Hydrogenasen sind essentielle Enzyme im mikrobiellen H2-Kreislauf und werden als vielversprechende Katalysatoren in biologisch basierten H2-Technologien angesehen. Ein entscheidender Nachteil vieler Hydrogenasen ist ihre hohe O2-Sensitivität. Die membrangebundene Hydrogenase (MBH) aus Ralstonia eutropha ist eines der wenigen Beispiele für Hydrogenasen, die in Gegenwart von O2 katalytisch aktiv sind. Die molekularen Ursachen dieser O2 Toleranz sind bislang ungeklärt. In bisherigen Studien wurde lediglich das [NiFe]-Zentrum und dessen Umgebung auf Faktoren untersucht, die die O2-Toleranz des Enzyms hervorrufen könnten. In dieser Arbeit wurde daher der Fokus auf die kleine Untereinheit der MBH gelegt, in der sich drei elektronentransferierende Fe-S Cluster befinden. Die ligandierenden Aminosäuren dieser Fe-S Cluster wurden mittels ortsspezifischer Mutagenese verändert und die resultierenden MBH-Varianten physiologisch, biochemisch, spektroskopisch und elektrochemisch charakterisiert. Dabei wurde gezeigt, dass die O2-Toleranz der MBH maßgeblich auf einer Modifikation eines dieser drei Fe-S Cluster beruht. In der direkten Umgebung des zum aktiven Zentrum nächstgelegenen Fe-S Clusters befinden sich sechs statt vier Cysteine, wie in O2 sensitiven [NiFe]-Hydrogenasen. Die beiden zusätzlichen Cysteine um dieses proximale Cluster wurden gegen Glycine ausgetauscht, die an der entsprechenden Position in O2-sensitiven Hydrogenasen zu finden sind. Der Austausch der zusätzlichen Cysteine führte in vivo und in vitro zu einer erhöhten O2 Sensitivität der MBH. In EPR-spektroskopischen Untersuchungen wurde beobachtet, dass diese MBH-Variante veränderte elektronische Eigenschaften aufweist. Statt des für O2-tolerante Hydrogenasen typischen EPR-Spektrums wurde ein Signal detektiert, welches in O2-sensitiven Hydrogenasen zu finden ist. Anhand der Ergebnisse wurde ein Modell erstellt, das erklärt, wie eine modifizierte Fe-S Clusterkette zur O2-Toleranz von Hydrogenasen beiträgt. / Hydrogenases are essential for H2 cycling in microbial metabolism and serve as valuable blueprints for H2-based biotechnological application. Like many metalloproteins, most hydrogenases are extremely oxygen-sensitive and prone to inactivation by even traces of O2. The O2-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha is one of the few examples that have established a mechanism enabling H2 uptake in the presence of ambient O2. The molecular mechanisms of this O2 tolerance are not yet unravelled. However, up to date, only the large subunit harbouring the [NiFe] active site has been in the focus of studies on O2 tolerance. In the present study, the role of the small subunit with its electron relay, consisting of three Fe-S clusters, was investigated. Amino acid residues involved in coordination of all three clusters were exchanged, and the resulting MBH variants were investigated with physiological, biochemical, electrochemical and spectroscopic methods. It is shown that the rare feature of O2 tolerance is crucially related to a modification of the electron transfer chain. The Fe-S cluster proximal to the catalytic centre is surrounded by six instead of the four conserved coordinating cysteines. Removal of the two additional cysteines renders the protein O2-sensitive in vivo and in vitro. Electron paramagnetic resonance spectroscopy of this MBH variant revealed a signal resembling the spectrum usually detected in O2-sensitive [NiFe]-hydrogenases. The data imply that the major mechanism of O2 tolerance is based on the reductive removal of oxygenic species guided by the unique architecture of the electron transport chain rather than a restricted access of O2 to the active site.

