Photosystem I (PSI) from plants, algae, and cyanobacteria can mediate H2 evolution in vivo and in vitro. A simple, self-platinization procedure that permits stable PSI-mediated H2 evolution in vitro has been developed. The H2 evolution capabilities of PSI from Thermosynechococcus elongatus have been characterized. This organism utilizes cytochrome c6 (cyt c6) as the e- donor to P700. Using a solution-based, self-organized platinization of the PSI nanoparticles, this study demonstrates a sodium ascorbate-cyt-PSI-Pt-H2 electron transport and proton reduction system that yields light-dependent H2. The system was thermostable with H2 evolution increasing up to 55°C. In addition, stability studies have shown the H2 evolution to be very stable, with no significant decrease over the 80 days investigated. Through simple optimization a H2 production rate of ~5.5 mol H2/h/mg Chl [micro-mole H2 per hour per milligram chlorophyll] was attained. To further optimize the H2 production Asc-cyt-PSI-Pt-H2 system, response surface methodology (RSM) was employed. The process parameter studied included temperature, light intensity and platinum salt concentration. The results showed that experimental data had a good fit to the proposed model (R2=0.99 and p < 0.001). Platinum salt concentration, temperature and the interaction between platinum salt concentration and temperature showed significant effects on the total H2 yield. Light intensity had minimal effect of the total H2 yield within the region studied. The optimum parameters for H2 photoproduction were light intensity of 240 μE/m2/s, [micro-eistien per square meter per second], platinum salt concentration of 636 μM [micro-mol/liter] and temperature of 310C. Finally, studies that will improve the H2 yield by increasing the kinetics of electron transfer were done. A hybrid protein was formed by engineering a gene to express a fusion of the membrane-bound [Ni-Fe] hydrogenase from Ralstonia eutropha H16 and the stromal-exposed subunits PsaE and PsaD of PSI from T. elongatus. A PsaE-free mutant of PSI was simultaneously formed by genetically disrupting the expression of the PsaE subunit of a native PSI; that will allow in vitro reconstitution of the desired PsaE-hydrogenase fusion protein with PsaE-free PSI.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Alimentos. / Made available in DSpace on 2013-07-15T22:41:58Z (GMT). No. of bitstreams: 1 211457.pdf: 782449 bytes, checksum: d435cd88c2395a1093a9452faf9e9c9a (MD5) / Poli(3-hidroxibutirato), P(3HB), é um polímero de reserva de carbono e energia acumulado intracelularmente por diversos microrganismos, sob condições desbalanceadas de crescimento. Além da vantagem de ser biodegradável e biocompatível, possui propriedades termoplásticas compatíveis aos plásticos convencionais. Embora apresente estas vantagens, apresenta um elevado custo de produção frente aos polímeros petroquímicos, o que dificulta o seu uso. Neste trabalho estudou-se a produção de P(3HB) em resíduos de indústrias de alimentos, objetivando reduzir os custos de produção. Primeiramente, verificou-se a capacidade de crescimento da bactéria Ralstonia eutropha nos resíduos das indústrias amiláceas e indústrias processadoras de maçã. Testou-se três modelos primários de crescimento, e o modelo Logístico, foi o que melhor descreveu o crescimento da bactéria nas diferentes fontes de carbono testadas. Após verificar a capacidade de crescimento da bactéria, foi realizada a produção de P(3HB), nos resíduos das indústrias de alimentos, e também a suplementação do meio de cultura com ácido oléico e óleo de soja, visando aumentar a produção de polímero. O resíduo da indústria amilácea mostrou-se uma boa fonte de carbono para a produção de P(3HB), onde os conteúdos de polímero acumulado foram de 46, 50 e 56% com produtividades de 0,22; 0,37 e 0,49 g.L-1.h-1 para as culturas sem suplementação, suplementação com ácido oléico e suplementação com óleo de soja. Quando se realizou o crescimento no resíduo da indústria processadora de maçã os conteúdos de P(3HB) acumulados foram de 14, 34 e 22% do total da massa celular seca, para as culturas sem suplementação, suplementação com ácido oléico e suplementação com óleo de soja. Apesar do baixo conteúdo de polímero acumulado, a utilização do bagaço de maçã como fonte de carbono para a produção de polihidroxialcanoatos é uma boa alternativa, pois é uma matéria-prima barata que pode contribuir na redução dos custos de produção deste biopolímero. A adição dos suplementos nutricionais ao meio de cultura favoreceu a produção de P(3HB) aumentando o conteúdo de polímero acumulado e a produtividade.

