abstract: The work described in the thesis involves the synthesis of a molecular triad which is designed to undergo proton coupled electron transfer (PCET) upon irradiation with light. Photoinduced PCET is an important process that many organisms use and the elucidation of its mechanism will allow further understanding of this process and its potential applications. The target compound designed for PCET studies consists of a porphyrin chromophore (also a primary electron donor), covalently linked to a phenol-imidazole (secondary electron donor), and a C60 (primary electron acceptor). The phenol-imidazole moiety of this system is modeled after the TyrZ His-190 residues in the reaction center of Photosystem II (PS II). These residues participate in an intermolecular H-bond between the phenol side chain of TyrZ and the imidazole side chain of His-190. The phenol side chain of TyrZ is the electron transfer mediator between the oxygen evolving complex (OEC) and P680 (primary electron donor) in PSII. During electron transfer from TyrZ to P680*+, the phenolic proton of TyrZ becomes highly acidic (pKa~-2) and the hydrogen is preferentially transferred to the relatively basic imidazole of His-190 through a pre-existing hydrogen bond. This PCET process avoids a charged intermediate, on TyrZ, and results in a neutral phenolic radical (TyrZ*). The current research consists of building a molecular triad, which can mimic the photoinduced PCET process of PSII. The following, documents the synthetic progress in the synthesis of a molecular triad designed to investigate the mechanism of PCET as well as gain further insight on how this process can be applied in artificial photosynthetic devices. / Dissertation/Thesis / M.S. Chemistry 2011

The PsbQ protein (16.5 kDa) is an extrinsic protein found in the thylakoid membrane of higher plants and green algae. As a member of the Psb protein family, it is situated in the oxygen evolving center and takes part in the water splitting reaction. The stable oxygen production in photosystem II depends on the cooperation of PsbQ with other photosynthetic proteins, mainly PsbP. In order to identify the possible interaction sites, the tertiary structure in solution has to be determined. Although the X-ray crystallographic structure of PsbQ was determined previously, the conformation of residues 14-33 (so-called "missing link") was still unknown at the onset of this work. The initial backbone assignment as well as a secondary structure estimation were achieved recently. In this thesis the resonance assignment was extended and 15N as well as 13C NOESY-HSQC spectra were recorded to obtain structural constraints. The solution structure was determined using the program CYANA. The results obtained show that, while the four helix bundle domain is nearly identical compared to the available X-Ray crystallographic structure significant deviations occur in the N-terminal region. In particular, the residues 37-41, where a short ?-strand had been proposed in the crystal structure, exhibit high ?-helical propensity.

abstract: There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo. / Dissertation/Thesis / Ph.D. Biochemistry 2013

The photosynthetic process has the unique ability to capture energy from sunlight and accumulate that energy in sugars and starch. This thesis deals with the light driven part of photosynthesis. The aim has been to investigate how the light-absorbing protein complexes Photosystem I (PS I) and Photosystem II (PS II), react upon illumination of light with lower energy (far-red light; 700-850 nm) than the absorption peak at respective primary donor, P700 and P680.  The results were unexpected. At 295 K, we showed that both PS I and PS II were able to perform photochemistry with light up to 130 nm above its respective primary donor absorption maxima. As such, it was found that the primary donors’ action spectra extended approximately 80 nm further out into the red-region of the spectrum than previously reported.  The ability to perform photochemistry with far-red light was conserved at cryogenic temperatures (< 77 K) in both photosystems. By performing EPR measurements on various photosystem preparations, under different illumination conditions the origin of the effect was localized to their respective reaction center. It is also likely that underlying mechanism is analogous for PS I and PS II, given the similarities in spatial coordination of the reaction center pigments. For PS II, the results obtained allowed us to suggest a model involving a previously unknown electron transfer pathway. This model is based upon the conclusion that the primary cation from primary charge separation induced by far-red light resides primarily on ChlD1 in P680. This is in contrast to the cation being located on PD1, as has been suggested as for visible light illumination. The property to drive photochemistry with far-red wavelengths implies a hither to unknown absorption band, probably originating from the pigments that compose P700 and P680. The results presented here might clarify how the pigments inside P680 are coupled and also how the complex charge separation processes within the first picoseconds that initiate photosynthetic reactions occur.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.