In times with elevated CO2 levels and global warming there is a need of finding alternatives to carbon based energy carriers. One such environmental friendly solution could be H2 produced by living organisms. Cyanobacteria are good candidates since they can produce H2 from sunlight and water through the combination of photosynthesis and H2 producing enzymes i.e. nitrogenases and/or [NiFe]-hydrogenases. This thesis investigates the maturation and transcriptional regulation of [NiFe]-hydrogenases in cyanobacteria, with a special focus on hydrogenase specific proteases. The core of all hydrogenases consists of the small and large subunit. The large subunit in which the catalytic site is located goes through an extenstive maturation process which ends with a proteolytic cleavage performed by a hydrogenase specific protease (HupW/HoxW). This thesis shows that within the maturation process of hydrogenases, the proteolytic cleavage is probably the only step that is specific with respect to different types of hydrogenases i.e. one type of protease cleaves only one type of hydrogenase. Further in-silico analysis revealed that these proteases and the hydrogenases might have co-evolved since ancient time and that the specificity observed could be the result of a conserved amino acid sequence which differs between the two types of proteases (HupW/HoxW). A number of different transcription factors were revealed and shown to interact with the promoter regions of several of the genes encoding maturation proteins. The results indicate that the hydrogenase specific proteases are regulated on a transcriptional level in a similar manner as the hydrogenases they cleave. This thesis contributes with knowledge concerning transcriptional regulation and protein regulation of hydrogenases which will be useful for designing genetically engineered cyanobacteria with an improved and adjustable H2 production. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 722

Hydrogenases are a class of metalloenzymes which catalyse H₂ oxidation and its reverse reaction, H⁺ reduction. There is interest in investigating how H₂ as an energy carrier is cycled in biology. Hydrogenases have also been studied extensively because there are potential applications for them as catalysts for H₂ oxidation in fuel cells or H₂ production via light-driven water splitting. For these applications, the ability for the hydrogenase to work in the presence of O₂ is an important issue. The microorganism Ralstonia eutropha is a well-studied model aerobic H₂ oxidiser: it can adopt H₂ as the sole energy source to grow cells in the presence of O₂. It produces at least three distinct O₂-tolerant NiFe hydrogenases: the membrane-bound hydrogenase (MBH), the NAD⁺-reducing soluble hydrogenase (SH) and the regulatory hydrogenase (RH). This Thesis employs protein film electrochemistry (PFE) to study the SH and RH. It is found that the SH is able to work in both direction (H₂ oxidation and H⁺ reduction) with minimum overpotential, which is critical in coupling 2H⁺/H₂ cycling with the closely spaced NAD⁺/NADH potential. Reactions of the SH with O₂ have been investigated, revealing at least two distinct O₂-inactivated states, but consistent with the requirement for the SH to function in air, it can be reactivated in the presence of O₂ at low potentials which could be provided by the NAD⁺/NADH pool in vivo. The affinity of the RH for H₂ was determined by PFE and found to be slightly higher than that of the SH and MBH. This may provide a way for the microbe to regulate hydrogenase expression in response to the H₂ availability. Carbon monoxide and O₂-inactivated states of the RH have been identified for the first time, confirming that a constricted gas channel is not sufficient to explain its O₂ tolerance. Observation of potential dependent reactions in hydrogenases means that it is important to have spectroscopic methods for characterising states triggered by inhibitors and potential. An Infrared spectroelectrochemical approach suitable for studying metalloenzymes has been developed and preliminary spectra on RH recorded. This method should provide many opportunities for future studies of redox states of hydrogenases.

Hydrogenases catalyse the interconversion of H₂ and H⁺. Protein Film Electrochemistry (PFE), a technique in which a redox enzyme is adsorbed directly onto an electrode, enables a detailed description of the catalytic function of these metalloenzymes to be obtained. Unlike small-molecule electrocatalysts, the hydrogenase active site is surrounded by a protein structure ensuring that it is relatively unperturbed by the electrode surface. In this thesis, PFE is used alongside mathematical modelling to explain differences between [NiFe]- and [FeFe]-hydrogenases, highlighting some important considerations for efficient, reversible electrocatalysis. This thesis probes the unusual reaction between [NiFe]-hydrogenases and cyanide. Through a detailed study utilising PFE, Electron Paramagnetic Resonance (EPR) and Attenuated Total Reflection Infrared spectroelectrochemistry (ATR-IR), it is demonstrated that cyanide promotes the formation of the inactive Ni-B state. Preferred formation of the Ni-B state over more slowly reactivating Unready states is considered an important characteristic of the O₂-tolerant class of [NiFe]-hydrogenases. The nature of the Ni-L state, commonly thought to be an artefact formed when a [NiFe]-hydrogenase is exposed to visible light, is probed via EPR and ATR-IR. In this thesis, the Ni-L state is shown to occur in samples of Hydrogenase-1 from Escherichia coli that have not been exposed to visible light, calling into question the true nature of this state. Finally, this thesis details the first study in which PFE is used to investigate the spontaneous incorporation of a synthetic active site mimic complex into apo-hydrogenase. Incorporation into apo-hydrogenase from Chlamydomonas reinhardtii and Clostridium pasteurianum is discussed, in both cases resulting in fully functional [FeFe]-hydrogenase, electrochemically indistinguishable from the native enzyme.