L’accumulation de biopolymère de réserve (PolyHydroxyAlcanoates ou PHA) par la souche Cupriavidus necator, à partir de substrats de type acides gras volatils (acide butyrique, acide propionique et acide acétique) a été étudiée. Elle est induite par une limitation phosphore. Les performances atteintes lors des cultures se situent parmi les meilleures de la littérature pour ce type de substrat : jusqu’à 66 g.L-1 de biomasse totale avec un pourcentage d’accumulation massique de 88% en PHB –PolyHydroxyButyrate- ou en PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- comportant jusqu’à 52% de motifs d’HV.Pour chaque source carbonée, une caractérisation cinétique et stœchiométrique de la souche a été réalisée en l’absence d’effets inhibiteurs dus aux substrats acides grâce à des cultures de type Fed-Batch avec des apports non limitants et non inhibiteurs en carbone. Il a été dégagé :- un taux de croissance maximal de la souche de 0,33 h-1 pour les trois acides étudiés- une relation entre vitesse spécifique de production de PHA et taux de croissance fixée par la disponibilité et les flux de production de NADPH2 avec un découplage inverse pour les taux de croissance supérieurs à 0,05 h-1 et un couplage partiel pour les taux de croissance inférieurs- un optimum de 0,35 Cmole.Cmole-1.h-1, associé à un taux de croissance de l’ordre de 0,05 h-1.- une amélioration de la production de PHB en termes de vitesses spécifiques mais également en termes de rendements si une faible croissance résiduelle est maintenueLa réponse de la souche à un excès de substrat acide a été caractérisée via l’étude de régimes transitoires induits par des pulses sur des cultures continues préalablement stabilisées en régime permanent. Il a été montré qu’en excès de phosphore, face à un brusque excès de substrat, la souche est incapable d’adapter rapidement son taux de croissance. L’excès est donc dirigé vers la production de PHA dont les voies sont plus rapidement mobilisables. En conditions limitantes de phosphore, le substrat excédentaire est utilisé pour la production de PHA. L’inhibition par les acides se traduit par une diminution des capacités de biosynthèse de la biomasse et des PHA entrainant une réduction de l’assimilation du carbone puis une diminution des rendements de conversion. D’autre part la sensibilité d’un système continu à un excès de substrat dépend du point de fonctionnement choisi : plus il est optimal en termes de vitesse, moins le système est robuste. L’acide propionique est très inhibiteur comparé aux autres acides étudiés (dès 3-4 mM contre 30-40 mM). Il n’agit pas simplement via une accumulation excessive dans le cytoplasme mais il exerce également une inhibition spécifique des voies métaboliques.Un antagonisme entre les substrats (acide acétique et butyrique) a été constaté et expliqué grâce à une analyse des flux métaboliques. L’acide acétique est assimilé préférentiellement pour produire la biomasse, l’énergie et les cofacteurs nécessaires à la production de PHA, alors que l’acide butyrique est utilisé pour la synthèse de PHB. La proportion maximale d’acide acétique admise dans l’alimentation en fonction des conditions fixées en régime permanent est calculée et peut être limitée à 40% du carbone.Enfin il a été déterminé que si une croissance résiduelle est assurée grâce à un apport en phosphore, le pourcentage maximal d’HV dans le polymère dépend du taux d’acide propionique dans l’alimentation et ne peux dépasser 33 ± 5% sur acide propionique pur. Par contre, si aucune croissance résiduelle n’est assurée, il est possible de convertir l’acide propionique en motifs d’HV uniquement / Reserve Biopolymer (PolyHydroxyAlkanoates or PHA) accumulation by the strain Cupriavidus necator, from Volatile Fatty Acids (VFA, like butyric acid, propionic acid and acetic acid) was investigated. This production is induced by a phosphorus limitation. For this type of substrates, performances reached during cultures are among the best listed in the literature: up to 66 g.L-1 of total biomass with 88% (w/w) of PHB –PolyHydroxyButyrate- or PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- with a HV content up to 52 Mole%.For each carbon source, kinetic and stoechiometric characterization has been carried out thanks to Fed-Batch cultures with non-limiting and non-inhibitory carbon feed. It has been established:- a maximal growth rate of 0,33 h-1 for the three acid investigated- a relationship between specific PHA production rate and growth rate which is set by the availability and production flux of NADPH2. For growth rate above 0,05 h-1, there is an inverse coupling. For growth rate under 0,05 h-1, there is a partial coupling.