Dissertations / Theses on the topic 'Oxygen evolving complex'

Oxygenic photosynthesis in plants, algae and cyanobacteria converts sunlight into chemical energy. In this process electrons are transferred from water molecules to CO2 leading to the assembly of carbohydrates, the building blocks of life. A cluster of four manganese ions and one calcium ion, linked together by five oxygen bridges, constitutes the catalyst for water oxidation in photosystem II (Mn4CaO5 cluster). This cluster stores up to four oxidizing equivalents (S0,..,S4 states), which are then used in a concerted reaction to convert two substrate water molecules into molecular oxygen. The reaction mechanism of this four-electron four-proton reaction is not settled yet and several hypotheses have been put forward. The work presented in this thesis aims at clarifying several aspects of the water oxidation reaction by analyzing the mode of substrate water binding to the Mn4CaO5 cluster. Time-resolved membrane-inlet mass spectrometric detection of flash-induced O2 production after fast H218O labelling was employed to study the exchange rates between substrate waters bound to the Mn4CaO5 cluster and the surrounding bulk water. By employing this approach to dimeric photosystem II core complexes of the red alga Cyanidoschyzon merolae it was demonstrated that both substrate water molecules are already bound in the S2 state of the Mn4CaO5 cluster. This was confirmed with samples from the thermophilic cyanobacterium Thermosynechococcus elongatus. Addition of the water analogue ammonia, that is shown to bind to the Mn4CaO5 cluster by replacing the crystallographic water W1, did not significantly affect the exchange rates of the two substrate waters. Thus, these experiments exclude that W1 is a substrate water molecule. The mechanism of O-O bond formation was studied by characterizing the substrate exchange in the S3YZ● state. For this the half-life time of this transient state into S0 was extended from 1.1 ms to 45 ms by replacing the native cofactors Ca2+ and Cl- by Sr2+ and I-. The data show that both substrate waters exchange significantly slower in the S3YZ● state than in the S3 state. A detailed discussion of this finding lead to the conclusions that (i) the calcium ion in the Mn4CaO5 cluster is not a substrate binding site and (ii) O-O bond formation occurs via the direct coupling between two Mn-bound water-derived oxygens, which were assigned to be the terminal water/hydroxy ligand W2 and the central oxo-bridging O5. The driving force for the O2 producing S4→S0 transition was studied by comparing the effects of N2 and O2 pressures of about 20 bar on the flash-induced O2 production of photosystem II samples containing either the native cofactors Ca2+ and Cl- or the surrogates Sr2+ and Br-. While for the Ca/Cl-PSII samples no product inhibition was observed, a kinetic limitation of O2 production was found for the Sr/Br-PSII samples under O2 pressure. This was tentatively assigned to a significant slowdown of the O2 release in the Sr/Br-PSII samples. In addition, the equilibrium between the S0 state and the early intermediates of the S4 state family was studied under 18O2 atmosphere in photosystem II centers devoid of tyrosine YD. Water-exchange in the transiently formed early S4 states would have led to 16,18O2 release, but none was observed during a three day incubation time. Both experiments thus indicate that the S4→S0 transition has a large driving force. Thus, photosynthesis is not limited by the O2 partial pressure in the atmosphere.

Protein dynamics play a key role in enzyme-catalyzed reactions. Vibrational spectroscopy provides a method to follow these structural changes and thereby describe the reaction coordinate as a function of space and time. A vibrational spectroscopic technique, reaction-induced FTIR spectroscopy, has been applied to the study of the oxygen-evolving complex (OEC) of photosystem II (PSII). In plant photosynthesis, PSII evolves oxygen from the substrate, water, by the accumulation of photo-oxidizing equivalents at the OEC. Molecular oxygen and protons are the products of this reaction, which is responsible for the maintenance of an aerobic atmosphere on earth. The OEC is a Mn4CaO5 cluster with nearby bound chloride ions. Sequentially oxidized states of the OEC are termed the S states. The dark-stable state is S1, and oxygen is released on the transition from S3 to S0. Using short laser flashes, individual S states are generated, allowing vibrational spectroscopy to be used to study these different oxidation states of the OEC. In current X-ray crystal structures, hydrogen bonds to water molecules are predicted to form an extensive network around the Mn4CaO5 cluster. In the OEC, four peptide carbonyl groups are linked to the water network, which extends to two Mn-bound and two Ca-bound water molecules. This dissertation discusses a vibrational spectroscopic method that uses these peptide carbonyl frequencies as reporters of solvatochromic changes in the OEC. This technique provides a new, high-resolution method with which to study water and protein dynamics in PSII and other enzymes.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9003号
農博第1185号
新制||農||821(附属図書館)
学位論文||H13||N3522(農学部図書室)
UT51-2001-F333
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 佐藤 文彦, 教授 關谷 次郎, 教授 大山 莞爾
学位規則第4条第1項該当

