This thesis describes the development and study of catalysts for redox reactions, which either utilize oxygen or hydrogen peroxide for the purpose of selectively oxidizing organic substrates, or produce oxygen as the necessary byproduct in the production of hydrogen by artificial photosynthesis. The first chapter gives a general introduction about the use of environmentally friendly oxidants in the field of organic synthesis, and about the field of artificial photosynthesis. The second chapter describes a computational study of the mechanism of palladium-catalyzed oxidative carbohydroxylation of allene-substituted conjugated dienes. The proposed mechanism, which was supported by DFT calculations, involves an unusual water attack on a (π-allyl)palladium complex. The third chapter describes a computational study of the oxidation of unfunctionalized hydrocarbons, ethers and alcohols with hydrogen peroxide, catalyzed by methyltrioxorhenium (MTO). The mechanism was found to proceed via rate-limiting hydride abstraction followed by hydroxide transfer in a single concerted, but highly asynchronous, step as shown by intrinsic reaction coordinate (IRC) scans. The fourth chapter describes the use of a new hybrid (hydroquinone-Schiff base)cobalt catalyst as electron transfer mediator (ETM) in the palladium-catalyzed aerobic carbocyclization of enallenes. Covalently linking the two ETMs gave a fivefold rate increase compared to the use of separate components. The fifth chapter describes an improved synthetic route to the (hydroquinone-Schiff base)cobalt catalysts. Preparation of the key intermediate 5-(2,5-hydroxyphenyl)salicylaldehyde was improved by optimization of the key Suzuki coupling and change of protecting groups from methyl ethers to easily cleaved THP groups. The catalysts could thus be prepared in good overall yield from inexpensive starting materials. Finally, the sixth chapter describes the preparation and study of two catalysts for water oxidation, both based on ligands containing imidazole groups, analogous to the histidine residues present in the oxygen evolving complex (OEC) and in many other metalloenzymes. The first, ruthenium-based, catalyst was found to catalyze highly efficient water oxidation induced by visible light. The second catalyst is, to the best of our knowledge, the first homogeneous manganese complex to catalyze light-driven water oxidation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Accepted. Paper 6: Submitted.</p>

Life on Earth is dependent on the oxygen produced through photosynthesis. The thylakoid membrane is the site for the light-driven reactions of photosynthesis, which oxidize water and supply energy in the form of ATP, mainly for carbon fixation. The utilization of ATP in the lumenal space of the thylakoid has not been considered in the past. In the latest years, increasing evidence for nucleotide metabolism in the thylakoid lumen of plant chloroplasts has been presented; ATP transport across the thylakoid membrane, and GTP binding to the PsbO extrinsic subunit of the water-oxidizing photosystem II (PSII) complex. In this thesis, various methods for prediction, identification, and characterization of novel plant proteins, are described. Nucleotide-binding motifs and nucleotide-dependent processes are reviewed, and the experimental data is discussed. 1) A thylakoid ATP/ADP carrier (TAAC) in Arabidopsis thaliana was identified and functionally characterized, and 2) the spinach PsbO protein was characterized as a GTPase. The Arabidopsis At5g01500 gene product is predicted as a chloroplast protein and to be homologous to the well-studied mitochondrial ADP/ATP carrier. The putative chloroplast localization was confirmed by transient expression of a TAAC-green fluorescent protein fusion construct. Immuno detection with peptide-targeted antibodies and immunogold electron microscopy showed the thylakoid as the main localization of TAAC, with a minor fraction in the chloroplast envelope. TAAC is readily expressed in etiolated seedlings, and its level remains stable throughout the greening process. Its expression is highest in developing green tissues and in leaves undergoing senescence or abiotic stress. It is proposed that the TAAC protein supplies ATP for energy-dependent reactions during thylakoid biogenesis and turnover. Recombinant TAAC protein was functionally integrated in the cytoplasmic membrane of Escherichia coli, and was shown to specifically transport ATP/ADP in a protonophore-sensitive manner, as also reported for mitochondrial AACs. The PsbO protein stabilizes the oxygen-evolving complex of PSII and is ubiquitous in all oxygenic photosynthetic organisms, including cyanobacteria, green algae, and plants. So far only the 3D-structure of the cyanobacterial PsbO is available. Four GTP-binding motifs in the primary structure of spinach PsbO were predicted from comparison with classic GTP-binding proteins. These motifs were only found in the plant PsbOs, in the -barrel domain of the homologous 3D-structure. Using circular dichroism and intrinsic fluorescence spectroscopy, it was shown that MgGTP induces specific structural changes in the PsbO protein. Spinach PsbO has a low intrinsic GTPase activity, which is considerably stimulated when associated with a dimeric PSII complex. GTP stimulates the dissociation of PsbO from PSII under both inhibitory and non-inhibitory light conditions. A role for PsbO as a GTPase in the function of the oxygen-evolving complex and PSII repair is proposed.

