{"id":{"repo_id":"tu-berlin","oai_identifier":"oai:depositonce.tu-berlin.de:11303/21345"},"canonical_url":"https://search.dev.ndltd.org/etd/tu-berlin/oai:depositonce.tu-berlin.de:11303/21345","repository":{"repo_id":"tu-berlin","name":"Technische Universität Berlin","base_url":"https://api-depositonce.tu-berlin.de/server/oai/request"},"display":{"title":"Vibrational spectroscopic studies on [NiFe] hydrogenases: insights into structure and function","abstract":"Molecular hydrogen has significant potential as an energy carrier in sustainable industrial production aiming to eliminate carbon dioxide emission. This potential, however, relies on the development of electrolyzers for energy storage through water splitting and fuel cell technologies for converting H2 and O2 back into electricity, using cheap, abundant, and efficient catalysts. In nature, H2 cycling is orchestrated by metalloenzymes known as hydrogenases, which operate with remarkable catalytic rates using exclusively nickel and/or iron metals. The discovery of hydrogenases has been a breakthrough towards novel catalytic strategies to replace noble metals like platinum. Additionally, hydrogenases have also been employed in H2-dependent biotechnological applications such as NAD(P)H-cofactor regeneration systems and biosensors. Despite these achievements, these enzymes also display a few drawbacks limiting their application. First, they are not easy to produce as their maturation depends on a complex biosynthetic machinery. Secondly, most hydrogenases are extremely sensitive to oxygen, which inhibits the catalytic sites. Thirdly, as hydrogenases are biomolecules with large molecular weights, the achievement of high catalyst densities at electrode surfaces is often problematic. To overcome these drawbacks, a profound understanding of the catalytic mechanism and the maturation of these enzymes is required, thereby defining the objective of this work which employs a combination of infrared (IR) spectroscopic techniques. In the first part, the catalytic subunit HoxG of the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator, lacking the [Fe-S] cluster-containing small subunit HoxK, has been subjected to detailed spectroscopic investigations. This project revealed so far unknown maturation intermediates of the stepwise assembled [NiFe] cofactor. Among them, the metal-free (preHoxGΔFeNi) and Ni-depleted (preHoxGΔNi) large subunit intermediates offer novel possibilities to introduce different active site metals aiming to develop in the future chemzymes with alternative catalytic functions. Subsequently, the HoxG subunit containing a fully equipped NiFe(CN)2CO cofactor was analyzed in detail in solution and subsequently also immobilized on surfaces using surface-enhanced infrared spectroscopy to understand its supramolecular arrangement, stability and (redox) reactivity. Experimental results were complemented by theoretical calculations by the group of Prof. Mroginski to achieve a comprehensive insight regarding orientation of the immobilized proteins and their distance from the electrode surface. In the second part, the soluble NAD+-reducing [NiFe]-hydrogenase from Hydrogenophilus thermoluteolus and the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator were used as model enzymes for IR spectroscopic investigations targeting the elucidation of certain catalytic and (oxygen-) inhibited intermediates. Both enzymes were investigated in a broad temperature range, focusing on the effect of temperature and light induced perturbations of the enrichment of certain redox species. One of the new discoveries was a light triggered conversion of the fully reduced state of the active site, Nia-SR, to the one-electron oxidized Nia-L. The light-driven Nia-SR → Nia-L reaction represents a photochemical shortcut of the catalytic cycle and may be a milestone for the manipulation of hydrogenases with light. Finally, we additionally resolved the unexpected IR spectral contributions of protonated cysteine residues during the conversion of certain hydrogenase redox states. These findings, backed up by biochemical and computational data performed in collaboration with other researchers highlight the importance of careful interpretation of IR signals.","abstract_html":"Molecular hydrogen has significant potential as an energy carrier in sustainable industrial production aiming to eliminate carbon