Chemistry
Ph.D.
The aim of this work is to synthesize and study novel multinuclear manganese systems to model the structure and function of the oxygen-evolving complex (OEC). With the synthesis and study of these model complexes, a greater understanding of nature’s OEC mechanism and processes will come to fruition as well as a viable homogenous water-oxidation catalyst. In addition, small molecule activation was investigated using an FeI precursor. A tetramanganese “pinned-butterfly” cluster with the chemical formula Mn4(µ3-N2Ph2)2(µ-N2Ph2)(µ-NHPh)2(L)4, was synthesized via self-assembly with the addition of N, N’-diphenylhydrazine to Mn(N(SiMe3)2)2). The reaction proceeds over the course of a few hours with a visible color change pale yellow to yellow, black and finally red. The self-assembly mechanism was elucidated with methods such as ligand labeling, kinetic isotope effect, IR spectroscopy, X-ray diffractometry (single crystal and powder), UV/vis kinetic studies and absorbance studies, hydrogen-atom transfer (HAT) competition studies, NMR studies, GC studies and freezing point depression studies. The “twisted basket” cluster is a two-hydrogen-atom reduced analogue of the aforementioned “pinned-butterfly” cluster with a chemical formula of Mn4(µ-NHPh)4(µ-PhNNPh-2N,N’)2(py)4. Conversion between the clusters was investigated and achieved with the addition of an equivalent of N,N’-diphenylhydrazine and heat to a solution of the “pinned-butterfly” complex. This conversion between the clusters displays similarities to the OEC in the sense that it is undergoing proton-coupled electron transfer (PCET), cluster rearrangement and N-N bond formation. While these novel tetramanganese clusters provide us unique, reactive, and flexible clusters, they are far too sensitive to air and water to perform any useful catalysis. Due to the ligands’ lack of stabilization, alternate ligand platforms were investigated that would be able to form more rigid complexes, but retain lability. Bi- and tridentate ligands were investigated that resulted in the synthesis of several novel multinuclear homo- and hetero- metallic complexes. The ligands include polyoligimeric silsesquioxanes and substituted pyridines. These multinuclear Mn clusters show similarities to the OEC in their composition and structures. Upon exposure to air, a color change is observed without the precipitation of a manganese oxide insoluble species. This observation supports the increased stability, yet retained reactivity of the chelated clusters. Lastly, an FeI precursor was reacted with CS2 in attempts to isolate an Fe-S carbide complex and model the iron-molybdenum cofactor (FeMoco). Instead, a CS2 bridging dimer was formed and isolated. The activation of CS2 led us to attempt the reaction of the FeI precursor with other analogues such as CO2, diisopropylcarbodiimide, methylisothiocyanate, and phenylacetylene. CO2 and acetylene have been shown to be reactive substrates to the native FeMoco. These small molecule activated Fe complexes were characterized using X-ray diffraction technqiues, UV/visible spectroscopy, electron paramagnetic resonance spectroscopy and infrared spectroscopy.
Temple University--Theses

