphate

PSII Photosystem II

P680 Primary electron donor chlorophyll molecule in PSII

QA Primary plastoquinone electron acceptor in PSII

QB Secondary plastoquinone electron acceptor in PSII

RC Reaction center

Si states Oxidation states of the Mn4CaO5 cluster, where ‘i’ is the

number of stored oxidizing equivalents

WOC Water oxidizing complex

XES X-ray emission spectroscopy

XRD X-ray diffraction crystallography

YZ Redox active tyrosine 161 of the D1 protein of PSII

α Miss probability during flash-induced oxygen evolution

v
β Double hit parameter during flash-induced oxygen
evolution

vi
List of Publications
I) Jan Kern, Roberto Alonso-Mori, Rosalie Tran, Johan Hattne,
Richard J. Gildea, Nathaniel Echols, Carina Glöckner, Julia
Hellmich, Hartawan Laksmono, Raymond G. Sierra, Benedikt
Lassalle-Kaiser, Sergey Koroidov, Alyssa Lampe, Guangye Han,
Sheraz Gul, Dörte DiFiore, Despina Milathianaki, Alan R. Fry, Alan
Miahnahri, Donald W. Schafer, Marc Messerschmidt, M. Marvin
Seibert, Jason E. Koglin, Dimosthenis Sokaras, Tsu-Chien Weng,
Jonas Sellberg, Matthew J. Latimer, Ralf W. Grosse-Kunstleve,
Petrus H. Zwart, William E. White, Pieter Glatzel, Paul D. Adams,
Michael J. Bogan, Garth J. Williams, Sébastien Boutet, Johannes
Messinger, Athina Zouni, Nicholas K. Sauter, Vittal K. Yachandra,
Uwe Bergmann, Junko Yano. (2013) Simultaneous Femtosecond
X-ray Spectroscopy and Diffraction of Photosystem II at Room
Temperature. Science 340, 491-495

Reprinted with permission from Science 340, 491-495, 2013.

Copyrights 2013 American Association for the Advancement of Science

II) Dmitriy Shevela, Birgit Nöring, Sergey Koroidov, Tatiana

Shutova, Göran Samuelsson, Johannes Messinger (2013)
Efficiency of photosynthetic water oxidation at ambient and
depleted levels of inorganic carbon. Photosynth Research 117, 401-
412

Reprinted with permission from Photosynthesis Research 340, 491-

495, 2013. Copyrights 2013 Springer

III) Sergey Koroidov, Dmitriy Shevela, Tatiana Shutova, Göran

Samuelsson, Johannes Messinger (2014) Mobile hydrogen
carbonate acts as proton acceptor in photosynthetic water
oxidation Submitted Manuscript

IV) Dmitriy Shevela, Sergey Koroidov, M. Mahdi Najafpour,

Johannes Messinger, Philipp Kurz (2011) Calcium manganese
oxides as oxygen evolution catalysts: O2 formation pathways
indicated by 18O-labelling studies.  Chemistry - A European
Journal 17, 5415-5423

vii
Reprinted with permission from Chemistry - A European Journal 17,
5415-5423 Copyright 2011 WILEY-VCH Verlag GmbH & Co. KGaA,
Weinheim

V) Sergey Koroidov, Magnus Anderlund, Stenbjörn Styring, Anders

Thapper, Johannes Messinger (2014) Mechanism of water
oxidation by cobalt-oxides Manuscript

Author’s contribution:

Paper I: Conceived, setup and performed O2 evolution

measurements

Paper II: Data analysis and interpretation. Helped with the

revision of the manuscript.

Paper III: Design and conduction of experiments, data analysis and

interpretation with contribution in writing of the manuscript.

Paper IV: Design and conduction of experiments, data analysis and

interpretation. Helped with the revision of the manuscript.

Paper V: Design and conduction of experiments, data analysis and

interpretation. Assisted in writing of the manuscript.

Publications not covered in the thesis

VI) Rolf Mitzner, Jens Rehanek, Jan Kern, Sheraz Gul, Johan Hattne,
Taketo Taguchi, Roberto Alonso-Mori, Rosalie Tran, Christian
Weniger, Henning Schröder, Wilson Quevedo, Hartawan
Laksmono, Raymond G. Sierra, Guangye Han, Benedikt Lassalle-
Kaiser, Sergey Koroidov, Katharina Kubicek, Simon Schreck,
Kristjan Kunnus, Maria Brzhezinskaya, Alexander Firsov, Michael
P. Minitti, Joshua J. Turner, Stefan Moeller, Nicholas K. Sauter,
Michael J. Bogan, Dennis Nordlund, William F. Schlotter,
Johannes Messinger, Andrew Borovik, Simone Techert, Frank M.
F. de Groot, Alexander Föhlisch, Alexei Erko, Uwe Bergmann,
Vittal K. Yachandra, Philippe Wernet, and Junko Yano (2013) L-
Edge X-ray Absorption Spectroscopy of Dilute Systems Relevant to
Metalloproteins Using an X-ray Free-Electron Laser. The Journal
of Physical Chemistry Letters 4, (21):3641-3647.

viii
VII) Jan Kern, Rosalie Tran, Roberto Alonso-Mori, Sergey Koroidov,
Nathaniel Echols, Johan Hattne, Julia Hellmich, Sheraz Gul,
Hartawan Laksmono, Raymond G. Sierra, Richard J. Gildea,
Guangye Han, Benedikt Lassalle-Kaiser, Ruchira Chatterjee,
Mohamed Ibrahim, Aaron Brewster, Carina Glöckner, Alyssa
Lampe, Dörte DiFiore, Despina Milathianaki, Alan R. Fry, M.
Marvin Seibert, Jason E. Koglin, Dimosthenis Sokaras, Tsu-Chien
Weng, Ralf W. Grosse-Kunstleve, Petrus H. Zwart, William E.
White, Michael J. Bogan, Marc Messerschmidt, Pieter Glatzel,
Garth J. Williams, Sébastien Boutet, Paul D. Adams, Athina Zouni,
Johannes Messinger, Nicholas K. Sauter, Uwe Bergmann, Junko
Yano, Vittal K. Yachandra (2013) Taking Snapshots of
Photosynthetic Water Oxidation Using Femtosecond X-ray
Diffraction and Spectroscopy Submitted Manuscript

VIII) Rosalie Tran, Jan Kern, Johan Hattne, Sergey Koroidov, Julia
Hellmich, Roberto Alonso-Mori, Nicholas K. Sauter, Uwe
Bergmann, Johannes Messinger, Athina Zouni, Junko Yano, Vittal
K. Yachandra (2014) The Mn4Ca Photosynthetic Water-Oxidation
Catalyst Studied by Simultaneous X-ray Spectroscopy and
Crystallography Using an X-ray Free Electron Laser Submitted
Manuscript

ix
1. Introduction
1.1. Solar energy

World energy consumption is projected to increase from 13 TW in

2001 to 27 TW by 2050 and to 43 TW by 2100 (Hoffert et al., 1998;
Barber, 2009). Currently, 85% of all energy comes from burning fossil
fuel (U.S. Energy Information Administration, What is Energy: Energy
Basics, 2011). These fossil fuels power our technologies and produce the
wide range of products that support our life and heat our homes. The
main problem in the current situation is not only limitation of fossil fuel,
but also the consequences of combustion that leads to the accumulation
of CO2 in the atmosphere. Notwithstanding the uncertainty about the
precise effects of CO2 emissions, we can certainly look at the CO2 level
from an historical perspective. For the last 800,000 years, for example,
atmospheric CO2 concentration has been estimated to be between 210-
300 ppm (Petit et al., 1999; Luthi et al., 2008). Over the past 100 years,
however, the CO2 level has risen from 300 ppm to 395 ppm (Current
Data for Atmospheric CO2), an increase that is most probably the result
of man-made CO2 emission. If one imagines that all fossil fuel that exist
is burnt then the level of carbon dioxide in our planet would reach to
values equivalent to those prior to photosynthesis (Barber, 2009).
Despite this fact, it is certain that fossil fuels will remain an important
energy carrier for some time, but it is essential to stepwise reduce its
usage and vital to develop technology that can derive energy form CO2-
free or CO2-neutral sources.

One modern approach is to use renewable energy and from various

renewable energy sources. By far the largest renewable energy resource is
the sun. Solar energy is clean, abundant and economically very attractive
energy source. The Earth is receiving solar energy at a rate of
approximately 120000 TW (Hoffert et al., 2002; Blankenship et al., 2011)
or 4.3 1020 J/hr (Lewis and Nocera, 2006). Sunlight provides thereby
more energy in one hour than all of the energy consumed by the entire
planet in one year. In order to satisfy the energy needs of humans until
the end of the 21st century storage of only 0,03% from 120000 TW are
needed. Nature has developed a way for utilization solar energy called
photosynthesis.

1
 Photosynthesis is the unique fundamental biological process that
converts CO2 (about 380 Gtonnes of CO2 is fixed annually with release of
260 Gtonnes of O2 (Barber, 2004)) into organic compounds, for example
sugars, using the energy of sunlight. Thus solar energy is converted into
chemical forms of energy. Nearly all life depends on this reaction either
directly as a source of energy, or indirectly as the ultimate source of their
food. Oxygenic photosynthesis occurs only in plants, algae and
cyanobacteria. Around 2.32 billion years ago, photosynthetic organisms
started to be dominating and increased the O2 level of our atmosphere
(Bekker et al., 2004). The development of oxygenic photosynthesis
drastically changed the Earth by creating and presently by maintaining a
level of about 20% Molecular oxygen in the atmosphere. This laid the
basis for the formation of the protective ozone layer that absorbs a large
part of the UV radiation from the sun. The increasing levels of
atmospheric O2 allowed the development of an aerobic metabolism and
more-advanced life forms (Olson and Blankenship, 2004).

1.2. Oxygenic photosynthesis

1.2.1. Photosynthetic energy storage

Solar energy supports life on Earth via photosynthesis, and this

process evolved very early in the history of our planet. First organisms
with ability to fix carbon appeared about 3.8 billion years ago
(Schidlowski, 1988; Mojzsis et al., 1996). The earliest fossils of
anoxygenic photosynthetic bacteria were found in 3.5 billion years old
sediments (Schopf, 1993) that formed in the environment of low
atmospheric oxygen (Awramik, 1992) and high level of reducing
molecules, such as CH4, H2S, H2 and others. Later on, H2O started to be
used by some photosynthetic organisms as the source of electrons and
protons for CO2 reduction. This process released O2 as a by-product, and
is hence called oxygenic photosynthesis. The organism that first
performed this revolutionary process (cyanobacteria) were ancestors of
the anoxygenic photosynthetic bacteria (Xiong et al., 2000). The O2
released was toxic for many organisms and only those organisms
survived that devolved protective mechanism and/or the ability to use O2
for burning organic matter with mitochondria. Oxygenic photosynthesis
performed by cyanobacteria algae and plants is the major process of solar
energy conversion on the Earth today (Figure 1.1)

2
Figure 1.1. Global distribution of photosynthesis and production of
organic matter showing values for land plants and ocean algal and
cyanobacterial communities. Adapted from SeaWiFS Project,
NASA/Goddard Space Flight Center and ORBIMAGE.

The basic equation of oxygenic photosynthesis can be written as:

6 CO2  6 H 2 O 
Light energy
enzymes
 C 6 H 12 O6  6 O2 (1.1.)
chlorophyll

The photochemical reaction in photosynthesis is a redox reaction,

with CO2 (removed from atmosphere) acting as an electron acceptor and
H2O as electron donor. It results in the formation of glucose, which
stores part of the solar energy in its chemical bonds, and in oxygen as a
byproduct.

In higher plants photosynthesis occurs in organelles known as

chloroplasts. Thylakoids, which span the inside of the chloroplasts, are
the structural unit of the primary light-converting reactions in
photosynthesis. The thylakoid membrane is a closed membrane system
that separates the luminal (inner) space of thylakoids from the stromal
(cytoplasmic) space of chloroplasts where CO2 fixation takes place.
Figure 1.2 shows the protein complexes that participate in the light
absorption, energy conversion and electron transfer reactions of oxygenic
photosynthesis. These protein complexes, which are integral parts of the
thylakoid membrane, are photosystem II (PSII) with light-harvesting

3
complex II (LHC-II), the Cyt b6f complex, photosystem I (PSI) with light-
harvesting complex I (LHC-I), electron carriers ferredoxin (Fd) and
ferredoxin-NADP-reductase (FNR) at a stromal, and plastocyanin (PC) at
luminal sides, respectively, and the adenosine triphosphate (ATP)
synthase.