With a surge in future demand for hydrogen as a renewable fuel, the specific aim of this study was to develop a novel strategy in photosynthetic hydrogen production from green algae, which is one of the cleanest processes among existing hydrogen-production methodologies currently being explored. The novel strategy designed was a spectral-selective PSI-activation/PSII-deactivation protocol that would work to maintain a steady flow of electrons in the electron transport system in the light-dependent part of photosynthesis for delivery of electrons to hydrogenase for photo-hydrogen production. The strategy would work to activate PSI to assist in driving the electron flow, while partially deactivating PSII to a degree that it would still supply electrons, but would limit its photosynthetic oxygen production below the respiratory oxygen consumption so that an anoxic condition would be maintained as required by hydrogenase. This study successfully showed that the implementation of the spectral-selective PSIactivation/ PSII-deactivation strategy resulted in actual and relatively sustained photohydrogen production in Chlamydomonas reinhardtii cells, which had been dark-adapted for three hours immediately prior to exposure to a PSI-spectral selective radiation, which had a spectral peak at 692 nm, covering a narrow waveband of 681-701 nm, and was applied at 15 W m⁻². The optimal condition for the PSI-spectral-selective radiation (692 nm) corresponded with low cell density of 20 mg chlorophyll L⁻¹ ("chl" henceforth) with cells grown at 25⁰C. At this condition, the PSI-spectral-selective radiation induced the maximal initial hydrogen production rate of 0.055 mL H² mg⁻¹ chl h⁻¹ which statistically the same as that achieved under white light of 0.044 mL H² mg⁻¹ chl h⁻¹, a maximal total hydrogen production of 0.108 mL H² mg⁻¹ chl which significantly exceeded that under white light of 0.066 mL H² mg⁻¹ chl, and a maximal gross radiant energy conversion efficiency for hydrogen production of 0.515 μL H² mg⁻¹ chl L⁻¹ that statistically matched that under white light of 0.395 μL H² mg⁻¹ chl L⁻¹. The study also successfully demonstrated the reversibility feature of the novel strategy, allowing for the cells to alternately engage in photo-hydrogen production and to recover by simply switching on or off the PSI-spectral-selective radiation.

Little is known of the physiological and biochemical mechanisms underlying competitive interactions between alien invasive grasses and native taxa, and how these are affected by resource supply. Consequently, this study compared photosystem II (PS II) function, photosynthetic gas and water exchange, enzyme and pigment concentrations, flowering and biomass accumulation in an indigenous geophyte, Tritonia crocata (L.) Ker. Gawl., grown in monoculture and admixed with the alien grass, Lolium multiflorum Lam., at different levels of water and nutrient supply. Diminished stomatal conductances were the primary cause of reduced net C02 assimilation rates, and consequent biomass accumulation in T. crocata admixed with L. multiflorum at all levels of water and nutrient supply with one exception. These corresponded with decreased soil water contents induced presumably by more efficient competition for water by L. multiflorum, whose biomass was inversely correlated with soil water content. Biochemical impairments to photosynthesis were also apparent in T. crocata admixed with L. multiflorum at low levels of water and nutrient supply. These included a decline in the density of working photosystems (reaction center per chlorophyll RC/ABS), which corresponded with a decreased leaf chlorophyll a content and a decreased efficiency of conversion of excitation energy to electron transport (¥0 / l-^o), pointing to a reduction in electron transport capacity beyond QA~, a decline in apparent carboxylation efficiency and Rubisco content. At low nutrient levels but high water supply, non-stomatal induced biochemical impairments to photosynthesis (decreased RC/ABS, chlorophyll a and Rubisco content) were apparent in T. crocata admixed with L. multiflorum. These attributed to a reallocation of fixed carbohydrate reserves to floral production which increased significantly in T. crocata under these conditions only and associated with a corresponding reduction in the mass of its underground storage organ (bulb). The results of this study did not support the hypothesis that under conditions of low water and low nutrient supply invasive annual grasses would have a lesser impact on the growth and physiology of native geophytes than under resource enriched conditions that favor growth of these grasses. Unresolved is whether resource limitation and allelopathic mechanisms functioned simultaneously in the inhibition of the native geophyte by the alien grass. / Thesis (M. Environmental Science (Ecological Remediation and Sustainable Utilisation))--North-West University, Potchefstroom Campus, 2008.