La photosynthèse est un processus biologique naturel qui convertit l'énergie lumineuse en énergie chimique par l'action de centres réactionnels photosynthétiques. L'énergie convertie est stockée sous forme de produits de haute énergie synthétisés par la branche réductive du processus photosynthétique. Les électrons nécessaires à ces réactions sont fournis par des molécules d'eau lors de leur oxydation par le centre de dégagement de l'oxygène (Oxygen Evolving Complex: OEC) pour le système de photosynthèse II (PSII). La photosynthèse artificielle cherche à reproduire les réactions qui se produisent dans les organismes naturels afin de i) de mieux comprendre les processus chimiques qui se déroulent dans les systèmes naturels, et ii) de parvenir à exploiter l'énergie solaire pour le développement de carburants propres et renouvelables. Chaque étape qui survient dans le processus de photosynthèse naturelle, telle que la capture de lumière, le transfert d'énergie, le transfert d'électron, la séparation de charge, l'activation du catalyseur et la réaction catalytique doit se produire au sein du système artificiel. La photosynthèse artificielle cherche à reproduire les réactions qui se produisent dans les organismes naturels afin de i) de mieux comprendre les processus chimiques qui se déroulent dans les systèmes naturels, et ii) de parvenir à exploiter l'énergie solaire pour le développement de carburants propres et renouvelables. Chaque étape qui survient dans le processus de photosynthèse naturelle, telle que la capture de lumière, le transfert d'énergie, le transfert d'électron, la séparation de charge, l'activation du catalyseur et la réaction catalytique doit se produire au sein du système artificiel. Avec ces concepts en vue, nous avons conçu, synthétisé et caractérisé des molécules qui imitent les réactions réalisées par les antennes et les centres réactionnels présents dans le photosystème II. Ces molécules sont capables de reproduire la séparation de charges induite par la lumière, le transfert d'électrons et l'accumulation d'équivalents oxydo-réducteurs observés pendant la photosynthèse naturelle. Les antennes artificielles se constituent de caroténoïdes et phthalocyanines. Ces molécules présentent des profiles d'absorption large avec des coefficients d'extinction élevés, et sont capables de supporter des transferts d'énergie ultra rapides qui permettent l'état de séparation de charges. En faisant varier la longueur de la chaine conjuguée des caroténoïdes de neuf à onze liaisons doubles, nous avons pu mettre en évidence comment ces molécules peuvent agir aussi bien comme donneurs que comme agents dissipateurs d'énergie, effet caractéristique qui s'apparente au processus de trempe non-photochimique (Non Photochemical Quenching: NPQ) qui se produit dans le cycle de la zéaxanthine. Les mimiques des agents donneurs du photosystème II ont aussi été étudiées. Ces systèmes supramoléculaires contiennent une partie photoactive liée de façon covalente par un intermédiaire à une cavité contenant un ion ou un agrégat d'ions métalliques. La photosensibilisateur utilisé est un complexe du ruthénium [Ru(bipy)3]2+ (bpy = 2,20-bipyridine), homologue du P680, qui absorbe la lumière dans le spectre visible et déclenche le transfert d'électron. Les espèces RuIII résultantes ont un potentiel d'oxydation réversible de 1.3 V vs SCE, comparables à celui de P680 (1.25 V vs NHE) et présentent donc la possibilité d'oxyder à la fois un complexe manganèse ainsi qu'une source d'électron. Concernant les molécules imitant le coté donneur du PSII, nous avons synthétisé des paires ruthénium-phénol, ainsi que des systèmes ruthénium-manganèse bimétalliques. Parmi ces dernières, nous avons étudié celles présentant des cavités de coordination constituées de terpyridines, vu qu'il a déjà été montré que les dimères Mn-di-μ-oxo-Mn de ce type peuvent catalyser l'oxydation de l'eau en oxygène moléculaire. Des salènes et salophènes ont aussi été examinés étant donné que de tels groupes peuvent accomplir l'oxydation à deux électrons de substrats organique. Dans la littérature, ces réactions sont toutes conduites par l'action d'oxydants chimiques externes, tandis que nous avons pour but d'utiliser des espèces oxydantes induites par l'action de la lumière.

The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.

Harness light from the sun is one of the 21st century’s major goals towards the substitution of fossil fuels for a renewable source of energy. Sustainable production of highly energetic molecules using sunlight as energy source can provide a recyclable fuel round the clock. In this regard, hydrogen from water is envisioned as an ideal cofactor as this energetic store. Viable production of solar fuels will require the use of earth-abundant based catalysts with high activity and efficiency. Long ago, Nature figured out how to take advantage of the sunlight by converting solar energy into chemical bonds, through water and carbon dioxide. This process has been perfected during millions of years and the development of an artificial system to replicate the natural photosynthesis is extremely challenging. Towards the design of these energy conversion schemes based on sunlight, CO2 and H2O, a key step is the water oxidation. The water oxidation provides the electrons needed for the production of fuel. An efficient catalyst is required to overcome the uphill energy multi-electron transformation. The main objective of this thesis is the design of artificial compounds that efficiently oxidizes water into O2, protons and electrons, as the first step towards the exploitation of the sunlight. The study of these complexes could contribute with valuable information about the oxidation mechanisms taking place during the photosynthesis. The results obtained in this thesis firstly show that readily available iron and iridium complexes can carry out the water oxidation in an efficient manner. Homogeneous high valent metal species (IrV/VI, FeV) are the responsible of this redox process. Furthermore, the characterization of a novel oxo-bridged iron-cerium complex constitutes the first direct observation of a heterodimetallic core in a synthetic water oxidation catalyst. These species can be construed as the closest structural and functional model for the essential heterodimetallic MnV–O–CaII center involved in the water oxidation in PSII.
L’aprofitament de la llum solar com a font d’energia és un dels objectius més prometedors alhora de substituir els combustibles fòssils per una font d’energia renovable. La producció sostenible de molècules energètiques mitjançant la llum del sol pot proporcionar un combustible reciclable durant les 24 hores del dia. En aquest aspecte, l’hidrogen obtingut de l’aigua s’entreveu com un cofactor ideal per aquest emmagatzematge energètic. L’ús de catalitzadors basats en materials abundants i amb una activitat i eficiència elevades seran elements indispensables per a la producció viable de combustibles solars. La natura va ser capaç de trobar un mecanisme per aprofitar l’energia solar convertint-la en enllaços químics mitjançant aigua i diòxid de carboni. Aquest procés ha sigut perfeccionat al llarg de milions d’anys i conseqüentment, el desenvolupament de sistemes artificials capaços d’imitar la fotosíntesi natural és extremadament complex. De camí cap al disseny de sistemes per a la conversió d’energia basats en la llum solar, el CO2 i l’H2O, un pas clau és l’etapa d’oxidació de l’aigua. Aquesta etapa proporciona els electrons necessaris per la producció de combustible. La presència d’un catalitzador és necessària per superar aquesta transformació multielectrònica, ja que requereix una elevada energia. L’objectiu principal d’aquesta tesi és el disseny de compostos artificials que oxidin l’aigua i alliberin oxigen, protons i electrons de manera eficient, com a primer pas cap a l’explotació de la llum. L’estudi d’aquests complexos pot contribuir amb informació valuosa sobre el mecanisme d’oxidació que tenen lloc durant la fotosíntesi. Els resultats obtinguts en aquesta tesi mostren que complexos de ferro i iridi fàcilment a l’abat són capaços de catalitzar l’oxidació de l’aigua de manera eficient. Espècies homogènies en alts estat d’oxidació (IrV/VI, FeV) són les responsables de dur a terme aquest procés redox. La caracterització d’un nou dímer de ferro-ceri unit per un pont oxo constitueix la primera observació directa d’un centre heterodimetàl•lic en un catalitzador artificial d’oxidació de l’aigua. Aquesta espècie constitueix el model estructural i funcional més semblant al centre de MnV-O-CaII present en el PSII.