- an optimum of 0,35 Cmole.Cmole-1.h-1 is associated with a growth rate of 0,05 h-1.- if a low residual growth rate is maintained, an improvement of PHB production is recorded in terms of specific production rate and yieldsThe response of the strain to an excess of acid substrate was characterized through the investigation of transient state induced by pulsed addition of substrate during continuous cultures stabilized in steady state. It was shown that in excess of phosphorus, when there is a substrate excess, the strain is unable to quickly adapt its growth rate, so the excess is directed to PHA production whose ways seem to be more easily mobilized. Under phosphorus limitation, an excess of substrate is used for PHA production. Acid inhibition results in a decrease in biomass and PHA production capacity which leads to a decrease in carbon assimilation and conversion yields. The sensitivity of a continuous system to an excess of substrate depends on the chosen operating point: the more it is optimal in terms of specific production rate, the less the system is robust. Propionic acid is highly inhibitory compared to the other acids studied (from 3-4 mM versus 30-40 mM). It does not act only via an excessive accumulation in the cytoplasm but also exerts a specific inhibition of metabolic pathways.An antagonism between substrates (acetic and butyric acid) has been established and explained thanks to the Metabolic Flux Analysis. Acetic acid is preferentially used to produce biomass, energy and cofactors for PHA synthesis, whereas butyric acid is used to product PHB. According to the conditions set during steady state, maximal content of acetic acid admitted in the feed can be calculated. It can be limited to 40% of the carbon in the feed.Finally if a growth rate is maintained thanks to a phosphorus supply, the maximal HV content in polymer is function of propionic acid in the feed and cannot exceed 33 ± 5 Mole% on pure propionic acid. Conversely, if there is no residual growth, a total conversion of propionic acid into HV is allowed

Zusammenfassung Das beta-Proteobakterium Ralstonia eutropha H16 besitzt zwei [NiFe]-Hydrogenasen, die dem Organismus das Wachstum mit H2 als alleiniger Energiequelle unter aeroben Bedingungen ermöglichen. Reguliert wird die Expression dieser [NiFe]-Hydrogenasen durch ein bakterielles Zweikomponentensystem. Die Signaltransduktionskette besteht aus einer H2-sensierenden, regulatorischen [NiFe]-Hydrogenase (RH), einer Histidin-Proteinkinase und einem Response-Regulator. Um Einblicke in die Struktur-Funktions-Beziehung der H2-sensierenden Komponente zu bekommen, wurden Aminosäure-Austausche in konservierten Bereichen nahe des NiFe-Zentrums der RH durchgeführt. Der Austausch des invarianten Glu13 (E13Q, E13L) resultierte im Verlust der regulatorischen, der H2-oxidierenden und der H/D-Austauschaktivitäten. Spektroskopische Daten wiesen auf ein vollständig assembliertes NiFe-Zentrum hin. Mit Hilfe der ortho-/para-Konversionsaktivität wurde gezeigt, dass dieses Zentrum nach wie vor H2 binden kann. Dies deutet darauf hin, dass eine H2-Bindung am aktiven Zentrum nicht für die regulatorische Funktion der RH ausreicht. Durch den Austausch des Asp15 in His wurde der Konsensus eines konservierten Motivs der Standard-Hydrogenasen hergestellt. Das entstandene RH-Mutantenprotein besaß nur noch eine sehr niedrige H2-oxidierende Aktivität, bei nahezu unveränderter H/D-Austauschrate sowie intakter regulatorischer Aktivität. Dies deutet darauf hin, dass der H2-Umsatz nicht entscheidend für die H2-Sensierung ist. Um Informationen über die Struktur des aktiven Zentrums der RH zu erhalten, war es notwendig, große Mengen RH zu isolieren. Hierfür wurde die Strep-tag-Technologie