Photosystem II (PSII) is the primary generator of electrons required for photosynthesis. The reaction center protein of PSII (RCII) is the most susceptible component of the photosynthetic machinery to damage. Photodamage can lead to long-term downregulation of photosynthesis. This occurs because plants are exposed to rapid light fluctuations and high light conditions, leading to the over accumulation of excess energy around PSII. Plants have developed a mechanism to dissipate this excess energy called nonphotochemical quenching (NPQ). In order to quantify the protectiveness of NPQ (pNPQ), a novel methodology was developed and employed. During methodology development, development, it is shown that a variable PSI fluorescence should be taken into account, and how it can be calculated. Application of the procedure assessed the contribution of xanthophylls lutein, violaxanthin, zeaxanthin, and the PsbS protein to pNPQ. Results show that the most important factors governing photoprotection are the PsbS protein and the correct xanthophyll composition in their natural binding sites. The more xanthophyll variation, the greater the photodamage at the end of the pNPQ assessment procedure. PsbS is essential to achieve the maximum pNPQ. PsbS increases the aggregation of LHCII. Arabidopsis with excess PsbS has three-times more aggregated LHCII than wild type levels of PsbS. The phototolerance and pNPQ required for Arabidopsis grown under different conditions and for leaves of different ages was also calculated. Plants grown under low light conditions accumulate disconnect antenna (LHCII), which is inefficient at protecting RCII, despite the high NPQ levels. Investigating plants of different ages, it was found that eight-week old Arabidopsis are the optimum age for pNPQ effectiveness. Younger and older leaves suffer photodamage at lower light intensities and form less pNPQ. This thesis demonstrates the novelty and adaptability of the pNPQ assessment procedure, and offers a sound case for its use in acclimation and photoinhibition experiments.

The aim of this research was to increase the rate of cell division in algae by exploring the effect of combinations of lasers of various wavelengths. Literature search has identified a gap in knowledge of the potential for increase in efficiency of the electron transition between photosystem II and photosystem I. This through the use of several wavelengths of blue and or red lasers, including 405 nm, 450, and 473 nm, 635 nm, 650 nm, 680 nm, 685 nm and 700 nm to generate photons with energies more closely matching the absorption spectra of algae receptors known as pigments. This investigation underpins the realisation that photons emanating from a specific laser are absorbed by algae pigments because there is a much closer match between the emission spectrum of the laser and the absorption spectrum of the pigments within the photosystems of algae. This research examined all of the available laser wavelengths in particular combinations; the resultant data contributed to the assembly of a matrix that illustrates the most appropriate laser combinations that promote cell division within algae. Chlamydomonas reinhardtii algae cells successfully grew and divided under exposure to both the blue laser, red laser and that of white light LED when each was applied individually or combined in a sequence. The order of the sequence of using the red and blue lasers in specific cases was important. The pH was maintained between 6.9 and 7.7, with temperatures maintained between 19.00 and 25.00 ºC. For the blue lasers, the laboratory results were as follows, (irradiation time was 12 hours every time): • 405 nm blue laser produced 1.8 x cell division of the white light LED. • For 450 nm blue laser: the white light LED produced 1.5 x cell division of the blue laser 450 nm. • 473 nm blue laser produced 2 x cell division of the white light LED. • 405 nm blue laser produced 3.6 x cell division of natural day light. • 450 nm blue laser produced 1.4 x cell division of natural day light. • 473 nm blue laser produced 4 x cell division of natural day light. For the red lasers, the laboratory results were as follows, (irradiation time was 12 hours every time): • For 635 nm red laser: the white light LED produced 4 x cell division of the red laser 635 nm. • 650 nm red laser produced 1.96 x cell division of the white light LED. • 680 nm red laser produced 2.3 x cell division of the white light LED. • For 685 nm red laser: white light LED produced 1.22 x cell division of the red laser 685 nm. • 700 nm red laser produced 1.35 x cell division of the white light LED. • For 635 nm red laser: the natural day light produced 2 x cell division of the red laser 635 nm. • 650 nm red laser produced 3.9 x cell division of natural day light. • 680 nm red laser produced 4.6 x cell division of natural day light. • 685 nm red laser produced 1.6 x cell division of natural day light. • 700 nm red laser produced 2.7 x cell division of natural day light. For the combination of blue and red lasers, the laboratory results were as follows, (irradiation time was 12 hours every time): • First combination: 405 nm blue laser followed by a combination of 680 nm and 700 nm red lasers produced 4.86 x cell division of the white light LED. • Second combination: 473 nm blue laser followed by a combination of 680 nm and 700 nm red lasers produced 4.66 x cell division of the white light LED. • Third combination: a combination of 680 nm and 700 nm red lasers produced 4.43 x cell division of the white light LED.