In the five chapters that follow, I delineate my efforts over the last five years to synthesize structurally and chemically relevant models of the Oxygen Evolving Complex (OEC) of Photosystem II. The OEC is nature’s only water oxidation catalyst, in that it forms the dioxygen in our atmosphere necessary for oxygenic life. Therefore understanding its structure and function is of deep fundamental interest and could provide design elements for artificial photosynthesis and manmade water oxidation catalysts. Synthetic endeavors towards OEC mimics have been an active area of research since the mid 1970s and have mutually evolved alongside biochemical and spectroscopic studies, affording ever-refined proposals for the structure of the OEC and the mechanism of water oxidation. This research has culminated in the most recent proposal: a low symmetry Mn₄CaO₅ cluster with a distorted Mn₃CaO₄ cubane bridged to a fourth, dangling Mn. To give context for how my graduate work fits into this rich history of OEC research, Chapter 1 provides a historical timeline of proposals for OEC structure, emphasizing the role that synthetic Mn and MnCa clusters have played, and ending with our Mn₃CaO₄ heterometallic cubane complexes.

In Chapter 2, the triarylbenzene ligand framework used throughout my work is introduced, and trinuclear clusters of Mn, Co, and Ni are discussed. The ligand scaffold consistently coordinates three metals in close proximity while leaving coordination sites open for further modification through ancillary ligand binding. The ligands coordinated could be varied, with a range of carboxylates and some less coordinating anions studied. These complexes’ structures, magnetic behavior, and redox properties are discussed.

Chapter 3 explores the redox chemistry of the trimanganese system more thoroughly in the presence of a fourth Mn equivalent, finding a range of oxidation states and oxide incorporation dependent on oxidant, solvent, and Mn salt. Oxidation states from Mn^II₄ to Mn^IIIMn^IV₃ were observed, with 1-4 O^2– ligands incorporated, modeling the photoactivation of the OEC. These complexes were studied by X-ray diffraction, EPR, XAS, magnetometry, and CV.

As Ca²⁺ is a necessary component of the OEC, Chapter 4 discusses synthetic strategies for making highly structurally accurate models of the OEC containing both Mn and Ca in the Mn₃CaO₄ cubane + dangling Mn geometry. Structural and electrochemical characterization of the first Mn₃CaO₄ heterometallic cubane complex— and comparison to an all-Mn Mn₄O₄ analog—suggests a role for Ca²⁺ in the OEC. Modification of the Mn₃CaO₄ system by ligand substitution affords low symmetry Mn₃CaO₄ complexes that are the most accurate models of the OEC to date.

Finally, in Chapter 5 the reactivity of the Mn₃CaO₄ cubane complexes toward O- atom transfer is discussed. The metal M strongly affects the reactivity. The mechanisms of O-atom transfer and water incorporation from and into Mn₄O₄ and Mn₄O₃ clusters, respectively, are studied through computation and ¹⁸O-labeling studies. The μ3-oxos of the Mn₄O₄ system prove fluxional, lending support for proposals of O^2– fluxionality within the OEC.