The photosynthetic reactions are initiated by the absorption of

light within the LHCs of PSII and PSI (Mullineaux, 1992; Nelson and
Yocum, 2006; Collins et al., 2012), where it excites electrons into a
higher molecular orbital. When the excitation energy generated in the
antenna reaches the PSII or PSI reaction center (RC), respectively, this
generates a charge separation, which is subsequently stabilized by
further electron transfer events that increase the distance between the
charged species and reduce their potential difference (Ben-Shem et al.,
2003; Dau and Haumann, 2008; Renger, 2012)

PS II (Wydrzynski and Satoh, 2005; Govindjee et al., 2010) uses

light in order to perform two chemical reaction: water oxidation and the
reduction of plastoquinone (Messinger and Renger, 2008; Renger and
Renger, 2008). In order to split water, four photochemical reactions are
needed to take out four electrons and four protons from two water
molecules. This leads to formation of one oxygen molecule. Protons form
water are released into the lumen (donor side of PSII) and electrons are
moved to the plastoquinone (QB) binding site. Fully reduced QB2- picks
up two protons from the stroma (acceptor side of PSII) side of the
medium, yielding plastohydroquinone (PQH2) (Figure 1.2). PQH2 then
leaves the PSII complex, and diffuses in the membrane to the Cyt b6f
complex. The empty QB-pocket is filled by another plastoquinone (PQ)
molecule from the PQ pool (for detail information look 1.2.2.).

4 h
2 H 2 O  2 PQ  4 H  stroma  O2  2 PQH 2  4 H  lumen (1.2.)

4
Figure 1.2. Diagram of the main protein complexes that perform oxygenic
photosynthesis in the thylakoid membrane of chloroplasts of higher
plants. Adapted from Shevela (2008)

The cytochrome b6f complex is an electron transfer and proton

translocating membrane enzyme acting as a redox link between PSII and
PSI. (Berry et al., 2000; Hervás et al., 2003). Plastoquinol PQH2 binds to
the Rieske iron-sulfur protein (FeS) where it is oxidized and
deprotonated. Protons are released into lumen and one of the two
electrons introduced into electron transport chain passes through Cyt f to
the electron carrier plastocyanin (PC). The other one is transferred to cyt
b6 and participates in the reduction of PQ molecules to regenerate PQH2.
This process is known as Q cycle and increases the proton concentration
difference between stroma and lumen (Bernat and Rögner, 2011; Cramer
et al., 2011):

PQH 2  2 H  stroma  2 PC ox  PQ  4 H  lumen  2 PC red (1.3.)

PSI is a large protein complex (Fromme, 1996; Amunts and

Nelson, 2009) that reduces on the stromal side NADP+ to NADPH
(nicotinamide adenine dinucleotide phosphate), and oxidizes PC on the
luminal side. This transmembrane electron transfer is energetically
driven by the light-induced excitation of the primary electron donor
P700 (Lubitz, 2006). P700* transfers its excited electron to the
chlorophyll molecule A0, which passes it on to the phylloquinone A1.
From here it is transferred to Fd via three different four-iron four-sulfur
[Fe4-S4] clusters known as Fx, Fa and Fb (Figure 1.3). The formed cation
radical P700•+ it is reduced directly by the reduced form of the mobile

5
electron transfer protein plastocyanin, PCred. The overall reaction of PSI
can therefore be written as:

h
PC red  Fdox   PC ox  Fdred (1.4.)

The reduced Fd transfers its electron to ferredoxin-NADP-

reductase (FNR), where the production of NADPH takes place (Vermaas,
2001).

2 Fdred  2 H  storma  NADP  

FNR

(1.5.)
2 Fdox  NADPH  H  stroma

The overall process of linear electron transfer form water to

NADP+ through PS II, Cyt b6f and PSI is presented in Z-scheme
(Figure1.3).

There is, however another pathway where electron form reduced

Fd is flowing back to Cyt b6f complex. This happens to increase an
amount of protons for ATP synthesis.

The trans-membrane potential generated by the trans-membrane

electron transfer reactions and via the accumulation of protons in the
thylakoid lumen is used by the ATPase for the synthesis of ATP from
ADP and Pi (adenosine triphosphate from adenosine diphosphate and
inorganic phosphate). (McCarty et al., 2000; Junge and Nelson, 2005;
Junge et al., 2009). In this process protons accumulated in the lumen
pass through the ATPase and generate a rotary motion of the F0F1 unit
that is required for ATP synthesis.

NADPH and ATP produced during photosynthesis provide the

energy for the synthesis of sugars form CO2 and H2O which are taken
form atmosphere and the roots, respectively. These processes of
photosynthesis can be divided into the light-reactions and dark (light-
independent) reactions:

2 H 2 O  2 NADP   3 ADP 3   3 HPO 4 2   H  

light
8 h

(1.6.)
O2  2 NADPH  3 ATP 4   3 H 2 O

6
CO2  2 NADPH  3 ATP 4   3 H 2 O 
dark

(1.7.)
CH 2 O   H 2 O  2 NADP   3 ADP 3   3 HPO42   H 

The reaction pathways by which CO2 gets fixed during the light-
independent reactions is known as the Calvin–Benson–Bassham cycle
(Bassham et al., 1950).

Figure 1.3. Representation of the oxygenic photosynthesis Z-scheme on

redox potential scale that trace electron transfer form water to NADPH.
Adapted from Shevela (2008)

1.2.2. Photosynthetic water oxidation

PSII is the only natural pigment-protein complex known to oxidize

water and release molecular oxygen. To drive this reaction it uses visible
light (400-700nm) (Lubitz et al., 2008; Pace and Krausz, 2012; Cox and
Messinger, 2013). However, recent measurements defined that 750 nm
will drive PSII electron transfer rather efficiently (Thapper et al., 2009).

4 h
2 H2O 
PSII
O2  4 H   4 e (1.8.)

PS II is embedded into thylakoid membrane and has a total

molecular mass of about 350 kDa. Crystal structures obtained from
thermophilic cyanobacteria (Thermosynechococcus elongatus and
Thermosynechococcus vulcanus) revealed the organization of PSII

7
(Zouni et al., 2001; Ferreira et al., 2004; Loll et al., 2005; Kawakami et
al., 2009; Guskov et al., 2010; Umena et al., 2011). Based on the latest 1,9
Å resolution structure (Umena et al., 2011), each PS II core complex
consists of 20 proteins, 35 Chl, 11 carotenoids, more than 20 lipids, 2
plastoquinons, two heme iron, one non-heme iron, 4 manganese atoms,
3 or 4 calcium atoms (one of which in Mn4CaO5 cluster), 3 chloride ions
and one bicarbonate ion (Figure 1.4.). All cofactors that are involved in
photosynthetic water oxidation and which are responsible for the
electron transport within the reaction center are bound by the PSII core
proteins D1 (PsbA) and D2 (PsbD) (Chua and Gillham, 1977; Debus, 1992,
2001; Nixon et al., 2005). The excitation energy generated by light
absorption in the LHCII is transmitted to RC via the inner chlorophyll-
binding proteins (CP43-PsbC and CP47-PsbB) (Bricker and Frankel,
2002; Eaton-Rye and Putnam-Evans, 2005).

In plants the extrinsic proteins PsbO (33kDa, also known as

manganese-stabilizing protein), PsbQ (17kDa), and PsbP (23kDa) are
located at lumen side and optimizing PSII activity, serve as enhancers of
O2 evolution and protect Mn4CaO5 cluster form outer reductants and
reactants in the surrounding aqueous phase (Seidler, 1996). The
Mn4CaO5 cluster together with its functionally important protein
environment and ligands forms a functional unit within PSII that is
known as water oxidizing complex (WOC) or oxygen evolution complex
(OEC) (Debus, 2005).

Absorption of the light by the inner or outer antenna of PSII leads

to the formation of an exited chlorophyll (Chl) state, Chl*, that is
transferred from the antenna to the primary electron donor in the RC,
P680, which in turn is excited to P680*. P680 is named after its
absorption maximum. The molecular equivalent is variously defined,
either as the special pair PD1PD2, or to also include the monomeric ChlD1
and ChlD2 molecules (Figure 1.5 B).

Chlantenna *  P 680  Chlantenna  1 P 680 * (1.9.)

8
Figure 1.4. Structure of PSII at a resolution of 1.9 Å. Reproduced from
Umena (2011).

9
Figure 1.5. Simplified architecture of PSII. Red arrows represent
excitation energy migration to P680, and black arrows show the path of
the electrons trough the electron transfer chain. Adapted from Messinger
and Shevela (2012).

After formation of the exited state P680* the primary electron

acceptor pheophytin (Pheo) accepts within few picoseconds the excited
electron from P680*. This charge separation leads to the formation of the
radical pair P 680 PheoD 1 (Klevanik et al., 1977; Klimov et al., 1977):

1
P 680* PheoD 1  P 680 PheoD 1 (1.10.)

10
 P680•+ has an oxidation potential of about +1.2 – 1.3V, which is the
highest known in nature (Jursinic and Govindjee, 1977; Klimov et al.,
1979; Rappaport et al., 2002). Thus PSII needs a protection against
oxidation and long-living triplet state 3P680* states that can be formed by
charge recombination. The carotenoids and the Cyt b559 (proteins PsbE
and PsbF) (Figure 1.5) are thought to be involved in this protection
mechanism (Vass et al., 1992; Lubitz et al., 2008).

The primary charge separation is stabilized by electron transfer

from Pheo•- to the primary plastoquinone molecule QA forming
P680•+QA•-. QA• is oxidized in a slower reaction by a second
plastoquinone molecule, QB, that after a second photo-chemical turnover
becomes fully reduced to QB2- and takes up two protons form the stroma,
forming PQH2. PQH2 is displaced from its binding site by a
plastoquinone molecule of dissolved in the thylakoid membrane, and is
subsequently oxidized again by the Cyt b6f complex (Messinger et al.,
2012). These ractions can be summarized as follows:

PheoD1 Q A  Pheo Q A

Q A  Fe QB  Q A Fe QB 
(1.11.)
Q A  Fe QB   2 H  stroma  Q A Fe QB H 2
Q A Fe QB H 2  PQ  Q A Fe QB  PQH 2

The non-heme iron between QA and QB plays a structural role and

also binds one hydrogen carbonate ion (HCO3-), which was suggested to
be involved in the protonation of QB-/2- (McConnell et al., 2012; Shevela
et al., 2012).

Tyrosine Z (D1-Tyr161, also termed YZ) (Ahlbrink et al., 1998;

Debus, 2001; Ananyev et al., 2002) is a part of the WOC and directly
involved in the light-induced electron transfer. It is an intermediate
redox-active electron carrier between P680 and the Mn4CaO5 cluster that
reduces P680•+ (Debus et al., 1988). Thus the Mn4CaO5 cluster at the
donor side of PSII is oxidized by P680•+ through YZ, which can donate an
electron much faster to P680•+ then the Mn4CaO5 cluster. His190 of D1 is
proposed to be the base that accepts the proton released from YZ upon
oxidation by P680•+. Proton may reversibly shift within a hydrogen bond
towards His190 resulting in a [YZ–O•…..H+–N–His190] complex

11
( P 680  Y Z  OH  N  His  P 6800  Y Z  O   H  N  His )(Debus, 2005;
Meyer et al., 2007; Dau and Haumann, 2008).

Four successive oxidations lead to the release of one oxygen

molecule. The quantum efficiency of the overall PSII chemistry described
by equation 1.2 (plastoquinone reduction and water oxidation) may be as
high as 90% under optimal light conditions (Dau and Zaharieva, 2009;
Dau et al., 2012; Cox and Messinger, 2013) .