Nicotinamide cofactors in their reduced and oxidised forms are important redox agents in biology. Of about 3000 dehydrogenases available to date, many require these cofactors for their activity. Dehydrogenases are of interest to chemists as they offer asymmetric catalysis to yield chiral products. The requirement of dehydrogenases for nicotinamide cofactors necessitates research into finding the best way of recycling the oxidised or reduced forms of these cofactors. Electrocatalytic NAD(P)H oxidation and NAD(P)⁺ reduction on standard electrodes is problematic due to unwanted side reactions and high overpotential requirements, but in Nature efficient enzyme catalysts are available to facilitate these reactions. The focus of this Thesis, the Soluble Hydrogenase of R. eutropha (SH) is a multimeric bidirectional hydrogenase that couples H2 oxidation to the reduction of NAD⁺ to NADH. Protein Film Electrochemistry (PFE) has been employed to study NAD⁺-reducing catalytic moieties of the SH for the first time. It is shown that SH subunits on an electrode are able to catalyse NADH oxidation and NAD⁺ reduction efficiently with minimal overpotential, which is significant because in vivo, NAD(H) cycling is coupled to 2H⁺/H₂ cycling and these reactions are closely spaced in potential. Substrate affinities and inhibition constants for the SH, determined using PFE are discussed in the context of the SH function and the related catalytic domains of respiratory Complex I. A range of molecules that are known to inhibit the related Complex I have been investigated for their ability to inhibit the SH moieties: the similarity between inhibition constants is consistent with structural and functional similarity between the SH and Complex I. The ability of the SH moieties to sustain NAD(H) catalysis in the presence of O₂ is also demonstrated and is consistent with the requirement for the SH to function under aerobic conditions and to reactivate the inactivated hydrogenase moiety by supplying low potential electrons from NADH. Engineered variants of the SH, designed to enhance the affinity towards NADP⁺, were investigated for the first time, using PFE. Electrochemical characterisation of the variants is presented and results are discussed alongside findings on the wild type SH. The variants are shown to exhibit NADP⁺ reduction, and to have higher affinity towards NADP⁺ than the wild type SH. The first efficient NADP⁺ reduction and NADPH oxidation is observed for one of the variants on a graphite electrode and the best variant showed a K_M of 1.7 mM for NADP⁺. This Thesis also provides evidence for the ability of moieties of the SH to be used in cofactor regeneration systems. Two novel systems are demonstrated. The first involves H₂ driven NADH recycling based on the NAD⁺-reducing moiety of the SH immobilised on graphite particles together with a hydrogenase or platinum, with electrons from H₂ passed from the hydrogenase through the graphite to the NAD⁺-reducing moiety. The second involves an electrode modified with the NAD⁺-reducing moiety of the SH, and is demonstrated as an electrochemical NADH recycling system coupled with NADH-dependent pyruvate reduction to lactate by lactate dehydrogenase. The ability of variants of the SH to catalyse NADP⁺ reduction suggests that it may also be possible to use these systems for recycling NADPH for catalysis of important biotransformation reactions by NADPH-dependent dehydrogenases.

The Cardiac Profile, a pattern of serum enzyme changes seen within seventy-two hours after an AMI, is diagnostic aid for detecting occurrence of infarcts. The effects of exercise stress on the Cardiac Profile aid clinicians in avoiding diagnostic errors in patients immediately after exercise. Five male volunteers ran from six to ten miles. Serum enzyme levels were monitored serially three days before and five days after stress. Enzyme activity was determined spectrophotometrically and electrophoretically. Significant increases in total CPK and LDH were seen. An LDH 'one-two flip' occurred eight hours after exercise. No MB-CPK was found following the run.