The following two chapters delineate several endeavors to isolate and characterize functional models of the oxygen-evolving complex (OEC) of photosystem II. Understanding the electronic structure and the precise mechanism of the O–O bond coupling step in the Kok cycle affords insight into this fundamental process and will guide the design of new earth-abundant catalysts to perform water oxidation under environmentally benign conditions. Nature performs this transformation by a heterometallic CaMn₄O₅ cluster arranged a tetra-metallic cubane bridged to a dangling manganese ion. Although a myriad of synthetic inorganic complexes are capable of water oxidation, these structures significantly underperform the OEC in terms of turnover number and turnover frequency. The objectives of this thesis are (i) to construct multimetallic clusters using the OEC as inspiration, (ii) to explore the reactivity of these clusters with oxygen-atom transfer reagents, and (iii) to identify intermediates responsible for oxygen-based chemistry.

In Chapter 1, a series of pseudo-C₃ symmetric tetra-manganese clusters with an interstitial µ₄-oxygen was synthesized and characterized in several oxidation states. These clusters (of the general formula [LMn₃(PhPz)₃OMn][OT_x]x; x = 1, 2) are supported by pyridine and alkoxide donors connected by a 1,3,5-triarylbenzene spacer. A µ₄-oxygen coordinates all four metal centers that are also bridged by phenyl pyrazolate (PhPz) ligands. This arrangement furnishes a vacant coordination site at a site-differentiated (apical) metal center. Exposure of these clusters to oxygen-atom transfer reagents (OAT’s) results in the intramolecular oxygenation of a C(sp²)–H bond of the bridging phenyl pyrazolate. Similarly, using 2,6-difluorophenyl pyrazolate (F₂ArPz) as the bridging ligand results in the oxygenation of the C–F bond with concurrent F-atom transfer. This reactivity represents an unprecedented C–F activation for molecular manganese complexes. All hydroxylated and fluorinated clusters were independently prepared to confirm the observed reactivity upon exposure to OAT’s. The pathways responsible for arene activation – postulated to proceed through an iodosobenzene adduct and subsequent formation of a transient high-valent manganese-oxo motif – are discussed.

In Chapter 2, a series of pseudo-C₃ symmetric heterometallic Fe₃Mn clusters of the general formula [LMn₃(PhPz)₃OMn][OTf]_x (x = 1–3) was synthesized and characterized. Similar to their homometallic tetra-manganese and tetra-iron analogs (Chapter 1), these clusters contain four metal centers with a central bridging interstitial µ₄-oxygen atom and bridging phenyl pyrazolate ligands. These clusters are further supported by pyridine and alkoxide donors, linked through a 1,3,5-triarylbenzene spacer. All complexes were characterized by zero-field ⁵⁷Fe Mössbauer spectroscopy to confirm the presence of a manganese metal center in the apical position, illustrating that these clusters are stable with respect to metal scrambling and/or decomposition. Treatment of these clusters with 1-(tert-butylsulfonyl)-2-iodosylbenzene (sPhIO) resulted in the oxygenation of the C(sp²)–H bond of the proximal phenyl pyrazolate motif to afford [LMn₃(PhPz)₂(OArPz)OMn][OTf]_x (x = 2, 3). During these studies, an unusual iodosobenzene adduct of [Fe^III₃Mn^II]³⁺ was isolated prior to C–H activation. This adduct has been characterized both by single-crystal XRD and ¹H-NMR spectroscopy. In order to gain insight into the C–H bond oxygenation by this iodosobenzene adduct, preliminary computational studies are presented to discuss the viability of a transient manganese-oxo species responsible for arene hydroxylation.

All life on earth depends mainly on the presence of oxygen. Largest producers of oxygen are green plants, cyanobacteria and algae. Oxygen is released from the oxygenevolving complex of photosystem II during photosynthesis and it is used in cellular respiration of all life complexes. The oxygen-evolving complex of photosystem II has the same function in each photosynthetic organism, but it has a different composition and organization of extrinsic proteins; only PsbO protein is ubiquitous in all known oxyphototrophs. Until now only low resolution electron microscopy structural models of plant PSII and crystal structures of cyanobacterial PSII are available. Higher plant extrinsic proteins (PsbP, PsbQ and PsbR) are structurally unrelated, non-homologues to the cyanobacterial extrinsic proteins (PsbO, PsbU and PsbV) and this is the reason why it is not possible to predict arrangement of these proteins on the lumenal site of higher plant PSII. Recently, models differ mainly in the structure of the oxygen-evolving complex, which could be resolved by determination of the exact binding sites for extrinsic proteins. An other question evolves: if the difference in the oxygen-evolving complex composition is the result of evolution or adaptation of photosynthetic organisms to their environment. Structural knowledge of extrinsic proteins that could help to resolve the location and subsequently the function of extrinsic proteins is still incomplete. From this case,structural analysis, interactions and probably arrangement of proteins PsbP and PsbQ was studied and is described in detail in this thesis.