1.2.3. Structure of the Mn4CaO5 cluster.

The structure of the Mn4CaO5 cluster was recently resolved at a

resolution of 1,9 Å by Shen and colleagues (Umena et al., 2011). This
study identified five oxygen atoms that bridge the five metal atoms of the
cluster. This is in good agreement with structures derived by Extended
X-ray Absorption Fine Structure (EXAFS) spectroscopy and DFT
calculations (Yano et al., 2006; Siegbahn, 2009). In Mn4CaO5 cluster one
calcium, four oxygens and three manganese form an asymmetric
structure of a ‘‘distorted chair’’. The fourth Mn is located outside of the
cubane and is linked to Mn1 and Mn3 atoms of the cubane through two
di-µ-oxo bridges involving the O5 and O4 atoms of the cluster (Figure
1.6.). The cubane-like structure was reported previously (Ferreira et al.,
2004), but the oxo-bridges and the exact distances between atoms could
be resolved.

Figure 1.6. Structure of the Mn4CaO5 cluster where numbers between

atoms represent distances (in ångströms). Reproduced from Umena
(2011).

12
 The four closest water molecules to the Mn4CaO5 cluster are direct
ligands of the cluster. W1 and W2 are attached to Mn4 and the other two
(W3, W4) to Ca. Since no other water molecules ligate the Mn4CaO5
cluster, it has been suggested that some of these four waters can act as
the substrate for water oxidation (Umena et al., 2011).

The unusually long distances between oxygen atom O5 and the

metals atoms of the cluster have been interpreted to suggest that these
bonds are weak and that O5 may be one of the oxygen substrates for di-
oxygen formation. The W2 and W3 are within hydrogen bond distance
with O5 and therefore may serve as the other substrate. On the other
hand, W3 is close enough to YZ so that it is also an attractive candidate as
a proton release group (Kargul and Barber, 2012) Thereby W3 rather O5
may be a hydroxide ion in the S1 state and based on this one can assume
that the O-O bond formation may occur between W2 and W3 (Umena et
al., 2011). In summary, the results of Shen and colleagues suggest that O-
O bond formation occurs in two of these three species O5, W2, and W3.

However, the applied dose leads to a reduction of ~25% of the

MnIII/IV ions to MnII (Yano et al., 2005; Umena et al., 2011), which also
noticeably changes the atomic distances as compared to EXAFS (Yano et
al., 2005; Grabolle et al., 2006; Glöckner et al., 2013). Thus, even at
cryogenic temperatures SR-based XRD of the Mn4CaO5 structure in PS II
is fundamentally limited by the radiation damage to the redox active
metal site, making it difficult in obtaining structures of stable or
transient reaction intermediates. A more detailed description is provided
in Section 3.

1.2.4. Oxidation of the Mn4CaO5 cluster.

The splitting of two water molecules into molecular oxygen and

four protons (see Equation 1.8) requires the accumulation of four
oxidizing equivalents on the Mn4CaO5 cluster of the WOC. In 1969, Joliot
and colleagues demonstrated that the flash-induced oxygen evolution of
dark-adapted chloroplasts and cells of algae exhibits a periodicity of four
(Joliot et al., 1969). Their finding can be sum up as follows: (i) almost no
oxygen was seen in the first two flashes, (ii) maximum of oxygen release
occurs after 3rd flash, (iii) four flashes are required for the detection of
every next maximum of oxygen releases, (iv) the oscillation is damped
and eventually all flashes generate same amount of O2. (Figure 1.7. A).
On the basis of these observations, Kok and coworkers designed a model,

13
which is known as the Kok model (Figure 1.7. B). In this model the
different oxidation states through which the WOC cycles during water
oxidation are referred to Si states, where i represents the number of
stored oxidizing equivalents (i=0,1,2,3 and 4) (Kok et al., 1970).

Figure 1.7. (A) FIOPs of dark adapted PSII samples, and (B) the extended
Kok model. Adapted from Messinger and Renger (2008).

The state stable in the dark is the singly oxidized S1 state

(MnIII2MnIV2) (the other S states decay back to S1). That is why the first
maximum of oxygen evolution occurs after three flashes. The S0
(MnIII3MnIV) state converts into S1 in the dark due to electron transfer to
the oxidized form of tyrosine D (YDox) (Styring and Rutherford, 1987;
Vass and Styring, 1991; Messinger et al., 1993). YD is located on the D2
protein (D2-Tyr 160) of PSII in the symmetry related position to YZ. In
contrast to YZ it does not participate in the fast electron transfer
reactions. However, YD can be oxidized by P680•+ and it can act as
reductant during the decay reactions of S2 to S1 or S3 to S2. (Vermaas et
al., 1984; Vermaas et al., 1988; Messinger and Renger, 1993; Faller et al.,
2001). The meta stable S2 (MnIIIMnIV3) and S3 (MnIV4) states can also
decay to S1 if the electron created during light-induced charge separation
is not quickly enough removed from acceptor site (QA and/or QB) (Diner,
1977; Robinson and Crofts, 1983; Rutherford and Inoue, 1984). The S4
state is an intermediate state that decays to S0 in a fast (1 ms)
spontaneous reaction under the liberation of O2. Despite numerous
attempts, the S4 state was not detected so far (Haumann et al., 2008;
Shevela et al., 2011). However S3YZox intermediate that is only stable in
millisecond time range under certain conditions (O2 pressure and
lowering pH under air) has been kinetically identified (Clausen and
Junge, 2004, 2008)

14
 The damping of flash-induced oxygen evolution patterns (FIOPs)
was proposed to occur due to miss (α) and double hit (β) probabilities
(Forbush et al., 1971). The miss probability represents the percentage of
WOCs that have the same oxidation state before and after flash excitation
(Si – flash – Si). The miss does not reflect a constant percentage of
inhibited PSII centers, rather it is statistically distributed overall PSII
centers within a flash train. The origin of the miss probability are the
redox equilibria on the donor and acceptor side that lead to temporary
blockage of stable charge separation in PSII (Renger and Hanssum,
1988; Shinkarev and Wraight, 1993). Moreover, the miss parameter can
also originate from the kinetic mismatches of the rate of P680•+
reduction by YZ because it competes with charge recombination between
P680•+ with a primary quinone electron acceptor, QA- (Christen and
Renger, 1999; Christen et al., 1999). Furthermore there is experimental
data indicating that value of α is dependent on the redox state Si of the
Mn4CaO5 cluster (Renger and Hanssum, 1988; Isgandarova et al., 2003;
Han et al., 2012; Suzuki et al., 2012).

The double hit parameter reflects the percentage of PSII centers

that have been exited twice during one flash (Si – flash – Si+2). This
accounts for the small oxygen yield after the second flash. The double hit
strongly depends on flash profile of the light source and can be avoided if
laser flashes are used (Jursinic, 1981).

1.2.5. Substrate water binding to the WOC.

The first mass spectrometry measurements with good enough time

resolution to detect the exchange rate of the substrate water molecules
were done by Messinger and co-workers (Messinger et al., 1995). The
authors have thoroughly examined the incorporation of oxygen atoms
from bulk water into the dioxygen evolved by the WOC of PSII. Because
of their experimental design, the rate of incorporation reflects the rate at
which bulk water incorporates into the water-binding site of the WOC in
a given S state.

The following points emerged from the MIMS studies (Messinger

et al., 1995; Hillier et al., 1998; Hillier and Wydrzynski, 2000; Hendry
and Wydrzynski, 2002, 2003; Cox and Messinger, 2013): (a) the two
substrate water molecules bind to separate sites throughout the S state
cycle; (b) The two sites are characterized by different exchange rates. The
slow exchanging site exchanges ~ 20 times slower than the fast

15
exchanging site in the S3 state, ~ 60 times slower in the S2 state, and ~
6000 times slower in the S1 state; (c) The two substrate waters are
already bound to the WOC at the S2 state; (d) Ca2+ is involved in
substrate water binding at the slow exchanging site; (e) The rate
constants measured at 10 °C for the slow exchanging site range from 2×
10-2 s-1 in the S1 state to 10 s-1 in the S0 state, and those for the fast
exchanging site range from 40 s-1 in the S3 state to ≥ 120 s-1 in the S1 and
S2 states.

Information about water binding in the WOC has also been

obtained by several different techniques: X-ray crystallography (XRD)
(Umena et al., 2011), magnetic resonance (Rapatskiy et al., 2012), and
FTIR spectroscopy (Debus, 2008; Noguchi, 2008).

1.3. Hydrogen carbonate as a cofactor of the WOC

Plants cannot perform photosynthesis without CO2. CO2 is fixed by

Rubisco (ribulose-1.5-bisphosphate carboxylase/oxygenase) and further
reduced into carbohydrates in the Calvin–Benson–Bassham cycle.
Moreover, CO2 in the form of hydrogen carbonate (HCO3-) (Figure 1.8)
plays a direct role in the light reactions through a structural and
regulatory role within PSII. Numerous data show that HCO3- has effects
both the acceptor and the donor side of PSII (McConnell et al., 2012;
Shevela et al., 2012).

Figure 1.8 Conversions of inorganic carbon species (Ci). Adapted from

(Shevela et al., 2012)

Based on the latest crystal structures HCO3- is a direct ligand of the

non-heme iron and forms hydrogen bonds to several amino asides of the
D1 and D2 proteins (Figure 1.9). Its binding to acceptor side of PSII was
already previously proven by electron paramagnetic resonance (EPR)
and Fourier transform infrared spectroscopy (FTIR) (Diner et al., 1991;
Petrouleas et al., 1994; Hienerwadel and Berthomieu, 1995). All these

16
data are in good agreement with the suggestion that HCO3- may stabiles
the QA-Fe-QB structure and allow efficient electron transport and
protonation of QB-/2-. Thus HCO3- becomes carbonate ion (CO32-) after
protonation of QB2- and may get a proton back form the stroma through
D1-E244 (Müh et al., 2012). However future studies are needed for a
better understanding of this protonation event.

On the donor side of PSII hydrogen carbonate has been suggested

to play role in the assembly of Mn-cluster during phototactivation
(Klimov et al., 1995; Baranov et al., 2000). Moreover, HCO3- was
proposed to play a role of base or proton transporter (Ananyev et al.,
2005; Shutova et al., 2008).

A more detailed description of the role of HCO3- on the donor side

of PSII is provided in Section 4 and in the attached Papers II and III.

Figure 1.9. The ligand environment around Fe and HCO3- of the iron-
quinone center of PSII. The bonds between the non-heme iron and its
ligands are shown as solid line, and the hydrogen bonds as dashed lines.
Reproduced from Umena (2011).

1.4. Water splitting in artificial photosynthetic systems.

It is vital to develop technology which can derive energy from

renewable resources. This idea was presented as far back as in 1912 by
Giacomo Ciamician (Ciamician, 1912), when he stated:– “Solar radiation
may be used for industrial purposes… Photochemistry will artificially

17
put solar energy to practical uses. To do this it would be sufficient to be
able to imitate the assimilating processes of plants”.

Nowadays Ciamician’s idea for production of ‘solar fuel’ from

inexpensive and abundant material such as water that could be split into
oxygen and hydrogen is highly attractive. Often hydrogen is called fuel of
the future since its combustion generates pure product as water. A
promising way for the light driven water splitting in newly made device
would be to mimic the molecular and supramolecular organization of the
natural photosynthetic system, i.e. “artificial photosynthesis”. Today the
concept of solar energy conversion as alternative to fossil fuels is an
important field of research and artificial photosynthesis became as a
highly promising scientific direction (Kirch et al., 1979; Lubitz et al.,
2008; Dau et al., 2010; Nocera, 2012; Wiechen et al., 2012).

Figure 1.10 gives a basic overview of artificial photosynthesis.

Artificial photosynthesis includes both oxidation and reduction reactions
in which water molecule is directly involved (Equation 1.12). Similar to
natural photosynthesis, the reaction starts with absorption of light by
photoactive species (A and D), which reach their exited states (A* and
D*). A* act a strong oxidant (by releasing an electron) in its excited state
and D* yields a reduced state (by capturing an electron). Generated
electron and electron holes can be transferred to water oxidizing catalyst
(Catox) and to a catalyst forming hydrogen by proton reduction (Catred),
respectively. Water oxidation and proton reduction reactions are linked
by a relay molecule R that transfers a charge between the PSII-like and
the PSI/hydrogenase-like units (Figure 1.10).

