Unifying information on reactivity of NiFe hydrogenases from different sample handling and spectroelectrochemical techniques

Thesis

This thesis investigates the catalytic mechanism and reactivity of [NiFe]-hydrogenases from E. coli by developing and implementing integrated spectroscopic, structural, and electrochemical approaches.

By employing an in-situ infrared microspectroelectrochemical method to determine the redox dependence of catalytic intermediates, an ex-situ electrochemical system was designed for precise redox poising of single crystals while maintaining their accessibility for further measurements. This approach yielded high-resolution crystal structures of key catalytic intermediates, including the Nia-SI, Nia-R, Nia-C, and Nia-L states. Furthermore, the proximal cluster in E. coli Hyd-1 exhibits a distinctive conformational change associated with its redox activity. A collection of inhibitor-bound crystal structures is also reported, providing insights into the effects of inhibitors.

A novel spectroelectrochemical methodology enables in-situ X-ray absorption spectroscopy measurements of biological samples at ambient temperature. This technique was validated through XANES potential titration and EXAFS analysis on the model metalloprotein Cu-azurin.

Overall, this work highlights the value of integrated methodologies and constitutes a significant contribution to bioinorganic chemistry, offering a versatile paradigm for investigating mechanisms in a broad range of metalloenzymes under diverse conditions.

Authors: Li, W

• DOI: 10.5287/ora-amnjkkpwa
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: in-situ X-ray absorption spectroscopy; [NiFe]-hydrogenases; spectroelectrochemistry
• Subjects: Bioinorganic chemistry; Spectroelectrochemistry