| Agradecimentos |
| Resumo |
| Abstract |
| Résumé |
| Table of contents |
| Index of Figures |
| Index of Tables |
| List of Abbreviations |
| Outline of the thesis |
| 1 | The Metabolism of Pyrococcus furiosus | |
| 1.1 | The metabolism of carbohydrates | |
| 1.2 | The fate of the reducing equivalents | |
| 1.2.1 | Reduction of elemental sulfur | |
| 1.2.2 | Formation of alanine | |
| 1.2.3 | Proton reduction to H2 |
| 2 | The molecular basis of hydrogen metabolism | |
| 2.1 | Ecological fluxes of H2 | |
| 2.2 | Types of hydrogenases | |
| 2.2.1 | Catalytic Properties in Common | |
| 2.3 | Hydrogenases Containing only Iron-Sulfur Clusters | |
| 2.4 | Nickel-containing hydrogenases | |
| 2.4.1 | EPR Spectroscopy | |
| 2.4.2 | X-ray absorption spectroscopy | |
| 2.4.3 | Fourier Transform Infra Red Spectroscopy | |
| 2.5 | Catalytic mechanism of [NiFe] hydrogenases | |
| 2.5.1 | Electron transfer | |
| 2.5.2 | Hydrogen activation | |
| 2.6 | Potential applications of hydrogenase |
| 3 | Materials and Methods | |
| 3.1 | Growth of Organism | |
| 3.2 | Cell lysis | |
| 3.3 | Isolation of membranes | |
| 3.4 | preparation of cell extracts | |
| 3.5 | Protein purification | |
| 3.5.1 | The soluble sulfhydrogenase I | |
| 3.5.2 | Sulfide dehydrogenase | |
| 3.5.3 | Partial purification of the membrane-bound hydrogenase. | |
| 3.5.4 | Ferredoxin | |
| 3.5.5 | Pyruvate: ferredoxin oxidoreductase | |
| 3.6 | Assays. | |
| 3.6.1 | Hydrogen production | |
| 3.6.1.1 | Sensitivity to inhibitors | |
| 3.6.1.2 | H2 production from pyruvate | |
| 3.6.2 | Hydrogen uptake | |
| 3.6.3 | Sulfide dehydrogenase | |
| 3.7 | Protein determination | |
| 3.8 | Iron determination with ferene | |
| 3.9 | Flavin extraction and characterization | |
| 3.10 | Anaerobic oxidation of sulfhydrogenase | |
| 3.11 | Preparation of samples for EPR. | |
| 3.12 | Mediated redox titrations. | |
| 3.13 | EPR spectroscopy | |
| 3.14 | Sequence analysis | |
| 3.15 | Protein analysis. |
| 3.16 | Gel blotting | |
| 4 | The Soluble Sulfhydrogenase I | |
| 4.1 | Reported properties of Pyrococcus furiosus sulfhydrogenase | |
| 4.2 | Derivation of prosthetic group structures from sequence analysis | |
| 4.3 | Catalytic properties | |
| 4.4 | EPR measurements | |
| 4.5 | Reduction with NADPH | |
| 4.6 | Redox titration at physiological temperatures. | |
| 4.7 | T-jump experiments | |
| 5 | The Sulfide Dehydrogenase |
| 5.1 | Background |
| 5.2 | The primary structure of SuDH translated from the genome | |
| 5.3 | Characterization of prosthetic groups through sequence comparisons | |
| 5.4 | Isoenzymes of sulfide dehydrogenase and sulfhydrogenase | |
| 5.5 | Chemical analysis and activity of SuDH | |
| 5.6 | EPR monitored redox titration of the Fe/S clusters | |
| 5.7 | Discussion | |
| 6 | The Membrane-Bound Hydrogenase |
| 6.1 | The current model of hydrogen production by P. furiosus | |
| 6.2 | Kinetics of soluble sulfhydrogenase I and of sulfide dehydrogenase I | |
| 6.3 | Whole cell acetate fermentation and activities in H2 metabolism | |
| 6.4 | Membrane-bound hydrogenase activity. | |
| 6.5 | Purification | |
| 6.6 | Sequence analyses | |
| 6.7 | Description of the mbh gene products. | |
| 6.8 | N-terminal sequencing. | |
| 6.9 | Enzymology. | |
| 6.9.1 | General properties | |
| 6.9.2 | CO inhibition. | |
| 6.10 | Inhibition by DCCD. | |
| 6.11 | EPR spectroscopy. | |
| 6.12 | A putative fourth hydrogenase in P. furiosus | |
| 6.13 | Discussion | |
| 7 | The molecular diversity of hydrogenases | |
| 7.1 | The relevance of sequence comparisons | |
| 7.2 | Phylogenetic relationships between the selected hydrogenases | |
| 7.3 | NAD-linked hydrogenases | |
| 7.4 | Sulfhydrogenases | |
| 7.5 | F420-non-reducing hydrogenases | |
| 7.6 | F420-reducing hydrogenases | |
| 7.7 | Complex membrane-bound hydrogenases | |
| 7.8 | Atypical hydrogenases (MbxJ from P. furiosus and homologues) | |
| 7.9 | Discussion | |
| 8 | Conclusions | |
| 8.1 | Sulfhydrogenase | |
| 8.2 | Sulfide dehydrogenase | |
| 8.3 | The pathway of Hydrogen evolution from reduced ferredoxin | |
| 8.4 | Insights from sequence comparisons | |
| 9 | References | |
| Appendix | A short primer on sequence comparisons |