O uso intensificado de combustíveis fósseis como fonte de energia, vê-se a necessidade do desenvolvimento de novas tecnologias, principalmente as renováveis, como o hidrogênio, que possui vantagens por ser elemento abundante no universo, ser renovável e não poluente. A utilização de microalgas e cianobactérias é uma alternativa para a produção de biohidrogênio a partir da quebra da água e de compostos orgânicos. De acordo com isso, nesta pesquisa foram testados diversos fatores físico-químicos e nutricionais nas condições de cultivo de cepas de Chlamydomonas spp. e Anabaena spp. Para tanto, cepas selecionadas foram cultivadas em duas fases experimentais, a primeira aeróbia e a segunda anaeróbia, para proporcionar produção de hidrogênio por biofotólise direta anaeróbia, via hidrogenase, sob privação de enxofre para a clorofícea, e de nitrogênio para a cianobactéria, estimulando para esta também a produção por biofotólise indireta, via nitrogenase. A cepa com melhor produtividade de hidrogênio, de cada gênero, foi selecionada para a etapa de otimização das fases experimentais de cultivo. Durante os ensaios foram realizadas análises de produção máxima, velocidade de produção, volume e produtividade de hidrogênio, além de análises de concentração de biomassa, físico-químicas, bioquímicas e geração de subprodutos. O método utilizado foi eficiente para produção de hidrogênio e ficou comprovada a diferença de produção de hidrogênio entre diferentes cepas. Anabaena sp. obteve produtividade média de hidrogênio quatro vezes maior, aproximadamente de 76,8 µmol.L-1.h-1, comparada a C. reinhardtii, com média de 18,6 µmol.L-1.h-1. / The intensifying use of fossil fuels as energy source, one sees the need to develop new technologies, especially renewable, such as hydrogen. This has advantages because hydrogen is an abundant element in the universe, be renewable and non-polluting. The use of microalgae and cyanobacteria is an alternative for the production of bio-hydrogen of breaking water and organic compounds. Accordingly, in this study were tested several physic-chemical factors and nutrition in growing conditions of Chlamydomonas spp. and Anabaena spp. strains. For this purpose, strains selected were cultured in two experimental phases, first aerobic and second anaerobic, to hydrogen production by direct biofotolise anaerobic, via hydrogenase, under sulfur deprived to chlorofycea, and nitrogen to cyanobacterium, for this also to production by indirect biofotolise, via nitrogenase. The strain with highest productivity of hydrogen, of each gender, was selected for the optimization of the experimental stages of cultivation. During the tests were analyzes of maximum production, velocity, volume and productivity of hydrogen, and analysis of biomass concentration, physic-chemical, biochemical and generation of by-products. The method used was efficient for the production of hydrogen and was different between strains. Anabaena sp. obtained average yield four times highest, approximately 76.8 µmol. L-1.h-1compared to C. reinhardtii, averaging 18.6 µmol. L-1.h-1.