Catox
2H2O   O2  4H   4e
(1.12.)
4H   4e 
Catred
 2 H2

18
Figure 1.10. Scheme of artificial photosynthesis proposed by (Kirch et al.,
1979). Adapted from Lubitz (2008).

Thus, artificial photosynthesis (AP) has the same principle of

electron flow as natural photosynthesis: The reduction equivalents
obtained by PSII during water oxidation are transferred through the
redox carriers to PSI and are then used to produce “natural fuel” –
NADPH.

1.4.1. Concept of artificial photosynthetic devices.

1.4.1.1. Semiconductor particles.

Light absorption results in the formation of energetic charge-

separated states in both molecular donor-acceptor systems and
semiconductor particles. Semiconductors are able to excite electrons into
higher energy levels and overcome the band gap. Electron are exited
from the valence band to the conduction band and created electron-hole
pairs can be utilized for water splitting. The band gap of the catalyst
supposed to be suitable for the absorption of sunlight and the accessible
redox potentials must be suitable to the water-splitting reaction. Today
there are two approaches for the light driving water splitting by
semiconductor partials: (i) water splitting by single semiconductor
partial (one step photo-excitation system), where catalyst partialy acts as
Catox and Catred without need of electron retransfer (Maeda and Domen,
2010) and (ii) two different types of semiconductors (two step photo
excitation system) for water splitting and proton reduction, respectively
(Kudo, 2011).
Efficiency of 6.5% has been achieved with two step photo excitation
system using WO3/Pt (Catox) and ZrO2/TaON (Catred) and IO3-/I- as
mediator to facilitate electron transfer (Maeda and Domen, 2010, 2011).

19
One step photo-excitation system have been realized by GaN/ZnO with
Mn3O4 for O2 and Rh/Cr2O3 core/shell nano particles for H2 evolution
(Maeda et al., 2010)

1.4.1.2. Electrolyzers.

In modern electrolyzers the electrocatalytic water-splitting is

carried out by the catalysts, which are deposited on electrode surfaces. In
alkaline water electrolyzers, dimensionally stable anodes are used, thin
films of ruthenium or iridium oxide directly supported on metallic
titanium (da Silva et al., 1997; Lassali et al., 1998). In case of polymer
electrolyte membrane electrolyzers, porous carbon electrodes coated
with platinum as a cathode and with noble metal oxides, like iridium or
ruthenium as anodes, are the currently most widely used set-ups (Song et
al., 2008; Millet et al., 2010; Zeng and Zhang, 2010).

Nowadays, commercially available electrolyzers typically have

efficiencies of up to 80 % (Armaroli and Balzani, 2010; Dau et al., 2010).
However, major drawbacks of current electrocatalytic water-splitting
devices are the expensive metals (Ir and Ru) as well as to their complex
constructions. Hydrogen production by water electrolysis is expensive
today and only used to generate hydrogen of high purity (Armaroli and
Balzani, 2010; Dau et al., 2010). In order to make electrolyzes more
affordable and attractive for alternative energy production, one needs to
find more cost-effective approaches. One way to optimize electrolyzers is
to couple them to photovoltaic cells (PVs) (Pearce, 2002; Swanson,
2009). Here sunlight is converted in to electric energy by a photovoltaic
cell to provide the required redox potential for water oxidation and
proton reduction.

1.4.1.3. The artificial leaf

The artificial leaf is solar water-splitting cell including earth-

abundant elements that operate in near-neutral pH conditions, both with
and without connecting wires (Reece et al., 2011; Nocera, 2012). The cell
is based on triple junction, amorphous silicon photovoltaic interfaced to
Co catalyst (Catox as oxygen evolving catalyst), and NiMoZn catalyst
(Catred as hydrogen evolving catalyst). Efficiency of water splitting
reaction is 4.7% for a wired configuration and 2.5% for a wireless
configuration.

20
 1.4.1.4. Dye-sensitized solar cells

The light harvesting efficiency can be increased by immobilizing

molecular dyes at the semiconductor’s surface. When the dye is
absorbing light, it is transferred into its excited state. From the excited
state, the dye injecting an electron into the conduction band of the
semiconductor, generates a charge at the semiconductor-dye interface. In
dye-sensitized solar cells, dye-sensitized semiconductor particles are
immobilized at a conducting surface like indium doped tin oxide or
fluorine doped tin oxide as anodes (O'Regan and Gratzel, 1991; Hagfeldt
et al., 2010).
Today TiO2 (Lee et al., 2012), NiO (Li et al., 2012), Nb and W oxides
(Abe et al., 2009) are used as semiconductors modified with organic
molecules (Abe et al., 2009; Li et al., 2012) or Ru complexes as dyes (Lee
et al., 2012). Here Ir (Catox) is used as water splinting catalyst (Lee et al.,
2012) and Co (Catred) as a catalyst for hydrogen generation (Li et al.,
2012).

Despite the progress made in development of artificial

photosynthetic devises some problems remain. Major challenge is
identification of effective materials that can act as Catox and Catred based
on abundant elements which are stable for long period under working
conditions.

1.4.2. Molecular catalysts for artificial water splitting

Today molecular catalysts for water splitting have resided

substantial attention. This catalyst offers greate opportunity for
structural design and tuning the WOC for optimal performance.
However, relatively small amount of molecular water oxidation catalysts
that operate with low overpotentials are known. Majority of them have
been discovered in the last few years.

The first multinuclear ruthenium based water splitting catalyst

(“ruthenium blue dimer”) was discovered in the early 1980s by Meyer
and colleagues (Gersten et al., 1982; Gilbert et al., 1985; Concepcion et
al., 2008). Later number of dinuclear ruthenium complexes showed to be
more or less effective catalyst for water splitting (Wada et al., 2000;
Wada et al., 2001; Deng et al., 2008; Geletii et al., 2008; Sartorel et al.,
2008). This suggested that at least two metal centers are needed for

21
water oxidation. However, this suggestion was disproved and it is widely
known now that water splitting is also possible with ruthenium
mononuclear complexes (Zong and Thummel, 2005; Tseng et al., 2008;
Concepcion et al., 2010; Duan et al., 2012).

In contrast to rapidly developing ruthenium water splitting

catalyst, there are examples of catalyst containing other metal centers
both for multinuclear or mononuclear systems. The water splitting ability
of iridium catalysts was discovered by Bernhard and colleagues
(McDaniel et al., 2007). Based on complex initially prepared in 1973
(Weakley et al., 1973), Hill and colleagues showed that compound that
contains an active Co4 core and free of carbon-based organic ligands is
catalytically active for water splitting (Yin et al., 2010).

Immobilization of molecular catalysts on the electrode surface and

application of them in to artificial photosynthetic devises will not be
discussed here, since molecular catalysts are beyond scope of this thesis.
In this section and further, molecular catalysts are briefly discussed.

1.4.3. Cobalt and manganese oxides as a catalysts for

artificial water splitting

Water oxidation to molecular oxygen is a four-electron four-proton

reaction that requires an overall redox potential of 1.23. To be capable to
drive this reaction by visible light one needs to avoid high energy species
such HO• that would be formed by a one-electron oxidation of water. As
such, transitions metals, or clusters thereof, that are able to support
multi-electron reduction steps are most suitable as water oxidation
catalysts. It is also highly important that the catalysts operate with low
overpotentials.

A comparison of almost 20 different transition metals oxides by

Harriman et.al. (Harriman et al., 1988) revealed that IrO2, Co3O4, RuO2,
Mn2O3 function most efficiently as O2-evolving catalyst. Up to now
situation has not changed very much and the most successful catalysts
for water oxidation are made from the above mentioned oxides (Morris
and Mallouk, 2002; Kanan and Nocera, 2008; Duan et al., 2009;
Concepcion et al., 2010; Jiao and Frei, 2010; Najafpour et al., 2010;
Duan et al., 2012). Oxides of noble metals are the most efficient water
oxidation catalysts reported so far. However, these oxides are no
promising catalytic components for artificial photosynthetic devices for

22
large scale solar fuel generation due to the low abundance and high costs
of Ru and Ir

Mn and Co are favored by many chemists for synthesis of “PSII-

like” artificial photosynthetic systems for water splitting since they are
relatively abundant (especially Mn). Mn is found as free element in
nature, for example at the ocean floor and the bottoms of many fresh-
water lakes, as well as in many minerals (Post, 1999; Dismukes et al.,
2001; Sauer and Yachandra, 2002). Moreover, Mn has the ability to
attain a large number of different oxidation states. Both are likely
reasons why it was chosen by nature as active center for water splitting
(Armstrong, 2008). The structure of this center is in higher plants, algae,
and in cyanobacteria. Artificial complexes that mimic the WOC, with
multi-nuclear Mn connected through µ-oxo bridges were found to act as
highly active water oxidation catalyst (Brimblecombe et al., 2008;
Najafpour et al., 2010; Hocking et al., 2011; Wiechen et al., 2012). The
metal centers of artificial Mn-complexes are cycling between MnIII and
MnIV oxidation states during water oxidation process and that is why
multi nuclear species, at least 4Mn, are required.

An interesting similarity was found between artificial Mn-

complexes and Mn-cluster of the WOC: if calcium is added to synthetic
Mn-catalyst, the oxygen evolving activity of Ca-Mn-catalyst increase
more than ten times as compared to Mn-catalysts without calcium
(Najafpour et al., 2010; Shevela et al., 2011; Tsui et al., 2013). Moreover
study on Mn3X cluster containing Na+,Ca2+,Sr2+, Zn2+ or Y3+ showed that
the reduction potential of Mn3X clearly depends on the charge of the
redox–inactive metal ions (Tsui et al., 2013). The more highly charged
Lewis acidic metal ions withdraw more electron density of the Mn3X
cluster making it easier to reduce. The Mn3X cluster containing Ca2+ or
Sr2+ ion exhibit the same reduction potential. Interestingly, that the WOC
of PSII remains active without significant structural changes and still
maintain enzymatic function only if Sr2+ ion replaced by Ca2+ (Hendry
and Wydrzynski, 2003; Brudvig, 2008; Pushkar et al., 2008; Cox et al.,
2011; Yachandra and Yano, 2011). Agapie and colleagues suggested that
possible role of the calcium ion in the WOC is to modulate the redox
potential of the cluster. This tuning of the cluster’s redox properties is
essential for the transfer of four electrons that are needed for water
splitting.

23
 Cobalt oxide is known to be a good water oxidation catalyst for
long time (Baxendale and Wells, 1957; Anbar and Pecht, 1967;
Shafirovich et al., 1980; Elizarova et al., 1981). Co-based catalyst were
studied by many research groups (Kanan and Nocera, 2008;
Surendranath et al., 2010; Yin et al., 2010; Shevchenko et al., 2011; Li
and Siegbahn, 2013). Known advantages are the efficiency at neutral pH,
the stability under working conditions and abundance (though less
abundant than Mn). Moreover, Co-oxide catalyst self-assemble upon the
oxidation of Co2+ to Co3+ ion in aqueous solution and have a structure
analog to that of the Mn4CaO5 cluster in PSII. X-ray absorption
spectroscopy studies revealed that Co-catalyst structure consist of several
Co(III)O6 octahedra units in incomplete and complete cubane blocks.
(Risch et al., 2009; Kanan et al., 2010; Du et al., 2012). In the event of
Ca-Co-catalyst no difference was observed in water oxidation
activity(Risch et al., 2012).

In order to test artificial photosynthetic systems for their ability to

act as water oxidation catalysts either electrochemistry or sacrificial
oxidation agents (Parent et al., 2013) are used. In the latter case they
must have a potential high enough to oxidize the catalysts that are used.
However, oxidation agent such as Ce(IV) forms insoluble cerium oxide
materials upon reduction to Ce(III) (Hayes et al., 2002). Formed cerium
oxide can incorporate into the catalytically active metal (de Leitenburg et
al., 1996).