dioxide emission. This potential, however, relies on the development of electrolyzers for energy storage through water splitting and fuel cell technologies for converting H2 and O2 back into electricity, using cheap, abundant, and efficient catalysts. In nature, H2 cycling is orchestrated by metalloenzymes known as hydrogenases, which operate with remarkable catalytic rates using exclusively nickel and/or iron metals. The discovery of hydrogenases has been a breakthrough towards novel catalytic strategies to replace noble metals like platinum. Additionally, hydrogenases have also been employed in H2-dependent biotechnological applications such as NAD(P)H-cofactor regeneration systems and biosensors. Despite these achievements, these enzymes also display a few drawbacks limiting their application. First, they are not easy to produce as their maturation depends on a complex biosynthetic machinery. Secondly, most hydrogenases are extremely sensitive to oxygen, which inhibits the catalytic sites. Thirdly, as hydrogenases are biomolecules with large molecular weights, the achievement of high catalyst densities at electrode surfaces is often problematic. To overcome these drawbacks, a profound understanding of the catalytic mechanism and the maturation of these enzymes is required, thereby defining the objective of this work which employs a combination of infrared (IR) spectroscopic techniques. In the first part, the catalytic subunit HoxG of the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator, lacking the [Fe-S] cluster-containing small subunit HoxK, has been subjected to detailed spectroscopic investigations. This project revealed so far unknown maturation intermediates of the stepwise assembled [NiFe] cofactor. Among them, the metal-free (preHoxGΔFeNi) and Ni-depleted (preHoxGΔNi) large subunit intermediates offer novel possibilities to introduce different active site metals aiming to develop in the future chemzymes with alternative catalytic functions. Subsequently, the HoxG subunit containing a fully equipped NiFe(CN)2CO cofactor was analyzed in detail in solution and subsequently also immobilized on surfaces using surface-enhanced infrared spectroscopy to understand its supramolecular arrangement, stability and (redox) reactivity. Experimental results were complemented by theoretical calculations by the group of Prof. Mroginski to achieve a comprehensive insight regarding orientation of the immobilized proteins and their distance from the electrode surface. In the second part, the soluble NAD+-reducing [NiFe]-hydrogenase from Hydrogenophilus thermoluteolus and the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator were used as model enzymes for IR spectroscopic investigations targeting the elucidation of certain catalytic and (oxygen-) inhibited intermediates. Both enzymes were investigated in a broad temperature range, focusing on the effect of temperature and light induced perturbations of the enrichment of certain redox species. One of the new discoveries was a light triggered conversion of the fully reduced state of the active site, Nia-SR, to the one-electron oxidized Nia-L. The light-driven Nia-SR → Nia-L reaction represents a photochemical shortcut of the catalytic cycle and may be a milestone for the manipulation of hydrogenases with light. Finally, we additionally resolved the unexpected IR spectral contributions of protonated cysteine residues during the conversion of certain hydrogenase redox states. These findings, backed up by biochemical and computational data performed in collaboration with other researchers highlight the importance of careful interpretation of IR signals.","abstract_has_math":false,"creators":["Karafoulidi-Retsou, Chara"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Hildebrandt, Peter","Zebger, Ingo"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024","date_published":"2024","updated_at":"2026-07-27T21:28:29Z","subjects":[],"languages":["en"],"rights":[],"rights_urls":["https://creativecommons.org/licenses/by-nc-sa/4.0/"],"identifier_entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://doi.org/10.14279/depositonce-20145"],"render_values":[{"text":"https://doi.org/10.14279/depositonce-20145","href":"https://doi.org/10.14279/depositonce-20145","code":true}]}]},"links":{"outbound_url":"https://depositonce.tu-berlin.de/handle/11303/21345","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Hildebrandt, Peter","Zebger, Ingo"]},{"key":"dc:creator","label":"Author","values":["Karafoulidi-Retsou, Chara"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2024-06-10T11:43:29Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-06-10T11:43:29Z"]},{"key":"dc:date.issued","label":"Date","values":["2024"]},{"key":"dc:type","label":"Dc Type","values":["Doctoral Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights.uri","label":"Rights URI","values":["https://creativecommons.org/licenses/by-nc-sa/4.