No description available.

In the society of today the need for alternative energy sources is increasing. The construction of artificial devices for the conversion of sunlight into electricity or fuel seems very attractive from an environmental point of view, since these devices are based on processes that does not necessarily generate any harmful biproducts. In the oxygen evolving photosynthetic process highly efficient energy and electron transfer reactions are responsible for the conversion of the sunlight into chemically stored energy and if the same principles can be used in an artificial device, the only electron supply required, is water. This thesis describes energy and electron transfer reactions in supramolecular complexes where the reactions are intended to mimic the basic steps in the photosynthetic process. All complexes are based on ruthenium(II)-trisbipyridine as photosensitizer, that is covalently linked to electron donors or electron or energy acceptors. The photochemical reactions were studied with time resolved transient absorption and emission measurements. In the complexes that mimic the donor side of Photosystem II, where a manganese cluster together with tyrosine catalyses the oxidation of water, intramolecular electron transfer was found to occur from Mn(II) or tyrosine to photo-oxidized Ru(III). Studies of a series of Ru(II)-Mn(II) complexes gave information of the quenching of the Ru(II) excited state by the coordinated Mn(II), which is important for the development of multi-nuclear Ru(II)-Mn complexes. In the supramolecular triad, PTZ-Ru2+-Q, the charge separated state, PTZ+●-Ru2+-Q-●, was rapidly formed, and further development where a second electron acceptor is linked to quinone is planned. Ultra fast energy transfer τ&lt;200 fs), was obtained between ruthenium(II) and osmium(II) in a small artificial antenna fragment. Fast and efficient energy transfer is important in larger antennas or photonic wires where a rapid energy transfer is desired over a large distance.

In today’s society there is an increasing need for energy, an increase which for the most part is supplied by the use of fossil fuels. Fossil fuel resources are limited and their use has harmful effects on the environment, therefore the development of technologies that produce clean energy sources is very appealing. Natural photosynthesis is capable of converting solar energy into chemical energy through a series of efficient energy and electron transfer reactions with water as the only electron source. Thus, constructing an artificial system that uses the same principles to convert sunlight into electricity or storable fuels like hydrogen is one of the major forces driving artificial photosynthesis research. This thesis describes supramolecular complexes with the intention of mimicking the electron transfer reactions of the donor side in Photosystem II, where a manganese cluster together with a tyrosine catalyses the oxidation of water. All complexes are based on Ru(II)-trisbipyridine as a photosensitizer that is covalently linked to electron donors like tyrosine or manganese. Photochemical reactions are studied with time-resolved transient absorption and emission measurements. Electrochemical techniques are used to study the electrochemical behavior, and different photoelectrochemical techniques are used to investigate the complexes adsorbed onto titanium dioxide surfaces. In all complexes, intramolecular electron transfer occurs from the linked donor to photo-oxidized Ru(III). It is also observed that coordinated Mn(II) quenches the excited state of Ru(II), a reaction that is found to be distance dependent. However, by modifying one of the complexes, its excited state properties can be tuned in a way that decreases the quenching and keeps the electron transfer properties. The obtained results are of significance for the development of multinuclear Ru-Mn complexes that are capable of multi-electron transfer.

Oxygenic photosynthesis is the major producer of both oxygen and organic compounds on earth and takes place in plants, green algae and cyanobacteria. The thylakoid membranes are the site of the photosynthetic light reactions that involve the concerted action of four major protein complexes known as photosystem II (PSII), cytochrome b6f complex, ATP synthase and photosystem I (PSI). The function of PSII is of particular interest as it performs the light–driven water splitting reaction driving the photosynthetic electron transport. My thesis addressed different aspects of PSII assembly and the functions of its low molecular weight PSII subunits PsbX and PsbW. Photosynthesis in green algae and higher plants is controlled by the nucleus. Many proteins of nuclear origin participate in the regulation of the efficient assembly of the photosynthetic protein complexes. In this investigation we have identified one of these nuclear encoded auxiliary proteins of photosystem II, REP27, which participates in the assembly of the D1 reaction center protein and repair of photodamaged PSII in the green algae Chlamydomonas reinhardtii. Interestingly, PSII is specially enriched in Low Molecular Weight (LMW) subunits that have masses less than 10kDa. These proteins account for more than the half of the PSII subunits. Several questions remains poorly understood regarding the LMW: Which is their evolutionary origin? What function do they perform in the protein complex? Where are they located in the protein structure? In this investigation the functions of two of these LMW subunits (PsbX and PsbW) have been studied using antisense inhibition and T-DNA knockout mutant plants in Arabidopsis thaliana. Deficiency of the PsbX protein leads to impaired accumulation and functionality of PSII. Characterization of PsbW knock-out plants show that PsbW participates in stabilization of the macro-organization of PSII and the peripheral antenna (Light Harvesting Complex, LHCII) in the grana stacks of the chloroplast, also known as PSII-LHCII supercomplexes.