eingesetzt, die es ermöglichte, die RH als natives Doppeldimer und als homodimeres Protein zu reinigen. Röntgenabsorptionsspektroskopie ergab erstmals, dass sich die Koordination des Nickel-Atoms im aktiven Zentrum sowohl im oxidierten als auch im reduzierten Zustand der RH deutlich von der in Standard-Hydrogenasen unterscheidet. / The beta-proteobacterium Ralstonia eutropha H16 is capable of using H2 as a facultative energy source by means of two distinct, energy-converting [NiFe]-hydrogenases. Transcription of the hydrogenase genes is regulated in response to the availability of H2 via a histidyl-aspartyl phosphorelay comprising a heterodimeric, regulatory [NiFe]-hydrogenase (RH), a histidine protein kinase and a response regulator. In order to gain insights into the mechanism of H2-mediated signal transduction, conserved amino acid residues close to the NiFe active site of the RH were exchanged. Replacement of the strictly conserved Glu13 within the RH large subunit by glutamine and leucine resulted in the loss of the regulatory, H2-oxidizing and hydrogen/deuterium exchange activities. Infrared spectroscopic analysis revealed, that the RH E13Q and E13L derivatives contained a fully assembled NiFe active site and showed para-/ortho-H2 conversion activity. These results indicated that H2-binding at the active site is not sufficient for H2 sensing. Replacement of Asp15, a residue unique in H2 sensors, by histidine restored the consensus of energy-linked [NiFe]-hydrogenases. The resulting RH mutant protein showed only traces of H2-oxidizing activity, whereas the H/D-exchange activity and the regulatory activity were nearly unaffected. H2-dependent signal transduction in the respective mutant strain was less sensitive to O2 than in the wild type. These results suggest that the H2 sensing is independent of H2 turnover. To get insights into the structure of the RH it was necessary to isolate large amounts of RH. By establishing the Strep-tag technology, allowing a fast, mild and efficient purification, it was possible to isolate the native RH-double dimer as well as the modified monodimeric RHstop protein in sufficient amounts for spectroscopic analyses. X-ray absorption spectroscopy showed for the first time that the RH active site undergoes significant structural changes upon exposure to H2.

Im biogeochemischen Wasserstoffkreislauf erfolgt der überwiegende Teil der H2-Aufnahme aus der Atmosphäre durch die Böden. Erst seit kurzem ist bekannt, dass die Oxidation von Wasserstoff in Böden mutmaßlich durch eine Reihe von Bodenbakterien vermittelt wird, die zur Aufnahme von Wasserstoff in atmosphärischen Konzentrationen befähigt sind. Diese Bakterien codieren [NiFe]-Hydrogenasen einer neuen Gruppe, die als Gruppe 5 der [NiFe]-Hydrogenasen klassifiziert wurde. Auch das beta Proteobakterium Ralstonia eutropha besitzt die Gene einer derartigen Hydrogenase, die aufgrund ihrer Ähnlichkeit zu den sonst überwiegend in Actinobakterien gefundenen Vertretern der Gruppe 5 als „Actinobakterielle Hydrogenase“ (AH) benannt wurde. In der vorliegenden Arbeit wurde die AH aus R. eutropha als erste Gruppe 5-[NiFe]-Hydrogenase in reiner Form isoliert und eingehend durch unterschiedliche biochemische, spektroskopische und röntgenkristallographische Verfahren untersucht. Die hierbei erhaltenen Ergebnisse unterstützen die für Gruppe-5-[NiFe]-Hydrogenasen postulierte Funktion im Erhaltungsstoffwechsel der Organismen unter besonderen Bedingungen, schließen jedoch eine Beteiligung der AH an der hochaffinen Oxidation von Wasserstoff in Böden aus. Jedoch zeigt das Enzym die neuartige Eigenschaft der sauerstoffinsensitiven Wasserstoff-Oxidation, was auf die Anwesenheit eines ungewöhnlichen, durch 1 Aspartat und 3 Cysteine koordinierten [4Fe4S]-Clusters und der vermuteten Kopplung der Elektronentransportketten in der mutmaßlich physiologischen doppeldimeren Form des Enzyms zurückzuführen sein dürfte. Die Arbeit erweitert somit die Kenntnisse auf dem Gebiet der Sauerstofftoleranz von Hydrogenasen sowie der Eigenschaften der Gruppe 5-[NiFe]-Hydrogenasen und ihrer physiologischen Rolle in den betreffenden Organismen. / In the biogeochemical hydrogen cycle, the dominating process