In summary, both synthetic Co and Mn oxides are very promising

candidates for large scale applications of artificial water oxidation
chemistry, since they are abundant, inexpensive and non-toxic catalysts.
One condition for this is, however, that their activity (turnover number
and turnover frequency) can be improved. More detailed information
about Co and Mn catalysts as their commonly used oxidation agents can
be found in Section 5 and Papers IV and V.

1.5. Relevance

Knowledge of the basic principles of water oxidation and

hydrogen conversion in nature is a presently a major goal for basic
research, because it also promises to yield knowledge applicable for the
energy needs of our society. This knowledge may allow us to use either
use (modified) photosynthetic organisms – or the isolated enzymes – in
biotechnological processes, or to design biomimetic (artificial) catalysts

24
for large-scale water splitting and hydrogen production. Such systems
would combine the tasks of light-absorption, charge separation, water-
splitting, proton transport and hydrogen formation all in one ‘device’.
While biological systems can already be used, the artificial devices bear
the promise to reach significantly higher efficiencies in the future.

25
 2. Materials and Methods
2.1. Determination of the chlorophyll concentration

The Chl concentration of BBY fragments (Berthold et al., 1981) ,

PSII cores and PSII crystals (Kern et al., 2005) was determined after
extraction in 80% (v/v) acetone, recording absorption at 646,6, 663,6,
664 and 750 nm on UV/VIS spectrophotometer. The formula used for
estimation of Chl concentration (mg/ml) in BBY membrane fragment as
follows (Porra et al., 1989).

c(Chl )  17,76( A646,6  A750 )  7,34( A663,6  A750 )  k (2.1.)

and in PS II cores and crystals, according to formula:

( A664  A750 )  k
c(Chl )  (2.2.)
74000

where k is dilution factor.

2.2. Membrane Inlet Mass Spectrometry

Mass spectrometry is a powerful technique that is used for

experimental analysis of charged compounds from single atoms to mega-
Dalton mass units. Mass spectrometry allows to study kinetics and
dynamics in biological system with very high accuracy (Konermann et al.,
2008). In this study a membrane inlet isotope ratio mass spectrometer
(MIMS) was used (Thermo Scientific™ Delta XP and Delta V).

A huge impact for the development of isotope ratio MS

instruments had the discovery of JJ. Thomson that Ne has two different
isotopes, 20Ne and 22Ne (Thomson, 1910, 1912, 1913). Thereafter, it
became obvious that isotope ratio MS is suitable for the analysis of many
stable isotopes.

The MIMS set-up was developed by G. Hoch and B. Kok in the

beginning of the 1960th (Hoch and Kok, 1963). They developed the idea
to separate the liquid or gaseous medium in which the reaction of

26
interest takes place from the high vacuum of the MS by a semipermeable
membrane, which allows only small gas molecules to pass through
(pervaporate) into the MS. The high vacuum in the MS is needed in order
to prevent collisions of the analyte molecules/ions with atmospheric
molecules. Such collisions would change their trajectories and thus
reduce both resolution and sensitivity of the MS. MS’s are able to detect
charged molecules only. That is why neutral gases have to be ionized
after entering into the MS. In both the Delata XP and the Delta V the
ionization occurs by electron impact (Siuzdak et al., 1996). A heated
filament plus a suitable electrical potential work as electron gun. The
thus created high-energy electrons interact with the analyte gas
molecules, M, creating positively charged analyte ions:

M  e  M   2e (2.3.)

Reaction 2.3 shows that each ionized molecule has almost the same
mass as before (without one electron), and that the mass to charge ratio
represents the molecular mass of the analyte (M•+= m/z). After ionization
and further acceleration the ions travel through a magnetic field, which
separates them according to their occurs m/z ratio (Dempster, 1918).
Mathematically this can be presented by the following formula:

2 2
m B R (2.4.)
z 2V

Where the ionized molecule with m/z will travel on a path of radius
R when it reaches the magnetic field B after being accelerated by a
potential V. The thus separated ions are detected by an instrument
specific array of Faraday cups. Amount of a Faraday caps depend on a
model of isotope ratio mass spectrometer. The detection principle of
Faraday caps based on the impact of ions on a metal surface which leads
to the release of electrons, which are collected directly. Then detector
amplify the initial signal, before it is read out into a software.

27
 Figure 2.1. displays a simplified scheme of a MIMS set up, and also
describes different types of measurements, which were performed during
this study.

Figure 2.1. Simplified scheme of a MIMS set up, in which the production
of gaseous analyte is initiated by (A) illumination of degased photo-active
samples (B) illumination of non-degassed samples that were
subsequently injected into a degassed buffer and (C) the reaction of non-
degased chemicals injected into the degased sample. The gaseous
reaction products penetrate through the gas permeable membrane and
reach the high vacuum section where they are ionized by electron impact
(EI). The thus generated ions are separated by a magnetic field (MF)
according to their mass-to-charge ratios and detected by Faraday cups.
The resulting signals of the different isotopes are amplified and recorded
online simultaneously.

28
2.3. H218O – labeling

H218O labeled water was used to understand possible pathways of

reactions that lead to the evolution of the isotopologues of O2 (16O2,
16O18O, 18O ) and CO (12C16O , 12C16O18O, 12C18O ). The addition of H 18O
2 2 2 2 2
into the sample was done for MIMS measurements only. The lowest level
of isotopic enrichment is given by Earth’s natural abundance (0.2 %). For
a given 18O-enrichment b of water, the isotope composition of O2 or CO2
was calculated according to the binomial distribution function, which
assumes the absence of isotope effects:

(a  b)2  a2  2ab  b2
(2.4.)
a  16O2 ; b  18O2

or

m z  32: 34: 36
44: 46: 48
 a2 : 2ab : b2 (2.5.)

Where a 2  2ab  b2  1 is the total product.

Another way to follow changes in the level of enrichment (total

fraction of 18O atoms in the product) as a function of reaction time is to
apply the following formula (Mills and Urey, 1940; Silverman, 1982)

2ab  2b2
18
 (2.6.)
2(a2  2ab  b2 )

The contribution of 17O to the total O2 and CO2 can be neglected

due its very small natural abundance (10-5 %).

2.4. Joliot type electrode and flash induced oxygen

evolution patterns

The FIOPs were obtained at defined temperature with a home-

built Joliot type electrode (Joliot and Joliot, 1968; Joliot, 1972) that was
modified by Messinger (Messinger, 1993). All measurements were done
in the absence of electron acceptors. A scheme of the modified Joliot type
electrode is shown on Figure 2.2.

29
Figure 2.2. Schematic cross-section of home-build Joliot type electrode.
Adapted from (Shevela, 2008).

For each measurements 10 µl aliquot of the sample was transferred

onto the surface of Pt-cathode in dim green light and then always kept
there for 3 minutes in order to reach complete temperature adaptation
and sedimentation. Before illumination of the sample with saturating
flashes, which were provided by a xenon flash lamp, the polarization
voltage of -750 mV was applied for 40 seconds. This time was necessary
to achieve a constant signal background. The flash frequency was 2 Hz.
Signals form the oxygen oscillation pattern were amplified and recorded
with 3.6 points per ms sampling rate. The control of the flash sequence,
and the recording of the data was done with a program (LabView
software, National Instrument) developed by the author.

2.4.1. Data evaluation

The deconvolution of the oxygen oscillation pattern into the Si state

populations was performed with Excel spreadsheet (Messinger et al.,
1991) based on the method developed by (Delrieu, 1974; Lavorel, 1976)

S n  K  S n 1 (2.7.)

30
 Where transition between states by light flashes are described by
operation of a matrix K on a column vector S

 S 0   0  
  
S   0  
S n   1  and K  
 S 2     0 (2.8.)
   
 S3  0   

S 
i
i 1 (2.9.)
n

where α, β and γ are the transition probabilities for the Si state

transitions: α, misses (Si→Si); γ, single hit (Si→ Si+1); β, double hit (Si→
Si+2); with α+β+γ=1.

The oxygen yield produced by the nth flash Yn is thus related to

components of a state vector

Y n  (1   )  S 3 n 1    S 2 n 1 (2.10.)

 S2 n 1 and  S3 n 1 are the population of the redox states S2 and S3

before the nth flash.

2.5. Clark electrode measurements of oxygen evolution

In order to measure the oxygen evolving activity of either PS II

samples or artificial compounds a Clack-type electrode (Qubit Systems
and Rank Brothers Ltd.) was used (Clark et al., 1953). For this PSII
samples were illuminated with saturating continues white light of a slide
projector. The samples were measured at a concentration of 10-20 µg Chl
ml-1 in a reaction chamber filled with designated buffers (final volume of
1 ml). The measurements were performed in the presence of 250 μM
PPBQ (phenyl-p-benzoquinone) or 250 μM DCBQ (2.6-Dichloro-1.4-
benzoqiuinone) and 5 mM FeCy (K3[Fe(CN)6]) as artificial electron
acceptors. For measuring the oxygen evolution rate of artificial
compounds the reaction was started by the addition of oxidizing agents
such as CeIV ([(NH4)2[CeIV(NO3)6]]) or [RuIII(bipy)3]3+. To keep a
constant temperature, the reaction mixture was thermostated to 20°C
and stirred constantly during measurements with a magnetic stir bar.

31
The electrode was calibrated using air-saturated and nitrogen-saturated
water at atmospheric pressure.

2.6. X-ray spectroscopy

X-ray measurements at Stanford Linear Accelerator Center (SALC)

were conducted together with the members of YACHANDRA/YANO LAB
(Lawrence National Lab, Berkeley, California, USA).

All conditions and parameters regarding illumination setup (such

as light intensity, frequency, sample flow, S states population and so on)
which were obtained by author and Johannes Messinger were applied at
SLAC for the X-ray experiments (Figure 2.3.).

Figure 2.3. Setup of simultaneous x-ray spectroscopy and crystallography

experiment at the Coherent X-ray Imaging instrument of Linac Coherent
Light Source (left side). Scheme for the illumination setup used to
advance PS II in the catalytic cycle and measure simultaneously the XRD
and XES signal at LCLS (right side).

Whole setup of simultaneous x-ray spectroscopy and

crystallography (Figure 2.3.) will not be discussed in material and
method section, since the main task of the author was to develop new
method for illumination of PSII crystals and suspensions.

2.7. Illumination setup

The samples employed were PSII core complexes that were

isolated from Thermosynechococcus elongatus (T. elongatus) as
described elsewhere (Kern et al., 2005). The rates of O2 evolution for

32
PSII cores typically were ~ 3500 µmol (O2)·mg (Chl)-1·h-1, and the
number of Chl’s per reaction center was 29-34.

PSII crystals (2-25 µm) or PSII cores at Chl concentrations of 6-10

mg/ml were diluted with H218O (97.6 %) which contained 1.2 M sucrose
or 27 % (w/v) glycerol and 5 mM CaCl2 to give a final 18O-enrichment of
about 12-48 % and final salt concentration 5 mM CaCl2, 50 mM MES, 1.2
M Sucrose or 27 % glycerol. No electron acceptors were added. Samples
were kept in darkness or very dim green light during all steps, except
when illuminated with the laser. The 18O enriched samples were
illuminated by a home build illumination setup (Figure 2.4.)

Figure 2.4.Top view of home-build illumination setup.

The setup is designed for the illumination of samples that flow

inside of a silica capillary (inner diameter (ID) of 50-100 µm and outer
diameter (OD) of 150 or 360 µm). For illumination ns-laser light pulses
traveling through up to four optical fibers (400 µm core diameter) that
were directly attached to the de-coated region of the capillary. The fiber
tips were connected to the capillary in holder base (Figure 2.5.)

33
Figure 2.5. Picture illustrating the coupling of the fibers to the capillary
in the holder base.

For the illumination the samples were pumped from their reservoir
(gas-tight Hamilton syringe) at a constant rate through the capillary by a
syringe pump. The illuminated samples were collected at the other end of
the capillary with another gas-tight Hamilton syringe that was mounted
onto another syringe pump that operated in the sample withdrawing
mode. Both syringe pumps were synchronized to the same flow rate thus
providing constant laminar flow through all length of the capillary. The
flow rate was optimized so that at the given flash rate each sample region
was illuminated only once with a flash.

