Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors open conformations forming complexes transferring electrons We previously demonstrated that distinct features the hinge region linking domain (FD) modulate conformer poses their interactions with Specific FD residues contribute in CYP isoform-dependent manner recognition transfer mechanisms are additionally modulated by structure CYP-bound substrate. To obtain insights into underlying mechanisms, we analyzed how mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity coupling efficiencies were evaluated regard structural molecular dynamics bearing alternate substrate at active site. Studies reveal variants not only but surprisingly reactions. Computational approaches evidenced considered generally close contact FD–CYP interface, exhibiting induced fits during complexation modified depending on presence orientation. It was concluded dynamic mutations, complex interface site exist consistently observed regiospecific alterations.