for hydrogen uptake from the atmosphere is performed in soils. Only recently it was shown that hydrogen oxidation in soils is presumably mediated by a number of soil-dwelling actinobacteria, which are enabled in high-affinity hydrogen uptake. These bacteria encode [NiFe] hydrogenases of a novel group classified as group 5 of [NiFe] hydrogenases. A hydrogenase of this group is also found in the beta proteobacterium Ralstonia eutropha and was named „Actinobacterial Hydrogenase“ (AH) for its similarity to the group 5 [NiFe] hydrogenases found in actinobacteria. In this work, the AH from R. eutropha was, as the first group 5 [NiFe] hydrogenase, purified to homogeinity and thoroughly characterized by various biochemical, spectroscopic and X-ray crystallographic methods. The results obtained hereby support the function in maintaining a basal metabolism under challenging conditions, that was postulated for group 5 [NiFe] hydrogenases. Yet, the results also exclude the possibility of the AH contributing to high-affinity hydrogen uptake in soils. However, the enzyme shows the novel property of being able of oxygen-insensitive hydrogen oxidation. This property is obviously connected to an unusual [4Fe4S] cluster coordinated by 1 aspartate and 3 cysteines, as well as to a supposed coupling of the electron transport chains in the double dimeric native form of the enzyme. Hence, this work broadens the knowledge in the field of oxygen tolerant hydrogen oxidation and provides new insights in the function of group 5 [NiFe] hydrogenases and their physiological role in the organisms.

Polyhydroxyalkanoates (PHAs) are polyesters composed of (R)-3-hydroxy-fatty acids. A variety of gram-positive as well as gram-negative bacteria and some archaea are able to produce these biopolymers as energy and carbon storage materials. In times of unbalanced growth, when carbon is available in excess but other nutrients are limited, PHA inclusions are formed. These granules are water-insoluble, stored intracellularly and can be maintained outside the cell as beads. The key enzyme for the formation of PHA inclusions is the PHA synthase PhaC, which catalyses the polymerization of (R)- 3-hydroxyacyl-CoA to PHA with the concomitant release of CoA. The PHA synthase from Ralstonia eutropha (currently Cupriavidus necator), which is covalently bound to the PHA granule surface, tolerates fusions to its N terminus without loss of activity. In this study it was investigated if it would also tolerate translational fusions to its C terminus. A specially designed linker was employed, aiming at maintaining the hydrophobic surroundings of the R. eutropha synthase C terminus to allow proper folding and activity. Two reporter proteins were tested as fusion partners, the maltose binding protein MalE and the green fluorescent protein GFP. As GFP is a hydrophobic protein itself, no additional linker between the PHA synthase and the reporter protein was necessary to produce PHA granules displaying the functional fusion protein on the surface. Principally, the PHA synthase PhaC tolerates translational fusions to its C terminus but the nature of the fusion partner influences the functionality. Recently, PHA granules have often been acknowledged as bio-beads. A one-step production allows the formation of functionalised beads without the need for further cross-linking to impart desired surface properties. PHA beads displaying a gold- or silica-binding peptide at the N terminus of PhaC were constructed and tested for their applicability. Additionally, these beads were able to bind IgG due to the ZZ domain of the IgG binding protein A, which was employed as a linker sequence. These functionalised beads can be used as molecular tools in bioimaging and biomedicine, combining organic core with inorganic-binding shell structures. In a different biomimetic approach, the display of ten lysine residues at the granule surface was achieved using the phasin protein PhaP as the anchoring matrix. Extensive work was performed in an attempt to also employ the synthase protein, but was unsuccessful. These positively charged bio-beads can be used for dispersion or crosslinking experiments as well as silica binding.

