Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information
<p>The catalytic mechanism of [NiFe]-hydrogenases is a complex, multi-step process involving several spectroscopically well-defined intermediates. Although the chemistry of each stage of the reaction cycle is generally understood, the precise interplay between the protein and the active site c...
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| Format: | Thesis |
| Language: | English |
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2024
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| _version_ | 1826315171075719168 |
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| author | Wong, KL |
| author2 | Vincent, K |
| author_facet | Vincent, K Wong, KL |
| author_sort | Wong, KL |
| collection | OXFORD |
| description | <p>The catalytic mechanism of [NiFe]-hydrogenases is a complex, multi-step process involving several spectroscopically well-defined intermediates. Although the chemistry of each stage of the reaction cycle is generally understood, the precise interplay between the protein and the active site cluster that enables such efficiency (TOF >1000s-1) remains unresolved. To address this knowledge gap, this thesis investigates an extensive structural characterisation of catalytic intermediates of E. coli Hyd-1 and Hyd-2. Previously, the Vincent group has employed an approach that combines electrochemical control of the redox state of protein crystals with infrared (IR) microspectroscopy to track changes in the speciation of the active site.1, 2 This methodology allows the generation of "roadmaps" illustrating the potentials at which the crystals contain, or are highly enriched in, a specific intermediate (such as Nia C) of the reaction cycle.</p>
<p>Modifying this methodology has enabled the recovery of electrochemically poised protein crystals for structure determinations using a range of diffraction techniques: X-ray diffraction at synchrotron and X-ray free electron laser (XFEL) sources, neutron diffraction, and microcrystal electron diffraction (MicroED). Overall this work highlights that this combination of techniques present a versatile approach to elucidate the structures of intermediates within the catalytic cycle. These methodologies showcase how electrochemical methods can effectively manipulate crystals of [NiFe]-hydrogenase into specific redox states, followed by the collection of diffraction data to capture static "snapshots" of catalytic intermediates under both cryogenic and ambient conditions. This collective ensemble of structures provides unparalleled insights into catalysis, revealing intricate details of the mechanism at the atomic level.</p> |
| first_indexed | 2024-12-09T03:21:25Z |
| format | Thesis |
| id | oxford-uuid:cf9f3fe3-545a-4a1a-9976-013a386db353 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:21:25Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:cf9f3fe3-545a-4a1a-9976-013a386db3532024-11-12T09:48:40ZUnderstanding biological hydrogen oxidation catalysis - uniting activity, structure and function informationThesishttp://purl.org/coar/resource_type/c_db06uuid:cf9f3fe3-545a-4a1a-9976-013a386db353Time-resolved spectroscopyChemistry, InorganicElectrodes, EnzymeX-ray spectroscopyX-rays--DiffractionElectron paramagnetic resonance spectroscopyInfrared spectroscopyElectrochemistryEnglishHyrax Deposit2024Wong, KLVincent, K<p>The catalytic mechanism of [NiFe]-hydrogenases is a complex, multi-step process involving several spectroscopically well-defined intermediates. Although the chemistry of each stage of the reaction cycle is generally understood, the precise interplay between the protein and the active site cluster that enables such efficiency (TOF >1000s-1) remains unresolved. To address this knowledge gap, this thesis investigates an extensive structural characterisation of catalytic intermediates of E. coli Hyd-1 and Hyd-2. Previously, the Vincent group has employed an approach that combines electrochemical control of the redox state of protein crystals with infrared (IR) microspectroscopy to track changes in the speciation of the active site.1, 2 This methodology allows the generation of "roadmaps" illustrating the potentials at which the crystals contain, or are highly enriched in, a specific intermediate (such as Nia C) of the reaction cycle.</p> <p>Modifying this methodology has enabled the recovery of electrochemically poised protein crystals for structure determinations using a range of diffraction techniques: X-ray diffraction at synchrotron and X-ray free electron laser (XFEL) sources, neutron diffraction, and microcrystal electron diffraction (MicroED). Overall this work highlights that this combination of techniques present a versatile approach to elucidate the structures of intermediates within the catalytic cycle. These methodologies showcase how electrochemical methods can effectively manipulate crystals of [NiFe]-hydrogenase into specific redox states, followed by the collection of diffraction data to capture static "snapshots" of catalytic intermediates under both cryogenic and ambient conditions. This collective ensemble of structures provides unparalleled insights into catalysis, revealing intricate details of the mechanism at the atomic level.</p> |
| spellingShingle | Time-resolved spectroscopy Chemistry, Inorganic Electrodes, Enzyme X-ray spectroscopy X-rays--Diffraction Electron paramagnetic resonance spectroscopy Infrared spectroscopy Electrochemistry Wong, KL Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information |
| title | Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information |
| title_full | Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information |
| title_fullStr | Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information |
| title_full_unstemmed | Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information |
| title_short | Understanding biological hydrogen oxidation catalysis - uniting activity, structure and function information |
| title_sort | understanding biological hydrogen oxidation catalysis uniting activity structure and function information |
| topic | Time-resolved spectroscopy Chemistry, Inorganic Electrodes, Enzyme X-ray spectroscopy X-rays--Diffraction Electron paramagnetic resonance spectroscopy Infrared spectroscopy Electrochemistry |
| work_keys_str_mv | AT wongkl understandingbiologicalhydrogenoxidationcatalysisunitingactivitystructureandfunctioninformation |