Direct electrochemistry as a mechanistic tool for studying engineered myoglobins: implications on carbene transferase...

Direct electrochemistry tools offer exclusive insight into the redox properties of metalloenzymes. However, they have not found widespread use within the artificial enzyme community, despite their potential to help inform catalyst design. Herein, we describe the use of a simple and robust method for non-covalent immobilisation of a library of myoglobin-based (Mb) engineered and cofactor-substituted metalloenzymes onto carbon electrodes. This allowed the determination of the reduction potentials (E_1/2) spanning M(III)/M(II) redox states for Fe and Mn Mb systems. In Fe-containing systems, more positive E_1/2 values matched higher carbene transferase activity under the tested conditions. For example, when E_1/2 = -10.6 mV (vs. standard hydrogen potential, SHE), a total turnover number (TTN) of 230 was observed, whereas when E_1/2 = -72.9 mV (vs. SHE), TTN = 16 in styrene cyclopropanation reactions. Additionally, we investigated the effect of active-site mutations on E_1/2 and the enantioselectivity of the enzymes. We found that a single mutation of residue 64 from histidine to glycine (H64G) reversed the enantiopreference of the enzyme. This proof-of-concept study highlights the use of direct electrochemistry as a fast and efficient mechanistic tool for characterising and informing the engineering of ArMs for applications in sustainable catalysis and electrochemical transformations.