No description available.

Hydrogenasen sind komplexe Enzyme, die die reversible Oxidation von molekularem Wasserstoff zu Protonen und Elektronen katalysieren. Diese Enzyme erlauben ihrem Wirtsorganismus das Wachstum unter chemolithoautotrophen Bedingungen. Der Modellorganismus Ralstonia eutropha besitzt drei gut charakterisierte Hydrogenasen der [NiFe]-Klasse, die sich durch ihre Sauerstofftoleranz auszeichnen. Ihr aktives Zentrum besteht aus einer komplexen prosthetischen Gruppe, welche aus einem Nickel- und einem Eisenatom besteht. Letzteres koordiniert drei diatomare Liganden, zwei Cyanide und ein CO. Die Synthese der gesamten Ni(SR)2(µ-SR)2Fe(CN)2(CO)-Gruppe ist ein komplexer Prozess. Die sogenannte Maturation benötigt wenigstens sechs akzessorische Proteine, die sogenannten Hyp-Proteine. Das umfassende Verständnis dieser Maturationsprozesse ermöglicht eine Vielzahl von biotechnologischen Anwendungen. Die vorliegende Arbeit untersucht die Maturation unter verschiedenen Gesichtspunkten. Zentrale, offene Fragen sind die Herkunft des Carbonylliganden sowie die Prozesse, die zur Ligandierung des katalytischen Eisens führen. Dazu wurden molekularbiologische, biochemische und spektroskopische Methoden in Verbindung mit Isotopenmarkierung eingesetzt. Unter anderem konnte dabei gezeigt werden, dass das katalytische Eisen alle seine Liganden bereits im HypCD-Komplex, dem zentralen Element der Maturation, erhält. Ferner konnte in dieser Arbeit, erstmalig für [NiFe]-Hydrogenasen, eine konkrete Biosynthese des seltenen und toxischen diatomaren CO-Liganden beschrieben werden. Ausgehend vom Alpha-Kohlenstoff von Glycin wird der Tetrahydrofolat (THF)-abhängige C1-Metabolismus mit C1-Einheiten versorgt. Durch die enzymatische Aktivität von HypX wird die Formylgruppe von N10-Formyl-THF zu CO umgesetzt. / Hydrogenases are complex enzymes that catalyze the reversible oxidation of molecular hydrogen into protons and electrons. These enzymes allow their host organism to grow under chemolithoautotrophic conditions. The model organism Ralstonia eutropha has three well-characterized [NiFe]-hydrogenases, which exhibit an extraordinary high oxygen tolerance. Its active center is a complex prosthetic group which consists of a nickel and iron atom. The latter coordinates three diatomic ligands, two cyanides and one CO. The biosynthesis of the whole Ni(SR)2(μ-SR)2Fe(CN)2(CO)-group is a complex process. This so-called maturation process needs the activity of at least six accessory proteins, the Hyp-proteins. Understanding the maturation allows a variety of biotechnological applications. The present study examines the maturation of [NiFe]-hydrogenases under different aspects. The major questions concern the origin of the carbonyl ligand as well as the processes that lead to ligandation of the designated catalytic iron. To adress these tasks, molecular biological, biochemical and spectroscopic methods in combination with isotopic labeling were employed. Inter alia, it could be shown that the catalytic iron in the HypCD-complex, the central element of the maturation process, contains all three diatomic ligands. Furthermore, this study describes, for the first time in [NiFe]-hydrogenases, a specific biosynthetic route of the rare and toxic diatomic CO-ligand. Starting from the alpha-carbon of glycine the tetrahydrofolate (THF)-dependent one-carbon metabolism is replenished with one-carbon units. Subsequently the formyl group from N10-formyl-THF is hydrolyzed by the enzymatic activity from HypX and further converted to carbon monoxide as determined by isotopic labeling and infrared spectroscopy.

