Influence of inter-domain dynamics and surrounding environment flexibility on the direct electrochemistry and electrocatalysis of self-sufficient cytochrome P450 3A4-BMR chimeras

The linker region of multi-domain enzymes has a very important role for the interconnection of different enzyme modules and for the efficiency of catalytic activity. This is particularly evident for artificial chimeric systems. We characterised an artificial self-sufficient enzyme developed by genetic fusion of the catalytic domain of cytochrome P450 3A4 and reductase domain of Bacillus megateriurn BM3 (BMR). Here we report the direct electrochemistry of 3A4-BMR chimeras immobilised on glassy carbon electrodes and we investigated the effect of inter-domain loop length and immobilising environment flexibility on both redox properties and electrocatalysis. We observe that redox potential can be modulated by the linker length and the immobilising layer flexibility. In addition, enzyme inter-domain dynamics and environment flexibility also modulate 3A4-BMR turnover efficiency on electrode system. V-max values are increased up to about 100% in the presence of testosterone and up to about 50% in presence of tamoxifen by decreasing immobilising film rigidity. The effect on 3A4-BMR V-max values is dependent on inter-domain loop length with 3A4-5GLY-BMR chimera being the more affected. The underlying reason for these observations is the potential motion of the FMN domain that is the key to shuttle electrons from FAD to haem.