The laser employed in this study was a Continuum Inlite II-20

(Nd:YAG laser, 532 nm, 7ns pulse width). To obtain stable output
intensity of 3.5-7.0 µJ/fiber the laser was operated continuously at 20Hz.
While the flash frequency was controlled via the Q-switch divider (20Hz
or 10Hz). The illumination periods were set with the help of a fast shutter
(SH05 operated with SC10 Controller; both Thorlabs).

The efficiency of the illumination of PSII was tested by recording

the oxygen oscillation pattern. In contrast to the Joliot-type FIOP
measurements, here each point needs to be collected separately (see
below). For each point 13.5 µl of sample was pumped through the
capillary and illuminated with the indicated number of capillaries. At the
end of each illumination, the sample collected in the receiving syringe
was injected into a degassed buffer of a MIMS cell. Since all experiments
were performed in air-saturated buffers H218O enrichment (see above)

34
was employed to record the 34O2 signal, which has a comparatively small
natural background. The traces obtained are displayed in Figure 2.6 A.

In order to calculate the flash pattern from these traces, the O2

signal obtained with ‘x’ fibers was subtracted from that obtained with
’x+1’ fibers. For the 1st flash, the background 34O2 signal of the non-
illuminated sample was subtracted (Figure 2.6 B).

Figure 2.6. On-line MIMS measurements of light-induced O2 yield

detected as mixed labelled 16O18O specie after illumination of
photosystem II from T. elongatus with 0, 1, 2, 3 and 4 flashes at pH 6,5
and 20°C (panel A). Panel B displays the flash pattern derived from A by
subtracting the signal of x fibers from that obtained with x+1 fibers.

Before the illuminations each fiber optic was tuned with an energy
meter (OPHIR PE 10-C) so that the output energy of each individual fiber

35
did not vary more than ±5 % from a stable output intensity. A Newport
LBP sensor was used for measuring beam profile of each fiber to be sure
that the intensity in each point of fiber profile remains the same (Figure
2.7.). To monitor the flow rate of the samples inside the capillary a liquid
flow sensor (Sensirion SLG64-0075) was used.

Figure 2.7. Profile of fiber optic.

36
3. Femto-second serial X-ray
crystallography and spectroscopy:
new tools for studying the Mn4CaO5
cluster in different oxidation states.
The water oxidation reaction in nature is accomplished by the
WOC in PS II. This multi-subunit membrane protein found in green
plants, algae, and cyanobacteria, uses light to catalyze the oxidation of
water to oxygen. The WOC of PS II contains a heteronuclear Mn4CaO5
cluster that couples the four-electron oxidation of water with the one-
electron photochemistry at the PS II reaction center, by acting as the
locus of charge accumulation. Energy derived from the absorption of
successive photons drives the WOC through the Kok cycle (Kok et al.,
1970), a series of five intermediate Si-states, where i represents the
number of oxidizing equivalents stored in the WOC. After the S4 state is
reached, a spontaneous reaction occurs that releases O2 and returns to
the WOC into the S0 state (Renger, 2011) (Figure 3.1.).

Figure 3.1. The Si state scheme for oxygen evolution. The proposed Mn
oxidation states for each of the intermediate states are given (including

37
the proposed transient [S4] state). The structure of the S1 state proposed
by Umena et.al. is displayed in the inset. Blue spheres, water molecules;
magenta spheres, manganese ions; red spheres, μ-oxo bridges; yellow
sphere, calcium

In the past years extensive studies of the dark stable S1 state using
X-ray diffraction (XRD) have achieved enormous progress: the
resolution was improved from 3.8 to 1.9 Å (Zouni et al., 2001; Kamiya
and Shen, 2003; Ferreira et al., 2004; Loll et al., 2005; Guskov et al.,
2009; Umena et al., 2011). In addition various techniques also have been
used to elucidate the structure and mechanism of the WOC of PSII
(Messinger et al., 1995; Haumann et al., 2005; Noguchi, 2008; Yano and
Yachandra, 2008; Yeagle et al., 2008; Berthomieu and Hienerwadel,
2009; Cox et al., 2011). Between different method for obtaining detailed
structures of PSII and the Mn4CaO5 cluster, XRD gives the most direct
information about the overall arrangement of the atoms.

For XRD, however, the X-ray radiation damage (Yano et al., 2005)
to the redox active Mn4CaO5 cluster has been an issue. The damage of the
cluster leads to the reduction of Mn(III) and Mn(IV) present in the intact
WOC to Mn(II) at an X-ray dose that is two to three orders of magnitude
lower than the dose required to produce an observable decay in
diffraction quality of PS II crystals (Yano et al., 2005; Grabolle et al.,
2006). Such radiation damage occurs despite of the fact that the data
collection is carried out at cryogenic temperature (100-150 K). The
radiation damage and the cryogenic temperatures make it impossible to
study transient reaction intermediates of PSII before and during O-O
bond formation. Radiation damage does not only occur in PSII, but is a
problem encountered also with many other redox active metallo-enzymes
(Corbett et al., 2007; Meharenna et al., 2010; Antonyuk and Hough,
2011; Hersleth and Andersson, 2011; Daughtry et al., 2012; Sigfridsson et
al., 2013).

The recent study by Umena et al. (2011), used a significantly lower

X-ray dose than previous XRD studies. Nevertheless, this dose leads to
the reduction of ~25% of the Mn to MnII. Despite of that the atomic
structure of PS II could be resolved at 1.9 Å resolution. This structure
revealed the positions of the four Mn and one Ca ions, and for the first
time also the five oxo-bridges were visible (Umena et al., 2011). However,
noticeable differences exist between the crystal structure and the EXAFS
results (Yano et al., 2006; Yano et al., 2007; Pushkar et al., 2008; Yano

38
and Yachandra, 2008) in which the data were collected at much lower X-
ray dose than in the XRD experiments by Shen and coworkers.
Specifically it can be noticed that the Mn-ligand and Mn-Mn distances
are significantly longer in the 1.9 Å crystal structure than those
determined by EXAFS.

Recently a new crystallographic approach was developed: a femto

second serial X-ray crystallography. In this new technique, suspension of
microcrystals is injected into X-ray region. Femtosecond X-ray pulses,
which are short enough pass the sample before significant electronic
rearrangement and atomic displacement occur (diffraction before
distraction) allow of collecting diffraction pattern form single randomly
oriented crystal. Crystals have to be replaced after each exposure. Besides
having the advantage that much smaller crystals can be studied than
previously, this technique has two key advantages that make it very
interesting for PSII research: (i) it is performed near room temperature,
thus studies can be performed at transient states, and (ii) it over comes
the problem of radiation damage. (Chapman et al., 2011; Hunter et al.,
2011; Kirian et al., 2011; Lomb et al., 2011; Aquila et al., 2012; Barty et
al., 2012; Boutet et al., 2012; Johansson et al., 2012; Koopmann et al.,
2012).

The first X-ray Free-Electron Laser (XFEL) was realized at the

Linac Coherent Light Source (LCLS) near Stanford, USA. The intense
and ultra-short X-ray pulses (<70 fs) allow to collect diffraction data
before the start of radiation damage (Neutze et al., 2000; Caleman et al.,
2010). By combining serial X-ray crystallography with X-ray emission
spectroscopy it was recently shown that the ‘probe-before destroy’
approach also works for the highly radiation sensitive Mn4CaO5 cluster in
PS II (Alonso-Mori et al., 2012; Kern et al., 2012). This approach thereby
provides the possibility to obtain molecular motions of the catalyst at
work by recording snapshots at different time points in the catalytic cycle
(Figure 3.1.). In such a study crystallography and spectroscopy can give
complementary information: spectroscopy provides detailed information
about changes in the Mn oxidation states, and crystallography probes the
structural changes of the Mn4CaO5 cluster and the protein.

In order to study the Mn4CaO5 cluster at different oxidation states

using this new combined approach it is essential to know the exact
oxidation state of the cluster at each point of the measurements. The
various oxidation states are set by in situ illumination of the crystals

39
while they travel to the interaction region with the X-ray pulse.
Therefore, the setup that illuminates the samples and delivers them to
the interaction region becomes a crucial part of the whole experiment.

3.1. Optimization of parameters for the light-induced Si

state turnover of PSII samples in a flow-illumination set-up.

To advance PS II samples into the different S states during the

XFEL experiments a new illumination setup was developed that allowed
illuminating the PSII samples while they are flowing through a capillary
towards the interaction region with the fs-X-ray pulses. Since the XFEL
experiments do not allow obtaining a fast feedback about the oxidation
states that are reached by this new setup, an independent approach was
required to obtain the best parameters for Si state turnover under these
conditions. Parameters that need to be optimized include: laser light
intensity, flow rate/flash frequency, optimal diameter and surface
treatment of capillaries. Therefore, a copy of this setup was constructed
in the Messinger lab at Umeå University. With this replica set up PSII
crystal and core suspensions were illuminated inside a striped silica
capillary with laser flashes that were delivered via fiber optics that were
directly attached to the capillary. The turnover efficiency was determined
from the quality of the oxygen oscillation pattern that were measured by
MIMS. These measurements were done in the presence of H218O, since
this allows to detect the oxygen production at m/z 34, which has a low
natural background signal due to dissolved oxygen from the atmosphere
(for more detailed information can be found in Section 2.7 and attached
Paper I).

Before testing the PSII preparations in the new flow-illumination

set up, the turnover of the samples was probed by illuminating them with
Xe-flashes in presence and absence of added exogenous electron
acceptors in a MIMS cell. The data presented in Figure 3.2 show that the
PSII core preparations are able to perform one full Si state cycle in the
absence of added electron acceptors. The data also show that these
preparations have a normal miss parameter. Both findings are essential
prerequisites for the subsequent flow-illumination studies. The O2 yield
after the second flash was higher in presence of PPBQ due to reductant-
induced oxidation of the non-heme iron (Petrouleas and Crofts, 2005).

40
Figure 3.2. Oxygen yield (m/z=34) measured by illumination of PSII core
preparations of T. elongatus in a MIMS cell as a function of flash
number. The PSII suspensions were measured with (black; 40µM PPBQ)
and without (red) added electron acceptors. The traces are normalized to
maximum yield in the first turn over cycle. A series of thirteen saturating
Xe-flashes with intermittent dark times of 25 s was given at 20°C and pH
6.3. The H218O enrichment was 15 %, the chlorophyll concentration 0.1
mg Chl/ml.

It was the most critical to determine the effective width of the

illumination regions (Figure 3.3). This can vary from the diameter of the
optical fibers due to effective illumination area. This knowledge is
important for determining the optimal combination of flow rate and
illumination frequency. The optimal flow rate that is synchronized with
flash frequency so that an almost uniform S state advancement of the
whole sample is achieved. If the flow rate is too slow with regard to
illumination frequency, double or even more illuminations will occur; if
it is too fast, non-illuminated sections remain.

41
Figure 3.3. Schematic picture of PSII crystals that were illuminated
inside a silica capillary with laser flashes.

While the flash frequency can be easily controlled, the flow sensor
and the syringe pump speed represent the average flow rate only. Since
sample is traveling through a capillary a laminar flow profile is
generated. Under conditions of laminar flow, the nature of viscosity
dictates a flow profile in which the velocity of the sample increases
towards the center of the capillary, while it is zero at the walls. This is
illustrated in Figure 3.4.

Figure 3.4. The flow profile of a fluid in a capillary shows that the fluid
acts in layers that slides over one another. νm corresponds to a maximum
velocity and ν is a velocity at the walls. Reproduced from
(http://hyperphysics.phy-astr.gsu.edu/hbase/pfric.html#lam)

This non-uniform flow adds some complicity into the

synchronization of the flow rate with illumination frequency. This shows
that in a flow set up one needs to find the best compromise of all
variables, and that one can never expect to see the same low misses and
double hits as in a stationary sample (Figure 3.2).