This thesis describes a series of studies devoted toward the synthesis of model complexes that mimic aspects of structure, redox state, and spectroscopy of the oxygen evolving complex (OEC) of Photosystem II. The OEC is a unique metallocofactor featuring a heteronuclear CaMn₄ core that catalyzes water oxidation. While advances in spectroscopic and structural techniques offer an ever more detailed view of the structure of the S-state catalytic intermediates, the precise mechanism of O−O bond formation remains debated. Aspects such as (1) role of Ca²⁺, (2) the location of the substrate waters, and (3) the (electronic) structure of the S-state intermediates remain unclear. To obtain a better understanding of the OEC, systematic structure−function(property) studies on relevant model complexes may be necessary. Despite significant efforts to prepare tetra- and pentanuclear complexes as models of the OEC, relevant complexes in terms of structure, redox state, spectroscopy, and reactivity are rare, likely due to the synthetic challenges of accessing a series of isolable clusters that are suitable for comparisons.

Chapter 1 presents a survey of tetramanganese model compounds with an emphasis on redox state and electronic structure, as probed by magnetometry and EPR spectroscopy. Structurally characterized model complexes are grouped according to Mn oxidation states and the S-state that they are mirroring. In contrast to the vast number of spectroscopic studies on the OEC, studies that probe the effect of systematic changes in structure on the spectroscopy of model complexes are rare in the literature.

Chapter 2 presents ongoing synthetic efforts to prepare accurate structural models of the OEC. The synthesis of accurate structural models is hampered by the low structural symmetry of the cluster, the presence of two types of metals, and the propensity of oxo moieties to form extended oligomeric structures. Desymmetrization of the previously reported trinucleating ligand leads to the formation of tetranuclear Mn₄^II precursors. Oxidation in the presence of Ca²⁺ leads to a CaMn₄O₂ model of the OEC, underscoring the utility of low-symmetry multinucleating ligands in the synthesis of hitherto unobserved oxo-bridged multimetallic core geometries related to the OEC.

Chapter 3 presents a series of [Mn^IIIMn₃^IVO₄] cuboidal complexes as spectroscopic models of the S₂ state of the OEC. Such complexes resemble the oxidation state and EPR spectra of the S₂ state, and the effect of systematic changes in the nature of the bridging ligands on spectroscopy was studied. Results show that the electronic structure of tetranuclear Mn complexes is highly sensitive to even small geometric changes and the nature of the bridging ligands. Model studies suggest that the spectroscopic properties of the OEC may also react very sensitively to small changes in structure; the effect of protonation state and other reorganization processes needs to be carefully assessed.

Chapter 4 presents a series of [YMn₃O₄] complexes as models of the [CaMn₃O₄] subsite of the OEC. The effect of systematic changes in the basicity and chelating properties of the bridging ligands on redox potential was studied. Results show that in the absence of ligand-induced geometric distortions that enforce a contraction of metal-oxo distances, increasing the basicity of the ligands results in a decrease of cluster reduction potential. A small contraction of metal-oxo/metal-metal distances by ~0.1 Å enforced by a chelating ligand results in an increase of cluster reduction potential even in the presence of strong basic donors. Such small, protein-induced changes in Ca-oxo/Ca-Mn distances may have a similar effect in tuning the redox potential of the OEC through entatic states, and may explain the cation size dependence on the progression of the S-state cycle.

Chapter 5 presents a series of [CaMn₃O₄] and [YMn₃O₄] complexes as models of the [CaMn₃O₄] subsite of the OEC. The effect of systematic changes in cluster geometry, heterometal identity, and bridging oxo protonation on cluster spin state structure was studied. Results show that the electronic structure of the Mn₃^IV core is highly sensitive to small geometric changes, the nature of the bridging ligands, and the protonation state of the bridging oxos: the spin ground states of essentially isostructural compounds can be S = 3/2, 5/2, or 9/2. Interpretation of EPR signals and subsequent structural assignments based on an S = 9/2 spin state of the CaMn₃O₄ subsite of the OEC must be done very cautiously.

