Towards electrochemically triggered disassembly of a redox-active artificial metalloenzyme immobilised on gold electrodes

In an era marked by escalating concerns surrounding increasing pollution levels, there is significant pressure on developing chemical processes which prioritise sustainability, while not compromising on output efficiency and quality. Artificial metalloenzymes (ArMs) represent an emerging field of biocatalysts, enabling chemical transformations which go beyond the scope of natural enzymes to be performed in mild conditions and aqueous environments with comparable activity and selectivity. The recent development of an ArM, based on an iron periplasmic binding protein and its cognate iron(III) siderophore complex, for the first time allowed redox-triggered disassembly. The chemical reduction of the iron(III) centre released the metal cofactor and facilitated the recycling of the protein scaffold. This thesis details the efforts towards optimising the disassembly process and transferring it onto a conductive surface to establish an immobilisation platform which offers electrochemical control over the ArM disassembly without adverse effects on the catalytic performance.

Initial work focused on the interrogation of the redox chemistry exhibited by the iron(III) siderophore complex at the centre of the redox-active anchor. The structural and voltammetric characterisation revealed its uncommon preference for the formation of the coordinatively unsaturated complex. The ArM was immobilised onto gold electrodes functionalised with nitriloacetic acid (NTA) through self-assembled monolayer (SAM) formation, exploiting the affinity between the protein’s polyhistidine tag and nickel(II) NTA complex resulting in minimal non-specific binding. A direct electrochemical connection was not able to be established with the immobilised ArM, thus multiple approaches for indirect determination of the metal cofactor release were studied. A robust approach utilising native ESI-MS was implemented to successfully distinguish between the ArM and the corresponding apoprotein. The thesis concludes in preliminary work towards artificially wiring the non-electrochemically active ArM; the approach is envisioned to be of interest to the wider community for establishing a connection with other electrochemically silent proteins.