Studying the metal sites of metallo-proteins by electrochemistry and spectroelectrochemistry

Thesis

This thesis details the development and refinement of spectroelectrochemical methodologies aimed at advancing the study of redox-active protein complexes, particularly those containing iron-sulfur (FeS) clusters. Initially, a UV-Vis spectroelectrochemical cell (UV-Vis SEC) was engineered to facilitate in situ analysis of redox processes in FeS proteins, which proves crucial for understanding the redox behaviour of nitrogenase reductases and their homologues. Despite its utility, the complexity of certain catalytic components rendered UV-Vis SEC inadequate for all systems, hence the approach of electrochemical infrared microspectroscopy (ECIRM) was explored. This technique was applied to study the [FeFe] hydrogenase from Desulfovibrio desulfuricans (DdHydAB), serving as a model to explore application of this technique to oxygen-sensitive samples with intricate active sites that have the additional complication of catalysing proton reduction (H2 evolution). The findings demonstrated that ECIRM is particularly effective in elucidating the complex redox dynamics and active site changes under experimentally challenging conditions.

Through these advanced methods, significant insights were gained into the stability of all-ferrous states and the environmental factors influencing them in proteins like NifH and AnfH. Challenges encountered with redox mediators in the presence of nucleotides underscored the need for novel mediators to avoid unwanted interactions during redox titrations. Furthermore, this research has not only enriched our understanding of FeS cluster chemistry in enzymatic systems but also highlighted the critical need for innovative tools to accurately dissect the sophisticated mechanisms of redox proteins.

Overall, the establishment of UV-Vis SEC and ECIRM for these sensitive metalloproteins has opened new avenues for probing complex redox systems, marking a significant advancement in the field of bioinorganic chemistry.

Authors: Sun, Y

• DOI: 10.5287/ora-o5rwonopo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: FeS cluster; nitrogenase reductases; infrared spectroelectrochemistry; nitrogenases; [FeFe] hydrogenase; UV-Vis spectroelectrochemistry; dark-operative protochlorophyllide oxidoreductase; electrochemical infrared microspectroscopy
• Subjects: Electrodes, Enzyme; Nitrogenase; Reaction mechanisms (Chemistry); Protochlorophyllide oxidoreductases; Hydrogenase