Die lösliche, NAD+-reduzierende Hydrogenase (SH) aus Ralstonia eutropha H16 ist eine Pyridinnukleotid-abhängige Hydrogenase. Das heißt, der Umsatz von H2 im Hydrogenasemodul des Enzyms ist an die Reduktion von NAD(P)+ im NAD(P)H:Akzeptor-Oxidoreduktasemodul gekoppelt. Die SH ist Vertreter des Subtyps, der auch in Gegenwart von O2 katalytisch aktiv ist. Dies wird ermöglicht durch eine reduktive Entfernung von O2, die nach dem aktuellen Modell abhängig ist vom rückläufigen e--Transport vom NADH:Akzeptor-Oxidoreduktasemodul zum aktiven [NiFe]-Zentrum in der großen Hydrogenaseuntereinheit. Der Einfluss des FeS-Clusters in der kleinen Hydrogenaseuntereinheit HoxY auf diesen Prozess wurde hier untersucht. Dabei konnte gezeigt werden, dass die vier hochkonservierten Cysteine C41, C44, C113 und C179 in HoxY an der Koordination des FeS-Zentrums beteiligt sind. Außerdem wurde das nahegelegene Cystein C39 als relevant für die Sauerstofftoleranz identifiziert. Weiterhin wurde gezeigt, dass das Tryptophan W42 aus HoxY essentiell für die Hydrogenaseaktivität der SH ist. Damit bestätigt sich, dass die Kinetik des rückläufigen e--Transports durch die Aminosäureumgebung des FeS-Clusters in HoxY beeinflusst ist. Ferner wurden in dieser Arbeit Ansätze zum Einsatz der SH aus R. eutropha in einer H2-produzierenden cyanobakteriellen Designzelle weiterverfolgt. Dazu wurden Hybridsysteme aus SH und cyanobakteriellem Photosystem I in vitro hinsichtlich ihrer Fähigkeit zur lichtgetriebenen H2-Produktion untersucht. Außerdem wurde an einem heterologen Expressionssystem der SH für Cyanobakterien gearbeitet. Weiterhin wurde die SH aus Rhodococcus opacus MR11 als komplementäres Modellsystem für O2-tolerante Pyridinnukleotid-abhängige Hydrogenasen etabliert. Dieser zur SH aus R. eutropha homologe Komplex hatte in früheren Arbeiten Vorteile für spektroskopische Studien offenbart und wurde hier erstmals im direkten Vergleich zur SH aus R. eutropha biochemisch und spektroskopisch charakterisiert. / The soluble, NAD+-reducing hydrogenase (SH) from Ralstonia eutropha H16 is a pyridine nucleotide-dependent hydrogenase. In these types of enzymes the conversion of H2 in the hydrogenase module of the complex is coupled to the reduction of NAD(P)+ in the NAD(P)H:acceptor oxidoreductase module. The SH belongs to a subtype that is catalytically active also in the presence of O2. This O2 tolerance is enabled by a reductive removal of O2, which according to the current model depends on a reverse e- flow from the NADH:acceptor oxidoreductase module to the active [NiFe] site in the large hydrogenase subunit. The impact of the FeS cluster in the small hydrogenase subunit HoxY on this process was analyzed in this study. Thereby it was shown that the four highly conserved cysteines C41, C44, C113 and C179 in HoxY are involved in the coordination of the FeS center. Further the nearby cysteine C39 was identified to be relevant for the O2 tolerance of the SH. Additionally we found the tryptophan W42 to be essential for the hydrogenase activity of the SH. Thus it was confirmed that the kinetic of the reverse e- transport is affected by the amino acid environment of the FeS cluster in HoxY. In addition, approaches for using the SH from R. eutropha in H2 producing cyanobacterial design cells were pursued. On one hand hybrid systems consisting of the SH and cyanobacterial photosystem I were analyzed in vitro for their capacity to produce H2 in a light dependent manner. On the other hand work on a heterologous expression system of the SH for Cyanobacteria was continued. Furthermore the SH from Rhodococcus opacus MR11 was established as complementary model system for O2-tolerant pyridine nucleotide-dependent hydrogenases. This complex, which is homologous to the SH from R. eutropha, has revealed advantages for spectroscopic analysis in earlier studies. Here it was characterized biochemically and spectroscopically for the first time in direct comparison with the SH from R. eutropha.