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://depositonce.tu-berlin.de/handle/11303/21345","https://doi.org/10.14279/depositonce-20145"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Molecular hydrogen has significant potential as an energy carrier in sustainable industrial production aiming to eliminate carbon dioxide emission. This potential, however, relies on the development of electrolyzers for energy storage through water splitting and fuel cell technologies for converting H2 and O2 back into electricity, using cheap, abundant, and efficient catalysts. In nature, H2 cycling is orchestrated by metalloenzymes known as hydrogenases, which operate with remarkable catalytic rates using exclusively nickel and/or iron metals. The discovery of hydrogenases has been a breakthrough towards novel catalytic strategies to replace noble metals like platinum. Additionally, hydrogenases have also been employed in H2-dependent biotechnological applications such as NAD(P)H-cofactor regeneration systems and biosensors. Despite these achievements, these enzymes also display a few drawbacks limiting their application. First, they are not easy to produce as their maturation depends on a complex biosynthetic machinery. Secondly, most hydrogenases are extremely sensitive to oxygen, which inhibits the catalytic sites. Thirdly, as hydrogenases are biomolecules with large molecular weights, the achievement of high catalyst densities at electrode surfaces is often problematic. To overcome these drawbacks, a profound understanding of the catalytic mechanism and the maturation of these enzymes is required, thereby defining the objective of this work which employs a combination of infrared (IR) spectroscopic techniques. In the first part, the catalytic subunit HoxG of the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator, lacking the [Fe-S] cluster-containing small subunit HoxK, has been subjected to detailed spectroscopic investigations. This project revealed so far unknown maturation intermediates of the stepwise assembled [NiFe] cofactor. Among them, the metal-free (preHoxGΔFeNi) and Ni-depleted (preHoxGΔNi) large subunit intermediates offer novel possibilities to introduce different active site metals aiming to develop in the future chemzymes with alternative catalytic functions. Subsequently, the HoxG subunit containing a fully equipped NiFe(CN)2CO cofactor was analyzed in detail in solution and subsequently also immobilized on surfaces using surface-enhanced infrared spectroscopy to understand its supramolecular arrangement, stability and (redox) reactivity. Experimental results were complemented by theoretical calculations by the group of Prof. Mroginski to achieve a comprehensive insight regarding orientation of the immobilized proteins and their distance from the electrode surface. In the second part, the soluble NAD+-reducing [NiFe]-hydrogenase from Hydrogenophilus thermoluteolus and the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator were used as model enzymes for IR spectroscopic investigations targeting the elucidation of certain catalytic and (oxygen-) inhibited intermediates. Both enzymes were investigated in a broad temperature range, focusing on the effect of temperature and light induced perturbations of the enrichment of certain redox species. One of the new discoveries was a light triggered conversion of the fully reduced state of the active site, Nia-SR, to the one-electron oxidized Nia-L. The light-driven Nia-SR → Nia-L reaction represents a photochemical shortcut of the catalytic cycle and may be a milestone for the manipulation of hydrogenases with light. Finally, we additionally resolved the unexpected IR spectral contributions of protonated cysteine residues during the conversion of certain hydrogenase redox states. These findings, backed up by biochemical and computational