<p>The solar energy accumulated by photosynthesis over billions of years is the sole source of energy available on Earth. Photosystem II (PSII) uses the sunlight to split water, an energetically unfavorable reaction where electrons and protons are extracted from water and oxygen is released as a by-product. Understanding this process is crucial for the future development of clean, renewable and unlimited energy sources, which can use sunlight to split water and produce hydrogen and electricity. In order to do so we need to understand how this is solved in plants.</p><p>I have been focusing on the role of two lumenal proteins associated with the thylakoid membrane PsbO and Cah3, in the water oxidation process. Convincing evidences have been presented supporting the hypothesis that bicarbonate acts as a proton acceptor in the water splitting process in PSII and the lumenal carbonic anhydrase, Cah3, supplies bicarbonate required for this function. The PsbO protein, an important constituent of the water-oxidizing complex, however, its function is still unknown. The PsbO protein undergoes a pH dependent conformational change that in turn influences its capacity to bind calcium and manganese, forming a catalytic Mn4Ca cluster in PSII. We propose that light-induced structural dynamics of the PsbO is of functional relevance for the regulation of proton release and for forming a proton sensing - proton transporting pathway. The cluster of conserved glutamic and aspartic acid residues in the PsbO protein acts as buffering antennae providing efficient acceptors of protons derived from substrate water molecules. Both proteins, Cah3 and PsbO have a conserved S-S bridge, required for proper folding and activity; therefore they are potential targets for red-ox regulation in lumen.</p> / <p>Solenergi som omvandlats av fotosyntesen under miljarder av år är basen för nästan all energi på jorden. Fotosystem 2 använder solljuset till att oxidera vatten, ur energisynpunkt en ofördelaktig process, där elektroner och protoner extraheras från vattenmolekyler vilket ger upphov till syrgas som biprodukt. Förståelsen av denna process är viktig för att vi i framtiden skall kunna utveckla rena och förnyelsebara energislag i obegrensad mängd. Genom att efterlikna fotosyntesprocessen skulle vi i framtiden kunna utvecka artificiella system som använder solljuset till att sönderdela vatten för att producera vätgas eller elektrisitet. För att kunna göra det så måste vi kunna förstå hur dessa processer fungerar i växterna.</p><p>Min forskning har fokuserat på att förstå funktionen hos två av de proteiner, PsbO och Cah3, som deltar i sönderdelningen av vatten. Jag har visat, för första gången, att ett lumen karboanhydras, Cah3, deltar i regleringen av den process där vatten spjälkas. Jag postulerar att Cah3 underlättar bort transporten av protoner från det vattenoxiderande komplexet genom att generera bikarbonat lokalt, som kan fungera som proton transportör. PsbO proteinet genomgår en pH beroende konformationsförändring vilket påverkar dess kapacitet and binda calcium och mangan som i sin tur formar ett katalytiskt Mn4Ca center i fotosystem 2. Jag föreslår att en ljusberoende strukturförändring av Psbo är av funktionell betydelse för regleringen av protonfrigörandet och formar ett proton-avkännande och proton-transporterande system. Ett kluster av konserverande glutamat- och aspartat-aminosyror i PsbO proteinet fungerar som ett buffrande nätverk för protoner som frigörs vid oxidering av vatten. Båda dessa proteiner innerhåller S-S bryggor ock kan därför vara red-ox reglerade i lumen.</p>