Another important factor is the required light intensity for efficient

turnover through the Si state cycle. To low intensities can lead to only
partial turnover of the samples, while too high intensities increase the
miss parameter via light scattering along the capillary, and may also
inactivate the sample. The optimal light intensity can be found in two

42
ways: (i) by the quality of the O2 oscillation pattern (same as above; see
Fig. 3.7), and (ii) by the total O2 produced per PSII complex and flash
number. The first method should normally be sufficient, but a small
uncertainty remains if there can be a certain part of the sample that
never sees any light, and thus does not contribute to the oscillation
pattern. To address this question approach (ii) needs to be employed,
which requires the absolute calibration of the MIMS signals. This
calibration was achieved by the injection of known volumes of air-
saturated water into the MIMS cell. This value was used to determine the
micromoles of O2 produced by PSII core complexes (PSII cc) by the
illumination with 3 flashes. This number was then divided by the µmole
PSII reaction center, which resulted, after accounting for diffusion losses
of 34O2 out of the capillary (data not shown) in about 0.73 O2/RC. Since 3
consecutive flashes are required to produce one molecule of oxygen in a
dark-adapted PSII reaction center, a maximum of one O2/RC can be
expected under these conditions. The data in Figure 3.2 show that this is
even true if they move slow and see more flashes. The above numbers of
73% oxygen yield directly translate into light saturation. For 100% light
saturation we would expect a value of 73% (0.93), since even under
stationary conditions an average ‘miss’ of 10% occurs due to charge
equillibria within PSII. Thus, we conclude that light intensity of 7 µJ is
high enough to be saturated the entire PSII cc sample (Figure 3.5).

43
Figure 3.5. Relative O2 yield per PSII as detected by MIMS as a function
of flash number. Conditions were the following: for PS II solutions silicon
capillary (ID = 75 µm, OD 160 µm), flow rate 0.5 µl/min and frequency 4
Hz, light intensity of each fiber was 7 µJ.

Figure 3.6 shows an example of an early flash pattern, in which the flash
frequency was too fast as compared to the flow rate. Therefore the
maximal O2 yield is reached only after the fourth flash.

44
Figure 3.6. MIMS measurements of light-induced O2 yield (m/z=34)
after illumination of PS II microcrystals in a flow-illumination set up.
Conditions were the following: silicon capillary (ID = 100 µm, OD 360
µm), flow rate 2.2 µl/min and frequency 10 Hz. Light intensity of each
fiber was 7 µJ

After optimization of all parameters, the FIOPs for PSII crystals and
cores displayed in Figure 3.7 were obtained. The result shows, despite a
somewhat high O2 yield after the first flash, the typical O2 oscillation
pattern with a maximum after the 3rd flash. They thus demonstrate that
good enrichments of the PSII cc complexes in the various Si states can be
reached with the new flow-illumination set up, and that the XFEL
measurements with PSII samples are feasible.

45
Figure 3.7. MIMS measurements of light-induced O2 yield (m/z=34) after
illumination of PS II solutions (black) and PS II microcrystals (red) in a
flow-illumination set up. Conditions were the following: for PS II
solutions silicon capillary (ID = 50 µm, OD 160 µm), flow rate 1.055
µl/min and frequency 20 Hz, for PS II microcrystals silicon capillary (ID
= 100 µm, OD 360 µm), flow rate 2.0 µl/min and frequency 10 Hz. Light
intensity of each fiber was 7 µJ

These illuminated conditions were used in the XFEL approach

(discussed above) to collect the XES and XRD data of PSII presented in
Paper I. The final goal of this project is to understand the water oxidation
mechanism by probing the geometric structure, the oxidation state and
electronic structure, and the changes in the Mn4CaO5 complex while it
proceeds through the Si state cycle.

46
4. Role of hydrogen carbonate in PSII
CO2 is not only the most discussed greenhouse gas in the
atmosphere of the Earth, but also a key metabolite in all living organisms
that participate in the global biological processes such as photosynthesis.
As a metabolite it exists in equilibrium with HCO3–and carbonic acid
(H2CO3) (Equations 4.1. and 4.2.). These three species are referred to
collectively as inorganic carbon (Ci). Reversible conversion of Ci allows to
transport CO2 and HCO3- in all cells. HCO3– is poorly soluble in biological
lipid membranes compared to CO2, which can freely diffuse in and out of
the cell. Therefore, the conversion of Ci according to reaction 4.1.
facilitates the transport of HCO3– and protons (H+) into the intracellular
space, while reaction 4.2. helps trap carbon dioxide within the cell.
Carbonic anhydrases (CA) catalyze the interconversion of the Ci species,
and thus speed up reactions 4.1 and 4.2 up to 1000000 times (Henry,
1996; Krishnamurthy et al., 2008). In photosynthetic organisms
(cyanobacteria, green algae and higher plants) CA is important for both
carboxylation and decarboxylation reactions, ion transport and pH
regulation (Moroney et al., 2001; Coleman, 2004).

HCO3   H  
 H 2CO3 
1
 CO2  H 2O (4.1.)

CO2  H 2O  
2
 HCO3   H
 H 2CO3  (4.2.)

The role of CO2 or HCO3- in photosynthesis was discovered by

Senebier who found in 1782 that the production of oxygen by plants
depends on the presence of “fixed air” (CO2) (Bay, 1931). Today it is
obvious that plants cannot perform the process of photosynthesis
without CO2. CO2 is fixed by Rubisco and two photosystems facilitate to
drive CO2 fixation by light reactions.

The possible roles of Ci in photosynthetic water-splitting have been

a controversial issue ever since the first report by Otto Warburg and
Günter Krippahl in 1958 on the ‘bicarbonate effects’ on electron flow in
PSII. Warburg and Krippahl observed that CO2 is required for oxygen
evolution reaction and their view was that HCO3-, not water, is the source
of oxygen produced by PSII (Warburg and Krippahl, 1958). This
hypothesis has been disproven by time resolved 18O-labelling studies

47
(Hillier et al., 2006; Shevela et al., 2008). This was not easy, because the
CA activity of PSII samples leads to a fast equilibration between the 18O-
isotope label of water and CO2 (Figure 4.1)

Figure 4.1. MIMS measurements of a change in C18O2 concentration as a

function of time after the 5 µl-injection and rapid mixing (<10 ms) of
H218O (to final enrichment of 3.2%) into the 150 µl-MS cell at pH 6.3 and
10°C. The Chl concentration of spinach PSII samples was 0.0 mg ml–1
(only MNS buffer in the MS cell) (trace 1) and 0.3 mg ml–1 (trace 2). For
better comparison the initial rise of the 18O-labeled CO2 content caused
by the injection of H218O is not shown.

Since then studies by many research groups explored two sites for
the ‘bicarbonate effects’ within PSII. The role and binding site for HCO3–
on the electron acceptor side of PSII is known (for review, see (Van
Rensen, 2002; Guskov et al., 2010)). Here HCO3– is known to bind to the
non-heme iron (Fe2+ in Figure 4.1) and is thought to facilitate the
reduction and protonation of the secondary plastoquinone electron
acceptor (QB) (reviewed in (Govindjee and Van Rensen, 1993; Govindjee
et al., 1997; Van Rensen et al., 1999; Van Rensen, 2002)). In contrast, the
site of interaction of HCO3– on the donor side is not obvious (Figure
4.2.).

48
Figure 4.2. Schematic presentation of PSII in high plants and green algae
(only core proteins are shown) and two sites (donor and acceptor) where
HCO3- has effect. Arrows shows the pathway of electron flow through
PSII. Reproduced from Shevela (2012)

The view that bicarbonate also has an effect on the donor side of PS
II arose initially from a hypothesis made by Stemler (Stemler and
Govindjee, 1973; Stemler et al., 1974). He suggested that the water-
oxidizing side of PSII is a possible site for the HCO3 − effect. However,
the first reliable evidence that HCO3- has an effect on the donor side of
PSII was derived by Klimov et al. (Klimov et al., 1995; Klimov et al.,
1995). In the PS II structure from Thermosynechococcus elongatus
obtained at a resolution of 3.5 Å by Ferreira et al. (Ferreira et al., 2004)
HCO3- has been tentatively included to bind as a ligand to the Mn4CaO5
cluster. However, based on X-ray crystallography (Guskov et al., 2010;
Umena et al., 2011), FTIR spectroscopy (Aoyama et al., 2008), and mass
spectrometry (Shevela et al., 2008; Ulas et al., 2008) data, HCO3– does
not bind tightly at or near the Mn4CaO5 cluster.

At the same time, the possibility for a weakly bound, rapidly

exchanging HCO3– in the vicinity of the cluster cannot be excluded (Van
Rensen and Klimov, 2005). Thus, for instance, HCO3– was recently
proposed to act as a proton acceptor for the WOC (Ananyev et al., 2005),
(Karlsson et al., 1995) (Figure 4.2.).

49
 In this Ph.D. study the possible participation of HCO3– in the
proton removal from the water-oxidizing side of PSII is investigated in
higher plant preparations. For this we used two different techniques: (i)
isotope-ratio membrane-inlet mass spectrometry (MIMS) in
combination with 18O-labelling (Beckmann et al., 2009; Shevela and
Messinger, 2013), and (ii) Joliot-type electrode for measuring flash-
induced oxygen evolution patterns (FIOPs) (Renger and Hanssum,
2009). These experiments were performed both at ambient and 20-times
reduced levels of Ci. To achieve and maintain such low levels of Ci in both
gas phase around the electrode and in the aqueous phase around the
investigated sample, the FIOPs measurements were done inside of a
glove-box filled with N2 and in Ci-depleted buffers (Figure 4.3.). Unlike to
all previous reports, in the present FIOPs study we fully control the levels
of the residual Ci upon CO2/HCO3−-depletion procedure. Thus, the
concentration of CO2 in gas phase above the sample was continuously
monitored with an infrared CO2 analyzer, while the Ci levels in aqueous
phase (sample medium) were detected by MIMS.

Figure 4.3. Picture of the experimental set-up that was used in this study
for the measurements of FIOPs under various levels of HCO3–/CO2. The
Joliot electrode with the Xe-flash lamp used for the FIOPs measurements

50
as well as mini-centrifuge used for sample washings were places inside
the glove box “Belle”.

From FIOPs important information about the turnover efficiency

of PSII can be obtained. The increased magnitude of the α-values at Ci-
low conditions can be either due to the changes of all redox equilibria
and kinetics of the donor or the acceptor side of PSII (i.e., redox-
potential differences between QB/QB-/QB2-, P680/P680+•, YZ/YZox and the
Si states of the WOC) (Renger and Hanssum, 1988; Shinkarev and
Wraight, 1993) or due to the slower reduction of P680+• by YZ in Ci-
depleted samples (Christen et al., 1999). While the misses may reflect the
properties of both the donor and the acceptor side of PSII, the double
hits reflect the properties of only acceptor side and are related linearly to
the rate of electron transfer from QA–• to QB/QB–• (Messinger et al., 1993).
Comparison of misses with double hits would give information in
interpretation of the “bicarbonate effect” and represent the origin of PSII
side effect.

In case of MIMS measurements present proposals for the action of

free or weakly bound HCO3– in photosynthetic water-splitting should
result in a light-induced release of CO2 concomitant with O2 evolution. If
HCO3- is a base for protons produced by photosynthetic water oxidation
(Ananyev et al., 2005; Shutova et al., 2008), CO2 should be formed as a
result of H2CO3 formation and its disproportionation into H2O and CO2:


 H2CO3 
HCO3  H  
 
CO2  H2O
 (4.3.)

In Papers II and III it is shown that HCO3- is needed for efficient

turnover of the WOC. The data demonstrate that the lack of HCO3− leads
to a reduced oxygen evolution activity of PSII, and increase overall miss
factor under HCO3- depletion (Shevela et al., 2013). In Paper III it is
shown that HCO3− acts as mobile proton acceptor of PSII that leads to
light induced CO2 evolution.

51
5. Water splitting in artificial
photosynthetic systems.
Increased global attention to the effects of releasing carbon dioxide
into the atmosphere by burning fossil fuels has led to investigations of
alternative fuel sources. One of the most promising approaches is
artificial photosynthesis. In this process, solar energy is collected and
delivered to a water oxidizing catalyst that splits water into dioxygen,
protons, and electrons. The extracted electrons can be used for the
reduction of protons to form dihydrogen, or may be used to reduce either
organic molecules or CO2 to products that can serve as fuels (Youngblood
et al., 2009). The development of water oxidation catalysts from
inexpensive and abundant elements would make artificial photosynthetic
systems highly affordable and attractive for large scale applications of
solar fuel devices.