data performed in collaboration with other researchers highlight the importance of careful interpretation of IR signals.","Molekularer Wasserstoff besitzt ein erhebliches Potenzial als Energieträger in einer nachhaltigen industriellen Produktion zu wirken, die darauf abzielt, Kohlenstoffdioxidemissionen zu vermeiden. Voraussetzung dafür ist jedoch die Entwicklung von Elektrolyseuren zur Energiespeicherung durch Spaltung von Wasser und Brennstoffzellentechnologien zur Rückverwandlung von H2 und O2 in Elektrizität unter Verwendung preiswerter und effizienter Katalysatoren. In der Natur wird der H2-Zyklus von Metalloenzymen, den sogenannten Hydrogenasen, gesteuert, die mit bemerkenswerten katalytischen Geschwindigkeiten arbeiten und ausschließlich Nickel- und/oder Eisenmetalle verwenden. Die Entdeckung der Hydrogenasen war ein Durchbruch auf dem Weg zu neuen katalytischen Strategien, die darauf abzielen, Edelmetalle wie Platin zu ersetzen. Darüber hinaus wurden Hydrogenasen auch in H2-abhängigen biotechnologischen Anwendungen wie Systemen zur Regeneration von NAD(P)H-Kofaktoren und als Biosensoren eingesetzt. Trotz dieser Erfolge weisen diese Enzyme auch einige Nachteile auf, die ihre Anwendung einschränken. Erstens sind sie nicht einfach herzustellen, da ihre Reifung von einer komplexen Biosynthesemaschinerie abhängt. Zweitens reagieren die meisten Hydrogenasen extrem empfindlich auf Sauerstoff, der ihre katalytischen aktiven Zentren o􀅌 stark hemmt. Drittens sind Hydrogenasen große Biomoleküle, so dass die Erzielung hoher Katalysatordichten an Elektrodenoberflächen o􀅌 problematisch ist. Um diese Nachteile zu überwinden, bedarf es einer umfassenden Kenntnis der katalytischen Mechanismen der Enzyme und ihrer Assemblierung, wodurch die Ziele dieser spektroskopischen Untersuchung definiert sind. Im ersten Teil wurde die katalytische Untereinheit HoxG der membrangebundenen [NiFe]-Hydrogenase aus Cupriavidus necator, der die [Fe-S] Cluster enthaltende kleine Untereinheit HoxK fehlt, einer detaillierten spektroskopischen Untersuchung unterzogen, wobei bisher unbekannte Reifungsintermediate des schrittweise aufgebauten [NiFe]-Cofaktors entdeckt wurden. Unter ihnen bieten die metallfreien (preHoxG ΔFeNi) und Ni-verarmten (preHoxGΔNi) Zwischenstufen der großen Untereinheit neue Möglichkeiten, verschiedene Metalle in das aktive Zentrum einzuführen, um in Zukunft􀅌 sogenannte „Chemzyme“ mit alternativen katalytischen Funktionen zu entwickeln. Anschließend wurde die HoxG-Untereinheit, die einen vollständig ausgestatteten NiFe(CN)2CO-Cofaktor enthält, in Lösung und anschließend auch immobilisiert auf Oberflächen mit der oberflächenverstärkten Infrarot-Spektroskopie eingehend untersucht, um die supramolekulare Anordnung, Stabilität und (Redox-) Reaktivität der Enzyme zu verstehen. Die experimentellen Ergebnisse wurden durch theoretische Rechnungen in der Gruppe von Prof. Mroginski ergänzt, so dass umfassende Einsichten über die Orientierung der immobilisierten Proteine und deren Abstand zur Elektrodenoberfläche gewonnen werden konnten. Im zweiten Teil wurden die lösliche NAD+-reduzierende [NiFe]-Hydrogenase aus Hydrogenophilus thermoluteolus und die membrangebundene [NiFe]-Hydrogenase aus Cupriavidus necator als Modellenzyme für IR-spektroskopische Untersuchungen verwendet, die auf die Aufklärung bestimmter katalytischer und (Sauerstoff-) inhibierter Zwischenprodukte abzielten. Beide Enzyme wurden in einem weiten Temperaturbereich untersucht, wobei ein Schwerpunkt auf die Auswirkungen von Temperatur und lichtinduzierten Störungen auf die Anreicherung bestimmter Redoxspezies gelegt wurde. Bemerkenswert ist, dass eine durch Licht ausgelöste Umwandlung des vollständig reduzierten Zustands des aktiven Zentrums, Nia-SR, in das mit einem Elektron oxidierte Nia-L nachwiesen werden konnte. Die lichtgesteuerte Nia-SR → Nia-L-Reak􀆟on stellt eine photochemische Abkürzung des katalytischen Zyklus dar und könnte ein Meilenstein für die Manipulation von Hydrogenasen mit Licht sein. Schließlich konnten auch die unerwarteten IR Signale von protonierten Cysteinresten während der Umwandlung bestimmter Hydrogenase-Redoxzustände aufgeklärt werden. Diese Ergebnisse, die durch biochemische und rechnerische Daten anderer Arbeitsgruppen gestützt werden, unterstreichen die Bedeutung einer sorgfältigen Interpretation der IR Spektren."]