Die NAD+-reduzierende Hydrogenase aus Ralstonia eutropha (SH) katalysiert die reversible H2-Oxidation in Verbindung mit der Reduktion von NAD+ in Gegenwart von Sauerstoff. Die bemerkenswerte O2-Toleranz des Enzyms wurde zuvor auf eine für [NiFe]-Hydrogenasen ungewöhnliche Struktur des Wasserstoff-spaltenden Zentrums zurückgeführt. Diese Hypothese wurde in dieser Arbeit mittels in situ-Spektroskopie an SH-haltigen Zellen widerlegt. Um die folgende Untersuchung der aus sechs Untereinheiten und mindestens acht Kofaktoren bestehenden SH zu erleichtern, wurde das Enzym mittels genetischer Methoden in seine beiden Module aufgeteilt. Das die H2-Oxidation katalysierende Hydrogenase-Modul beinhaltete ein FMN-Molekül, welches für die reduktive Reaktivierung des oxidativ modifizierten Zentrums benötigt wird. Das Diaphorase-Modul besaß ebenfalls ein FMN, und die Reduktion von NAD+ wurde von der Anwesenheit von O2 nicht beeinträchtigt. Neben Wasserstoff reagierte das [NiFe]-Zentrum der SH auch mit Sauerstoff. Dabei wurde sowohl Wasserstoffperoxid- als auch Wasser im Hydrogenase-Modul freigesetzt. Die Sauerstofftoleranz der SH basiert auf einer kontinuierlichen Reaktivierung des durch Sauerstoff oxidierten [NiFe]-Zentrums. Aufgrund der außergewöhnlichen Sauerstofftoleranz stellt die SH ein vielversprechendes System für die wasserstoffgetriebene Regeneration von NADH in gekoppelten enzymatischen Reaktionen dar. In dieser Arbeit wurde ein SH-Derivat durch rationale Mutagenese konstruiert, das in der Lage war, ebenso den Kofaktor NADP+ wasserstoffabhängig zu reduzieren. Durch Ganzzellansätze kann die zeitaufwändige und kostenintensive Proteinreinigung vermieden werden. Um die wasserstoffabhängige in-vivo-Kofaktorregeneration zu ermöglichen, wurde die SH in Pseudomonas putida heterolog produziert. Die in dieser Arbeit erzielten Ergebnisse sind sowohl für das molekulare Verständnis der H2-abhängigen Katalyse als auch für die biotechnologische Anwendung der O2-toleranten SH relevant. / The NAD+ reducing hydrogenase from Ralstonia eutropha (SH) catalyzes the reversible oxidation of hydrogen in connection with the reduction of NAD+ in the presence of oxygen. The remarkable oxygen tolerance was previously related to an unusual [NiFe] active site with four instead of two cyanide ligands. This hypothesis was rejected in this study by using in situ spectroscopy on SH containing cells. To simplify the investigation of the six-subunit and at least eight cofactors containing SH, the enzyme was separated into its two modules by genetic methods. The hydrogen oxidizing hydrogenase module contained one FMN molecule, which was required for the reductive reactivation of the oxidatively modified active site. The diaphorase module carried a second FMN. The reduction of NAD+ was not affected by the presence of oxygen. In addition to hydrogen, the [NiFe] center of the SH reacted with oxygen. Both hydrogen peroxide and water were released by the hydrogenase module. The oxygen tolerance of the SH is based on a continuous reactivation of the oxidized [NiFe] center. Due to the oxygen tolerance, the SH is a promising system for hydrogen based NADH regeneration in coupled enzymatic reactions. In this study a SH derivative was constructed by means of rational mutagenesis. The SH derivative was able to reduce the cofactor NADP+ by hydrogen oxidation. The time consuming and costly protein purification can be avoided by using whole cell approaches. In order to allow the hydrogen dependent in vivo cofactor regeneration, SH was heterologously produced in Pseudomonas putida. The results obtained in this study are relevant for the molecular understanding of hydrogen dependent catalysis and for the biotechnological application of the oxygen tolerant SH.