While unfinished, appendices 1 and 2 present other important aspect in OEC model chemistry. Appendix 1 presents the synthesis of ¹⁷O-labeled [Mn^IIIMn₃^IVO₄] and [CaMn₃^IVO₄] complexes as models of the OEC. Ongoing characterization of μ³-oxos in such complexes provide valuable benchmarking parameters for future mechanistic studies. Appendix 2 presents the synthesis and characterization of [Mn₄^IVO₄] cuboidal complexes as spectroscopic models of the S₃ state of the OEC, the last observable intermediate prior to O−O bond formation at the OEC.

The first step of photosynthetic metabolism effects the facile oxidation of water to dioxygen and hydrogen cations. This is achieved through an incompletely-understood process of light-driven four-electron oxidation at the Mn4CaO5 cofactor of the Oxygen Evolving Complex (OEC) of the Photosystem II (PSII) holoenzymatic complex in photosynthetic autotrophs. Biomimesis of this reaction—artificial photosynthesis—may offer energy-efficient routes to industrial hydrogen generation and value-added derivatives, with implications for solar energy fixation. This thesis consists of a compilation of four publications relating to Density Functional Theory (DFT) studies of structural and spectroscopic aspects of the OEC of PSII. These publications consist of research resolving the basis of structural anomalies in metal-substituted PSII, combinatoric simulation of difference spectra corresponding to proton-coupled oxido-reduction scenarios of PSII models, simulation of the hyperfine and superexchange magnetic interactions in PSII models, and the development of a methodology for obtaining vibrational intensities in the Mobiel Block Hessian (MBH) approximation, with applications to accelerated modeling of the vibrational structure of complex models of PSII, as well as other large molecules. These publications are presented alongside explanatory introductions and preceded by a general survey of the state of the art of photosynthesis research, context for the relevance of this research, and methodological discussion. Concluding remarks are also presented.

碩士
東吳大學
化學系
93
Ammonia is an inhibitor of water oxidation and a structural analog for substrate water, making it a valuable probe for the structural properties of the possible substrate-binding site on the oxygen-evolving complex (OEC) in photosystem II (PSII). By using the NH3-induced upshift of the 1365 cm-1 IR mode in the S2QA-/S1QA spectrum and the NH3-modified S2 state EPR signals of PSII as spectral probes, we found that ethylene glycol has clear effects on the binding properties of the NH3 specific site on the OEC. Our results show that in PSII samples containing 30% (v/v) ethylene glycol, the affinity of the NH3-specific binding site on the OEC is estimated more than ten times lower than that in PSII samples containing 0.4 M sucrose. In addition, our results show that the NH3-induced upshift of the 1365 cm-1 IR mode in the S2QA-/S1QA spectrum is dependent on the concentration of ethylene glycol, but not dependent on the concentration of sucrose (up to 1.5 M) or methanol (up to 5.4 M). By comparing the concentration dependence of sucrose and ethylene glycol on NH3-induced spectral change and also by comparing the sucrose and ethylene glycol data at similar concentrations (~1 M), we conclude that ethylene glycol has a clear effect on the NH3-induced spectral changes. Furthermore, our results also show that ethylene glycol alters the steric requirement of the amine effect on the upshift of the 1365 cm-1 mode in the S2QA-/S1QA spectrum. In PSII samples containing 30% ethylene glycol, only NH3, not other bulkier amines (e.g., Tris, AEPD and CH3NH2), has a clear effect on the upshift of the 1365 cm-1 mode in the S2QA-/S1QA spectrum; in contrast, in PSII samples containing 0.4 M sucrose, both NH3 and CH3NH2 have a clear effect. On the basis of the above results, we proposed that ethylene glycol might act directly or indirectly to decrease the affinity or limit the accessibility of NH3 and CH3NH2 to the NH3-specific binding site on the OEC in PSII. Finally, we also applied the same approach to test whether or not methanol is able to compete with ammonia on its binding site on the OEC. We found that 4% methanol does not show any significant effect on NH3-induced up-shift of the 1365 cm-1 mode in the S2QA-/S1QA spectrum and the NH3-modified S2 state g=2 multiline EPR signal. Our results suggest that methanol is unable to compete with NH3 on binding to the Mn site of the OEC that gives rise to the altered S2 state g=2 multiline EPR signal.