},{"key":"dc:title","label":"Title","values":["Vibrational spectroscopic studies on [NiFe] hydrogenases: insights into structure and function"]}]}],"canonical_facts":{"dc:contributor.advisor":["Hildebrandt, Peter","Zebger, Ingo"],"dc:creator":["Karafoulidi-Retsou, Chara"],"dc:date.accessioned":["2024-06-10T11:43:29Z"],"dc:date.available":["2024-06-10T11:43:29Z"],"dc:date.issued":["2024"],"dc:description.abstract":["Molecular hydrogen has significant potential as an energy carrier in sustainable industrial production aiming to eliminate carbon dioxide emission. This potential, however, relies on the development of electrolyzers for energy storage through water splitting and fuel cell technologies for converting H2 and O2 back into electricity, using cheap, abundant, and efficient catalysts. In nature, H2 cycling is orchestrated by metalloenzymes known as hydrogenases, which operate with remarkable catalytic rates using exclusively nickel and/or iron metals. The discovery of hydrogenases has been a breakthrough towards novel catalytic strategies to replace noble metals like platinum. Additionally, hydrogenases have also been employed in H2-dependent biotechnological applications such as NAD(P)H-cofactor regeneration systems and biosensors. Despite these achievements, these enzymes also display a few drawbacks limiting their application. First, they are not easy to produce as their maturation depends on a complex biosynthetic machinery. Secondly, most hydrogenases are extremely sensitive to oxygen, which inhibits the catalytic sites. Thirdly, as hydrogenases are biomolecules with large molecular weights, the achievement of high catalyst densities at electrode surfaces is often problematic. To overcome these drawbacks, a profound understanding of the catalytic mechanism and the maturation of these enzymes is required, thereby defining the objective of this work which employs a combination of infrared (IR) spectroscopic techniques. In the first part, the catalytic subunit HoxG of the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator, lacking the [Fe-S] cluster-containing small subunit HoxK, has been subjected to detailed spectroscopic investigations. This project revealed so far unknown maturation intermediates of the stepwise assembled [NiFe] cofactor. Among them, the metal-free (preHoxGΔFeNi) and Ni-depleted (preHoxGΔNi) large subunit intermediates offer novel possibilities to introduce different active site metals aiming to develop in the future chemzymes with alternative catalytic functions. Subsequently, the HoxG subunit containing a fully equipped NiFe(CN)2CO cofactor was analyzed in detail in solution and subsequently also immobilized on surfaces using surface-enhanced infrared spectroscopy to understand its supramolecular arrangement, stability and (redox) reactivity. Experimental results were complemented by theoretical calculations by the group of Prof. Mroginski to achieve a comprehensive insight regarding orientation of the immobilized proteins and their distance from the electrode surface. In the second part, the soluble NAD+-reducing [NiFe]-hydrogenase from Hydrogenophilus thermoluteolus and the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator were used as model enzymes for IR spectroscopic investigations targeting the elucidation of certain catalytic and (oxygen-) inhibited intermediates. Both enzymes were investigated in a broad temperature range, focusing on the effect of temperature and light induced perturbations of the enrichment of certain redox species. One of the new discoveries was a light triggered conversion of the fully reduced state of the active site, Nia-SR, to the one-electron oxidized Nia-L. The light-driven Nia-SR → Nia-L reaction represents a photochemical shortcut of the catalytic cycle and may be a milestone for the manipulation of hydrogenases with light. Finally, we additionally resolved the unexpected IR spectral contributions of protonated cysteine residues during the conversion of certain hydrogenase redox states. These findings, backed up by biochemical and computational data performed in collaboration with other researchers highlight the importance of careful interpretation of IR signals.","Molekularer Wasserstoff besitzt ein erhebliches Potenzial als Energieträger in einer nachhaltigen industriellen Produktion zu wirken, die darauf abzielt, Kohlenstoffdioxidemissionen zu vermeiden. Voraussetzung dafür ist jedoch die