The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts.

[FeFe]-Hydrogenases (H2ases) are metalloenzymes that can catalyze the reversible reduction of protons to molecular hydrogen as part of the metabolism of certain cyanobacteria and green algae. Due to the low availability of the enzyme, synthetic complexes that mimic the natural active site in structure, function and activity are highly sought after. In this thesis, a number of [FeFe]-H2ases active site model complexes were synthesized to answer open questions of the active site and to develop unprecedented bio-inspired proton reduction catalysts. The first part describes the synthesis and the protonation properties of a [Fe2(μ-adt)(CO)4(PMe3)2] (adt = azadithiolate) complex which contains two basic sites that are similar to those found in the enzyme active site. Unusual kinetic factors give rise to four discrete protonation states. The twofold protonated state is the first model complex that simultaneously carries a proton at the azadithiolate nitrogen and a bridging hydride at the Fe-Fe bond. In the second part, a model complex with an unprecedented amine ligand was synthesized and studied. In analogy to the enzyme active site, the labile amine ligand is expelled after electrochemical reduction. The third part describes a series of model complexes with electronically different aromatic dithiolate ligands. It is demonstrated in one case that the tuning of the ligand by electron-withdrawing substituents results in proton reduction catalysis at an overpotential that is lower than that required by the non-substituted parent compound. The design and the synthetic work towards a new ruthenium-diiron dyad for light-driven hydrogen production are presented in the fourth part. In the final part, differently isotope-labelled mixed valent Fe(I)-Fe(II) model complexes were synthesized, in particular the unprecedented 15N labelled analogue, with the aim to provide EPR-spectroscopic references that will allow the elucidation of the nature of the central atom in the dithiolate bridge of the [FeFe] hydrogenase active site.

Oxidative stress is a natural consequence of photosynthetic oxygen evolution and redox enzyme processes. Trp oxidation to N-formylkynurenine (NFK) is a specific, reactive oxygen species (ROS)-mediated reaction. This thesis work describes the identification and functional characterization of NFK in oxygen evolving Photosystem II (PSII). Although proteomics studies have confirmed NFK modifications in many types of proteins, limited knowledge on the biochemical significance exists. In vitro studies in thylakoids and PSII membranes were used to establish a correlation between oxidative stress, NFK formation, and photoinhibition. The in vivo effect of preventing Trp oxidation to NFK was assessed by site-directed mutation in the cyanobacteria Synechocystis sp. PCC 6803. This work provides insight into the role of NFK in photosynthetic oxygen evolution and photoinhibition. Based on the current knowledge of NFK, ROS, and repair, a new model is described. In this modified model for photoinhibition and repair, NFK plays a role in signaling for turnover of damaged proteins. NFK may play a similar role in replacement of damaged proteins in other systems.

Photosynthetic electron transport produces ATP and NADPH which support carbon fixation by the Calvin Cycle. To avoid over-reduction of the electron transport chain, plants must balance absorption and consumption of light energy. Mitochondrial alternative oxidase (AOX) is a non-energy-conserving electron sink, making it an ideal candidate to oxidize excess reductant and regulate chloroplastic redox state. Wild-type (WT) and transgenic Nicotiana tabacum lines with enhanced or suppressed AOX protein levels were grown under low light (LL) and moderate light (ML). LL-grown plants were also shifted to ML. AOX transcript and protein levels were enhanced in WT plants under ML. Chlorophyll fluorescence, gas exchange, and contents of chlorophyll, carbohydrate, and malondialdehyde were measured. Lack of AOX protein decreased Photosystem II (PSII) quantum efficiency and CO2 assimilation rates while enhancing PSII excitation pressure compared to WT. These findings suggest a role for AOX in mediating the chloroplast-mitochondrion relationship during acclimation to higher irradiance.