Les centres FeS sont un des cofacteurs protéiques majeurs, ils se trouvent aussi bien chez les bactéries que chez les eucaryotes. Ils ont des rôles essentiels de transfert d'électron, liaison de substrat et son activation, régulation d'expression de gènes, donneur de soufre etc. Leur agencement est très varié, allant du centre [2Fe-2S] à l'agrégat plus complexe MoFe7S9X (X = C, N ou O) de la nitrogénase. L'assemblage de ces centres se fait par des machineries protéiques. Nous avons étudié le système ISC (Iron-Sulfur Cluster) chez les bactéries, qui fabrique des centres [2Fe-2S] et [4Fe-4S]. Il est composé des protéines IscS, IscU, IscA, HscA, HscB et d'une ferrédoxine. Deux de ces protéines, IscS, qui est une cystéine désulfurase et IscU, protéine dite échafaudage, sont le cœur de la machinerie puisque IscS apporte le soufre sur la protéine IscU, qui, avec le fer qu'elle aura obtenu d'une autre protéine (non clairement identifiée à ce jour), fabriquera le centre fer-soufre et le transfèrera à une apoprotéine. Nous avons isolé un complexe stable (IscS-D35A-IscU)2 contenant un centre [2Fe-2S] dans des conditions anaérobie. Différentes formes du complexe ont été obtenues et cristallisées afin d'obtenir leurs structures, résolues par remplacement moléculaire. Ces structures nous ont permis de proposer un mécanisme d'assemblage des centres [2Fe-2S] à l'échelle atomique et électronique. Nous avons d'autre part étudié la protéine HmdB probablement impliquée dans la maturation de l'hydrogénase à fer. HmdB fait partie de la superfamille des protéines à radical SAM. Des cristaux de l'apoprotéine ont été obtenus et sa structure a été résolue par remplacement moléculaire. Même si une partie de la structure n'est pas visible du fait de l'absence de centre [4Fe-4S], elle donne une première vue du site actif de la protéine. / FeS clusters are widely used protein cofactors, found both in bacteria and eukaryotes. They play key roles such as electron transfer, substrate binding and activation, regulation of gene expression, sulfur donor etc. They are really various, ranging from the [2Fe-2S] cluster to the more complex MoFe7S9X (X = C, N or O) agregate of nitrogenase. Clusters assembly is carried out by protein machineries. We studied the ISC (Iron-Sulfur Cluster) in bacteria, who assembles [2Fe-2S] and [4Fe-4S] clusters. It is composed of IscS, IscU, IscA, HscA, HscB proteins and a ferredoxin. Two of these proteins: the cysteine desulfurase IscS, and the scaffold protein IscU, represent the core of the machinery as IscS provides sulfur protein on IscU, which, with iron obtained from another protein (not clearly identified to date), assemble the iron-sulfur center. The latter transfers it to an apoprotein. We isolated under anaerobic conditions a stable (IscS-D35A-IscU)2 complex containing a [2Fe-2S] cluster. Different forms of the complex were obtained and their structures were solved by molecular replacement. These structures allowed us to propose a mechanism for the assembly of the [2Fe-2S] clusters at the atomic and electronic levels. We have also studied the HmdB protein, which is proposed to maturate the [Fe]-hydrogenase. HmdB is a member of the radical SAM proteins superfamily. Crystals of the apoprotein were obtained and its structure was solved by molecular replacement. Although part of the structure is not visible due to the absence of the [4Fe-4S] cluster, this structure gives a first view of the active site of the protein.

Il existe dans la Nature des métalloenzymes, les hydrogénases, capables de produire et consommer du dihydrogène de façon catalytique. La résolution des structures de ces hydrogénases a révélé la présence de sites actifs de nature organométallique, pouvant-être décrits comme des entités bimétalliques à base de fer et de nickel. Dès lors, de nombreux complexes dinucléaires du fer ont été synthétisés en vue de comprendre et de reproduire les processus de conversion H+/H2 réalisés par les hydrogénases [FeFe]. En particulier, des composés dithiolato-pontés dinucléaires du fer, possédant des ligands bidentates ont été élaborés suite aux résultats d'études théoriques ayant proposé qu'une disubstitution dissymétrique sur un des atomes de fer de ces molécules pouvait permettre de reproduire certaines caractéristiques structurales clés du site actif. Une partie des travaux décrits dans ce mémoire s'inscrit dans la suite des études entreprises dans le laboratoire concernant l'utilisation de ligands bidentates chélatants dans des complexes dinucléaires du fer inspirés par le site actif des hydrogénases [FeFe], comme la 1,10-phénanthroline ou encore les diphosphines. L'autre partie concerne le développement d'une voie de synthèse systématique pour l'obtention de complexes trinucléaires originaux du fer présentant un agencement quasi-linéaire des trois atomes de fer. Le comportement électrochimique des espèces synthétisées ainsi que leur réactivité en milieu acide ont été étudiés. / Hydrogenases are metalloenzymes found in Nature which are able to catalyse the production and the uptake of dihydrogen. Reports orr the structures of these hydrogénases have revealed the organometallic nature of their active sites, which are based on bimetallic assemblies containing nickel and iron atoms. Therefore, many diiron complexes have been synthesized in order to understand and to reproduce the high efficiency of [FeFe]- hydrogenases towards the reversible conversion of protons into dihydrogen. In particular, dithiolato-bridged diiron compounds, with bidentate ligands, have been developed in reason of results of theoretical studies which have suggested that an asymmetrical disubstitution at one iron atom of such bioinspired molecules may allow to reproduce some structural key features of the active site. One part of the works reported in this thesis concems pursuing studies previously undertaken in the laboratory on the use of bidentate chelating ligands with diiron bioinspired models of [FeFe] hydrogenases, such as 1,10- phenanthroline or diphosphines. Another part reports the development of a systematîc way for the synthesis of original trinuclear iron clusters having a quasi-linear arrangement of the three iron atoms. Electrochemical behaviours of the synthesized species as well as their reactivity in acidic medium have been studied.

<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>