Entwicklung von Elektrolyseuren zur Energiespeicherung durch Spaltung von Wasser und Brennstoffzellentechnologien zur Rückverwandlung von H2 und O2 in Elektrizität unter Verwendung preiswerter und effizienter Katalysatoren. In der Natur wird der H2-Zyklus von Metalloenzymen, den sogenannten Hydrogenasen, gesteuert, die mit bemerkenswerten katalytischen Geschwindigkeiten arbeiten und ausschließlich Nickel- und/oder Eisenmetalle verwenden. Die Entdeckung der Hydrogenasen war ein Durchbruch auf dem Weg zu neuen katalytischen Strategien, die darauf abzielen, Edelmetalle wie Platin zu ersetzen. Darüber hinaus wurden Hydrogenasen auch in H2-abhängigen biotechnologischen Anwendungen wie Systemen zur Regeneration von NAD(P)H-Kofaktoren und als Biosensoren eingesetzt. Trotz dieser Erfolge weisen diese Enzyme auch einige Nachteile auf, die ihre Anwendung einschränken. Erstens sind sie nicht einfach herzustellen, da ihre Reifung von einer komplexen Biosynthesemaschinerie abhängt. Zweitens reagieren die meisten Hydrogenasen extrem empfindlich auf Sauerstoff, der ihre katalytischen aktiven Zentren o􀅌 stark hemmt. Drittens sind Hydrogenasen große Biomoleküle, so dass die Erzielung hoher Katalysatordichten an Elektrodenoberflächen o􀅌 problematisch ist. Um diese Nachteile zu überwinden, bedarf es einer umfassenden Kenntnis der katalytischen Mechanismen der Enzyme und ihrer Assemblierung, wodurch die Ziele dieser spektroskopischen Untersuchung definiert sind. Im ersten Teil wurde die katalytische Untereinheit HoxG der membrangebundenen [NiFe]-Hydrogenase aus Cupriavidus necator, der die [Fe-S] Cluster enthaltende kleine Untereinheit HoxK fehlt, einer detaillierten spektroskopischen Untersuchung unterzogen, wobei bisher unbekannte Reifungsintermediate des schrittweise aufgebauten [NiFe]-Cofaktors entdeckt wurden. Unter ihnen bieten die metallfreien (preHoxG ΔFeNi) und Ni-verarmten (preHoxGΔNi) Zwischenstufen der großen Untereinheit neue Möglichkeiten, verschiedene Metalle in das aktive Zentrum einzuführen, um in Zukunft􀅌 sogenannte „Chemzyme“ mit alternativen katalytischen Funktionen zu entwickeln. Anschließend wurde die HoxG-Untereinheit, die einen vollständig ausgestatteten NiFe(CN)2CO-Cofaktor enthält, in Lösung und anschließend auch immobilisiert auf Oberflächen mit der oberflächenverstärkten Infrarot-Spektroskopie eingehend untersucht, um die supramolekulare Anordnung, Stabilität und (Redox-) Reaktivität der Enzyme zu verstehen. Die experimentellen Ergebnisse wurden durch theoretische Rechnungen in der Gruppe von Prof. Mroginski ergänzt, so dass umfassende Einsichten über die Orientierung der immobilisierten Proteine und deren Abstand zur Elektrodenoberfläche gewonnen werden konnten. Im zweiten Teil wurden die lösliche NAD+-reduzierende [NiFe]-Hydrogenase aus Hydrogenophilus thermoluteolus und die membrangebundene [NiFe]-Hydrogenase aus Cupriavidus necator als Modellenzyme für IR-spektroskopische Untersuchungen verwendet, die auf die Aufklärung bestimmter katalytischer und (Sauerstoff-) inhibierter Zwischenprodukte abzielten. Beide Enzyme wurden in einem weiten Temperaturbereich untersucht, wobei ein Schwerpunkt auf die Auswirkungen von Temperatur und lichtinduzierten Störungen auf die Anreicherung bestimmter Redoxspezies gelegt wurde. Bemerkenswert ist, dass eine durch Licht ausgelöste Umwandlung des vollständig reduzierten Zustands des aktiven Zentrums, Nia-SR, in das mit einem Elektron oxidierte Nia-L nachwiesen werden konnte. Die lichtgesteuerte Nia-SR → Nia-L-Reak􀆟on stellt eine photochemische Abkürzung des katalytischen Zyklus dar und könnte ein Meilenstein für die Manipulation von Hydrogenasen mit Licht sein. Schließlich konnten auch die unerwarteten IR Signale von protonierten Cysteinresten während der Umwandlung bestimmter Hydrogenase-Redoxzustände aufgeklärt werden. Diese Ergebnisse, die durch biochemische und rechnerische Daten anderer Arbeitsgruppen gestützt werden, unterstreichen die Bedeutung einer sorgfältigen Interpretation der IR Spektren."],"dc:identifier.uri":["https://depositonce.tu-berlin.de/handle/11303/21345","https://doi.org/10.14279/depositonce-20145"],"dc:language.iso":["en"],"dc:rights.uri":["https://creativecommons.org/licenses/by-nc-sa/4.0/"],"dc:title":["Vibrational spectroscopic studies on [NiFe] hydrogenases: insights into structure and function"],"dc:type":["Doctoral Thesis"]},"updated_at":"2026-07-27T21:28:29Z"}