For a better understanding of the water splitting reaction and the

mechanism of O-O bond formation it is a common approach to use
transition metal complexes and transition metal oxides (Kraatz and
Metzler-Nolte, 2006) as models to mimic the WOC in PSII (Barber and
Tran, 2013). However, is has been difficult to develop systems that
imitate closely the complex arrangement of the Mn4CaO5 cluster.
Therefore, a general strategy is the development of simplified structural
models (Meyer, 2008; Cook and Borovik, 2013). In addition, such
complexes are tested for their catalytic efficiency and theirby their
potential to be used in artificial photosynthesis devices (Figure 5.1.)

52
Figure 5.1. Possible O-O bond formation mechanisms. Nucleophilic
attack (red boxes), oxo/oxyl radical coupling (blue boxes). Reproduced
from Cox (2013)

In order to split water with ‘naked’ synthetic catalysts, sacrificial

oxidants are used that have a high-enough redox potential to oxidize the
catalysts into the required oxidation states. Alternatively, the catalysts
can be oxidized electrochemically or photoelectrochemically if attached
for example to a semiconductor surface. The two most commonly used
non-oxygen transferring, single-electron oxidation agents are CeIV and
[Ru(bipy)3]3+. They can be used in experiments to screen for “real” water
oxidation catalysts (equations 5.1., 5.2., 5.3.) (Xu et al., 2010; Shevela et
al., 2011). CeIV and [Ru(bipy)3]3+ providing redox potentials of 1,7 V vs
NHE (Hayes et al., 2002) and 1,3 V vs NHE (Harriman et al., 1981),
respectively.

4Ce IV  2 H 2O 
Catalyst
4Ce III  O2  4 H  (5.1.)

3
4  Ru(bpy)3   2 H2O 
Catalyst

2
(5.2.)

4  Ru(bpy)3   O2  4 H

53
In case of photochemically generated [Ru(bipy)3]3+

2 h 2 *
 Ru(bpy)3  
  400 nm
  Ru(bpy)3 
2* 3
 Ru(bpy)3   S2O82   Ru(bpy)3   SO42  SO4
2* 3
 Ru(bpy)3   SO4   Ru(bpy)3   SO42
(5.3)
or
2*
 Ru(bpy)3   Co III ( NH 3 )5 Cl  
3
 Ru(bpy)3   Co II (aq)  5 NH 3  Cl 

5.1. Manganese oxides

Dinuclear manganese compounds have been intensively used as a

model systems in order to understand water splitting reaction and
development of artificial catalysts (Yagi and Kaneko, 2001;
Mukhopadhyay et al., 2004; Cady et al., 2008; Mullins and Pecoraro,
2008; Collomb and Deronzier, 2009; Wiechen et al., 2012). All of them
have either a [Mn2(µ-O)] or a [Mn2(µ-O)2] core. The Mn-oxidation states
vary, and different combination of MnII, MnIII and MnIV have been
reported. It was shown that dinuclear Mn complexes in lower oxidation
states can be oxidized in single electron steps that increase the oxidation
state of one manganese center form MnIII to MnIV (Huang et al., 2002;
Huang et al., 2004; Kurz et al., 2008; Berends et al., 2012).

However, no dinuclear manganese complexes showed water

splitting activity in reaction with CeIV and [Ru(bipy)3]3+, but oxygen
evolution was observed with H2O2 and HSO5- (potassium
peroxymonosulfate, also known as oxone). Our 18O labeling experiment
in combination with MIMS provided information that neither H2O2 nor
HSO5- can act as oxidizing agents to drive ‘true’ water oxidation (see
Paper IV). The isotope labelling observed rather provides evidence that
the tested of Mn2-complexes can only catalyze oxygen formation with
these oxidants via oxygen transfer reactions. This also calls in question
an earlier study (Limburg et al., 1999) in which water oxidation by
another dimeric manganese complexes was reported to occur after HSO5-
addition.

54
 Present experiments on PSII and artificial catalysts indicate water-
oxidation chemistry involves a cycling between the oxidation states MnIII
and MnIV. It is therefore important to increase manganese nuclearity of
the compounds form dinuclear to tetranuclear complexes in order to
improve their efficiency. This is needed because four electrons must be
subtracted from water to allow O2 formation.

The groups of Kaneko (Ramaraj et al., 1987) and Yagi (Yagi and
Narita, 2004; Yagi et al., 2007) reported that the attachment of dinuclear
Mn complexes to the surface of the clay kaolin leads to the formation of
active water-oxidation catalyst. Subsequent catalytic studies (Yagi et al.,
2007) and EPR spectroscopy investigations (Li et al., 2009; Berends et
al., 2011) showed that the catalytic units formed at the clay surface
consist of more than two manganese ions, and that they have an
oxidation state of III or higher. X-ray crystallography of the attached
complexes demonstrated that Mn4-species are formed at the surface by
dimerization of two Mn2-units via µ-oxido ligands. Similarly, a recent
study found water oxidation for dimerized dinuclear manganese
complexes, supporting again that four manganese ions are needed for
efficient water oxidation at low overpotentials (Karlsson et al., 2011).
Attachment of monomeric MnII to the surface of the same clay does not
lead to catalytic activity (Berends et al., 2011).

It appears that a naturally occurring mineral discovered in

Morocco in 1963, CaMn2O4 which is nowadays known as marokite,
(Gaudefroy et al., 1963) is a very interesting model for the Mn4CaO5
cluster of PSII. It was concluded that the oxidation state of manganese is
+III and that marokite can be successfully synthesizes in laboratory
(Najafpour et al., 2010). The O2 formation pathways by CaMn2O4 and α-
Mn2O3 have been investigated in this Ph.D. project. In Paper IV it is
shown that entirely different pathways of dioxygen formation catalysis
exist for reactions involving different oxidants

5.2. Cobalt oxides

Another promising candidate for large scale water splitting are Co-
based water-oxidation catalysts. The potential of Co to support water
oxidation is known for a long time (Noyes and Deahl, 1937; El Wakkad
and Hickling, 1950; Baxendale and Wells, 1957; Anbar and Pecht, 1967;
Shafirovich and Strelets, 1978). In 2008 the photo-electrochemically
oxidation of water by cobalt oxide films was reinvestigated by Nocera and

55
co-workers (Kanan and Nocera, 2008). This study has attracted much
interest due to ability of the Co-oxide catalyst to split water at neutral pH
under ambient conditions. The catalysts also self-assembled from
relatively low cost materials (Surendranath et al., 2009). Further studies
showed: (i) water splitting occurs from the CoIV oxidation state (McAlpin
et al., 2010; McAlpin et al., 2011) (ii) cobalt catalyst self-heal (Lutterman
et al., 2009) (iii) does not require deionized water and even works in
brine and river water (Esswein et al., 2011) suggesting that for example
chloride ions do not inhibit oxygen evolution.

XAS measurements (Risch et al., 2009; Kanan et al., 2010) and

theoretical calculation (Hu et al., 2012; Li and Siegbahn, 2013; Mattioli et
al., 2013) revealed that the Co-catalyst structure forms by several CoIIIO6
octahedra units in incomplete and complete cubane blocks and that they
have structural similarity to the Mn4CaO5-cluster in PSII.

Despite on all successful studies and constant progress there is still

no agreement whether the O-O bond formation occurs at Co-oxides via
nucleophilic attack or by direct coupling, and whether for example
bridging oxygens are involved as substrate. Using time resolved 18O-
labelling MIMS and by employing the previously introduced
nanoparticular Co oxides (Co/M2P system) (Shevchenko et al., 2011) it is
shown in Paper V that water oxidation on amorphous Co-oxides occurs
via direct coupling of two pre-bound terminal water derived ligands.

56
6. Summary
The demonstration of light-induced CO2 formation by PSII and the
dependence of CO2 evolution on the concentrations of Ci and added
buffer in the medium demonstrate conclusively that HCO3− acts as
mobile proton acceptor of PSII and that HCO3- is needed for efficient
turnover of the WOC (see Papers II and III). The data demonstrate that
the lack of HCO3− leads to a reduced oxygen evolution activity of PSII,
and that the light-induced Si state transitions of the Kok cycle occur with
an increased overall miss factor under HCO3- depletion (Shevela et al.,
2013). Since strictly alternating electron and proton removals from the
WOC appear to be an inherent and crucial part of the mechanism of
water oxidation in PSII (Siegbahn, 2009; Klauss et al., 2012), it appears
very likely that this function of HCO3− developed early during evolution
and can be found in all O2 evolving organisms (Dismukes et al., 2001).
Paper III thus establishes the long sought mechanism for the CO2/HCO3−
dependence of O2 production by PSII. This finding adds a new
component to the understanding of PSII and to the regulatory networks
of photosynthesis in higher plants.

Illumination of PSII crystals and suspensions with the newly

developed illumination setup revealed that the illuminated samples cycle
through all S states with acceptable miss probability (Messinger and
Renger, 2008). A clear maximum of O2 release after the 3rd flash was
achieved by optimization of all parameters. This result enabled the
collection of XES and XRD data of PSII at room temperature using the
new approach of femto second serial X-ray crystallography combined
with X-ray emission spectroscopy at the X-ray free-electron laser at
LCLS. So far data were recoded from both the dark state (S1) and the first
illuminated state (S2) (Kern et al., 2013). With the resolution obtained so
far no indication of radiation damage could be found. Thus, using the
developed illumination conditions further measurements will be
performed that allow to characterize all other intermediates, including
the kinetically unstable S3YZ•, and possibly the S4 state, in which the O-O
bond formation and the evolution of molecular oxygen occurs.

The presented 18O-labelling results on calcium manganese-oxides

on oxygen formation show that such reactions can be grouped into three
categories:

57
1. catalase-type reactions involving hydrogen peroxide, in which the
bulk water is not involved in O2 formation, in agreement with (Haber
and Weiss, 1934; Kremer and Stein, 1959)
2. oxygen-transfer reactions involving oxone, in which one oxygen
atoms from the bulk water participates in the formation of dioxygen
3. water oxidation, in which both oxygen atoms of the O2 product carry
the 18O label signature, and for which the use of non-oxygen-
transferring oxidation agents is necessary.

Employing the previously introduced Co/M2P system (Shevchenko

et al., 2011) in combination with the chemical oxidant ([Ru(bipy)3]3+)
and time resolved 18O-labelling membrane-inlet mass spectrometry it is
demonstrated in Paper V that the O-O bond formation in these
amorphous Co-oxide catalysts occurs between two pre-bound, fast
exchanging oxygen species, i.e. via direct coupling between two terminal
water/hydroxide ligands. It is also shown that 2.35 Co form one catalytic
site. Since it was previously demonstrated that in the resting state Co has
an oxidation state of at least CoIII, (Kanan et al., 2010; McAlpin et al.,
2010; Artero et al., 2011; Nocera, 2012) we conclude that the direct
coupling mechanism involves at least one oxygen radical, in agreement
with recent theoretical studies (Li and Siegbahn, 2013; Mattioli et al.,
2013).

58
Acknowledgements

В первую очередь я хочу поблагодарить всех родных и близких, в

особенности родителей - маму и папу, а также любимую жену Таню
за их любовь, терпение, понимание и постоянную поддержку.

I also would like to express my special appreciation and thanks:

To my adviser Prof. Dr. Johannes Messinger, you have been a

tremendous mentor for me. I would like to thank you for constant
stimulation, useful discussion, patience and for allowing me to grow as a
research scientist.

To my friend Dr. Dmitry Shevela, for inestimable help at the

beginning of my Ph.D., grate discussions in different fields, bringing out
laughter, no matter what and of course for preparation of the figures for
my thesis.

To my lab-mats, for cozy atmosphere and enthusiasm in the laboratory

To all Members of Yachandra/Yano lab for nice environment and

care during my stay in Berkeley, opportunity to be involved into amazing
project and the fruitful collaboration.

To my friends for unforgettable time. I am lucky that I know all of you.

Thank you All !!!

59
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