Photosystem II or PS II, found in oxygenic photosynthetic organisms such as cynobateria or higher plants, is the catalyst for the most energetically demanding reaction in nature, the oxidation of water to molecular oxygen and protons. The water oxidase in PS II contains a Mn4Ca cluster (oxygen evolving complex, OEC), whose catalytic mechanism, despite extensive investigation, remains unresolved. The precise oxidation levels of the manganese is especially important in understanding the real catalytic mechanism of the OEC. Many experiemental techniques, such as EPR and ENDOR, have been adopted in historical studies, and also due to the more recent development in semiconductors, a higher level of computational analysis and simulation became available to study the system in theory. Here is the work completed to provide the first 55Mn pulsed ENDOR studies on the S2 state multiline spin ½ centre of the oxygen evolving complex (OEC) in Photosystem II (PS II), at temperatures below 4.2 K. These were performed on highly active samples of spinach PS II core complexes, developed previously in the laboratories using specific preparation procedure and experimental techniques to achieve 100% turnover rate, for photosystem spectroscopic use, at temperatures down to 2.5 K. Under these conditions, previously hindered observation, by relaxation effects, of most of the manganese ENDOR resonances from the OEC coupled Mn cluster are suppressed.55Mn ENDOR hyperfine couplings ranging from 50 to 680 MHz are now seen on the S2 state multiline EPR signal. These, together with complementary high resolution X-band CW EPR measurements and detailed simulations, reveal that at least two and probably three Mn hyperfine couplings with large anisotropy are seen, indicating that three MnIII ions are likely present in the functional S2 state of the enzyme. This suggests a low oxidation state paradigm for the OEC (mean Mn oxidation level 3.0 in the S1 state) and unexpected Mn exchange coupling in the S2 state, with two Mn ions nearly magnetically silent. Our results rationalize a number of previous ligand ESEEM/ENDOR studies and labelled water exchange experiments on the S2 state of the photosystem.

The photosynthetic water oxidation reaction is catalyzed by an inorganic Mn4OxCaClyHCO3-z cluster at the heart of the oxygen evolving complex (OEC) in photosystem II. In the absence of an atomic resolution crystal structure, the precise molecular organization of the OEC remains unresolved. Accordingly, the role of the protein and inorganic cofactors of PSII (Ca2+, HCO3- and Cl-) in the mechanism of O2-evolution await clarification. In this study, rapid 18O-isotope exchange measurements were applied to monitor the substrate-water binding kinetics as a function of the intermediate S-states of the catalytic site (i.e. S3, S2 and S1) in Triton X-100 solubilized membrane preparations that are enriched in photosystem II activity and are routinely used to evaluate cofactor requirements. Consistent with the previous determinations of the 18O exchange behavior in thylakoids, the initial 18O exchange measurements of native PSII membranes at m/e = 34 (which is sensitive to the 16O18O product) show that the ‘fast’ and ‘slowly’ exchanging substrate-waters are bound to the catalytic site in the S3 state, immediately prior to O2 release. Although the slowly exchanging water is bound throughout the entire S-state cycle, the kinetics of the fast exchanging water remains too fast in the S2, S1 [and S0] states to be resolved using the current instrumentation, and left open the possibility that the second substrate-water only binds to the active site after the formation of the S3 state. Presented is the first direct evidence to show that fast exchanging water is already bound to the OEC in the S2 state. Rapid 18O-isotope exchange measurements for Ex-depleted PSII (depleted of the 17- and 23-kDa extrinsic proteins) in the S2 state reveals a resolvable fast kinetic component of 34k2 = 120 ± 14 s-1. The slowing down of the fast phase kinetics is discussed in terms of increased water permeation and the effect on the local dielectric following removal of the extrinsic subunits. In addition, the first direct evidence to show the involvement of calcium in substrate-water binding is also presented. Strontium replacement of the OEC Ca2+-site reveals a factor of ~3-4 increase in the 18O exchange of the slowly exchanging water across the S3, S2 and S1 states while the kinetics of the fast exchanging water remain unchanged. Finally, a re-investigation of the proposed role for bicarbonate as an oxidizable electron donor to photosystem II was unable to discern any 18O enrichment of the photosynthetically evolved O2 in the presence of 18O-bicarbonate. A working model for O2-evolution in terms of these results is presented.

Conifers are important both ecologically and socioeconomically, however, same parts of their biology are not that well researched. This includes genetics and breeding and partly even physiology. Because quantitative genetic analyzes applied in breeding necessitate an analysis of a large number of samples, and conventional methods of analysis are quite time-consuming, certain parameters describing e.g. the activity of photosynthetic electron-transport chain (ETC) are considered for such use. Several methods of the measurement of the activity of photosynthetic ETC exist, but there are some problems with their usage in conifers. I studied this issue from different points of view in three parts of this thesis. 1) I compared the photosynthetic ETC activity in 8 species of conifers using chlorophyll (Chl) fluorescence measurements on intact needles and polarographic measurements in isolated chloroplasts. Each method brought different information. 2) I measured Chl fluorescence parameters, reflectance spectra and pigment content in 536 genetically defined trees of Pinus sylvestris L. Many parameters showed relatively high genetic variability and heritability. I have also determined the suitability of various reflectance indices to estimate pigment and water content of needles. 3) I have optimized the...