No description available.

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

The A-cluster of acetyl-CoA synthase (ACS) catalyzes the formation of acetyl- CoA from CO, coenzyme-A, and a methyl group donated by a corrinoid iron-sulfur protein. Recent crystal structures have exhibited three different metals, Zn, Cu, and Ni, in the proximal site, which bridges a square-planar nickel site and a [Fe4S4] cubane. Contradicting reports supported both the nickel and copper containing forms as representing active enzyme. The results presented here indicate that copper is not necessary or sufficient for catalysis and that copper addition to ACS is deleterious. Several proposed mechanisms exist for the synthesis of acetyl-CoA, the two most prominent are the ‘paramagnetic’ and ‘diamagnetic’ mechanisms. The ‘diamagnetic’ mechanism proposes a two electron activation that precedes methylation to produce an EPR silent Ni2+-CH3 species. This then reacts with CO and coenzyme-A to form acetyl- CoA and regenerate the starting species. The ‘paramagnetic’ mechanism assumes a one electron activation prior to the methylation of the paramagnetic Ni1+-CO state to form an unstable Ni3+-acetyl species. This is immediately reduced by an electron shuttle. Results are presented here that no shuttle or external redox mediator is necessary for catalysis. This supports the ‘diamagnetic’ mechanism, specifically that a two-electron reductive activation is necessary and that the Ni1+-CO species is not an intermediate. The two-electron reductive activation required by the ‘diamagnetic’ mechanism results in an unknown electronic state. Two proposals have been made to describe this form of the A-cluster. The first hypothesis from Brunold et al involves a one-electron reduction of the [Fe4S4]2+ cube and a one-electron reduction of the Nip 2+. This should result in a spin-coupled state that is S = integer. The Ni0 hypothesis requires both electrons to localize on the Nip 2+ forming a zero-valent proximal nickel. Mössbauer spectroscopy has been used to probe the oxidation state and spin state of the [Fe4S4] cube in the reduced active form. No integer spin system is found and this is interpreted as supporting the Ni0 hypothesis. Additionally, spectra are presented that indicate the heterogeneous nature of the A-cluster is not caused by the occupancy of the proximal site.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第18339号 / 農博第2064号 / 新制||農||1023(附属図書館) / 学位論文||H26||N4846(農学部図書室) / 31197 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 左子 芳彦, 教授 澤山 茂樹, 教授 菅原 達也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22485号 / 農博第2389号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5265(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 吉田 天士, 教授 澤山 茂樹, 教授 菅原 達也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

One of today’s most pressing scientific challenges is the conception, development and deployment of renewable energy technologies that will meet the demands of a rapidly increasing population. The motivation is not only dwindling fossil fuel reserves, but also the necessary curtailment of emissions of the greenhouse gas carbon dioxide (a product of burning fossil fuels). The sun provides a vast amount of energy (120,000 TW globally), and one major challenge is the conversion of a fraction of this energy into chemical energy, thereby allowing it to be stored. Dihydrogen (H₂) that is produced from water is an attractive candidate to store solar energy (a ‘solar fuel’), as are high energy carbon-containing molecules (such as CO) that are formed directly from carbon dioxide. One key aspect is the development of catalysts that are able to offer high rates and efficiencies. In biology, some microbes acquire energy from the metabolism of H₂ and CO. The biological catalysts - enzymes - that are responsible are hydrogenases (for the oxidation of H₂ to protons); and carbon monoxide dehydrogenases (CODHs, for the oxidation of CO to CO₂). These redox enzymes, containing nickel and iron as the only metals, are extraordinary in terms of their catalytic characteristics: many are fully reversible catalysts and offer very high turnover frequencies (thousands per second are common), with only tiny energy input requirements. This Thesis uses a hydrogenase from the bacterium Escherichia coli, and two CODHs from the bacterium Carboxydothermus hydrogenoformans, as the catalysts in H2 production and CO₂ reduction systems. Chapter 3 describes the concept and development not of a solar fuel system, but of a device that catalyses the water-gas shift reaction (the reaction between CO and water to form H₂ and CO₂) - a process of major industrial importance for the production of high purity H₂. Chapters 4, 5 and 6 detail photochemical CO₂ reduction systems that are driven by visible light. These systems, operating under mild, aqueous conditions, involve CODHs attached either to TiO₂ nanoparticles that are sensitised to visible light by the co-attachment of a ruthenium-based dye complex, or to cadmium sulfide nanomaterials that, having a narrow band gap, are inherently photoexcitable by visible light. The motivation here is not the construction of technological devices; indeed, the enzymes that are used are fragile, highly sensitive to oxygen, and impossible to scale to industrial levels. Rather, the drivers are those of scientific curiosity (can the incorporation of these remarkable biological catalysts enable the creation of outstanding solar fuel devices?), and of producing systems that serve as benchmarks and inspiration for the development of fully synthetic systems that are robust and scalable.

La CO déshydrogénase (CODH) de Desulfovibrio vulgaris est une métalloenzyme qui catalyse la réduction réversible du CO2 en CO. C’est un homodimère composé de deux sites actifs Ni-4Fe-4S et de trois centres fer-soufre. Durant ma thèse, nous avons étudié la maturation de la CODH à nickel et les propriétés catalytiques de la CODH à nickel de D. vulgaris.Pour comprendre le mécanisme de maturation de la CODH à nickel, nous avons caractérisé deux formes de la CODH à nickel produites en présence ou en absence de CooC par des approches biochimiques, spectroscopiques, électrochimiques et cristallographiques. Notre caractérisation montre que la présence de CooC est nécessaire à l’obtention d’une CODH mature et activable. Nous avons également mis en évidence un processus d’activation en présence de nickel dans des conditions réductrices qui n’implique apparemment pas de changement structural du site actif.Notre étude de la CODH à nickel par électrochimie nous a permis de mettre en évidence plusieurs phénomènes d’activations/inactivations de l’enzyme dans des conditions aérobies et anaérobies, et l’existence d’une hétérogénéité fonctionnelle : plusieurs formes de l’enzyme qui montrent des propriétés catalytiques différentes peuvent être présentes simultanément. Cette observation pourrait éclairer d’une façon nouvelle l’hétérogénéité structurale observée par cristallographie et remettre en question les mécanismes proposés sur la base de ces structures. / The monoxide carbon dehydrogenase (CODH) from Desulfovibrio vulgaris is a metalloenzyme which catalyses the reversible reduction of CO2 into CO. It is a homodimer containing two active sites and three iron-sulfur clusters. During my thesis, we studied the maturation of CODH nickel and catalytic properties of Ni-CODH from D. vulgaris.In order to understand, the maturation mechanism of Ni-CODH, we have characterized two forms of Ni-CODH produced in the presence or absence of CooC by biochemical, spectroscopic, electrochemical and crystallographic approaches. Our characterisation shows that the presence of CooC is necessary to obtain a mature Ni-CODH which can be activated. We have also identified an activation process in the presence of nickel in reducing conditions that apparently involves no structural change in the active site.Our study of the Ni-CODH by electrochemistry has shown several phenomena of activation/inactivation of the enzyme under aerobic and anaerobic conditions, and the existence of a functional heterogeneity : several forms of the enzyme which show different catalytic properties may be present simultaneously. This observation could illuminate the structural heterogeneity observed by crystallography and question the proposed mechanisms on the basis of these structures.