Expanding the structural landscape of cytochrome P450 reductases and the effect of redox mediators on CYP-catalyzed reactions

Cytochrome P450 reductases (CPRs) are microsomal diflavin oxidoreductases that transfer electrons from nicotinamide cofactors to class II cytochrome P450 monooxygenases (CYPs). CPRs can also reduce other proteins, including cytochrome b 5 , heme oxygenase, and components of the fatty acid elongation system, as well as nonphysiological acceptors such as cytochrome c , ferricyanide, and various drugs. Although structural and functional studies have provided valuable insights into CPRs, the available data remain insufficient to fully elucidate their electron transfer mechanism and interactions with diverse redox partners. Here, we report the structural and functional characterization of two fungal CPRs from Candida albicans ( Cal CPR) and Mycosarcoma maydis (formerly Ustilago maydis , Uma CPR) and compare their properties with previously characterized CPRs. N -terminal truncation enabled soluble expression in Escherichia coli , and kinetic parameters were determined using cytochrome c and ferricyanide. Although Uma CPR shares greater sequence similarity with ascomycete CPRs than with mammalian CPRs, its overall structure displays features more closely resembling mammalian CPRs, including its FAD-binding topology. We further investigated the ability of both CPRs to support C. albicans CYP52A21 activity and evaluated the effects of reaction environment and redox mediators on CYP–CPR reconstitution. Both truncated CPRs supported CYP activity, with CPR performance strongly influenced by the reaction environment. Addition of the redox mediator cytochrome b 5 from M. maydis significantly enhanced product formation, including when a nonnative CPR was used. These findings highlight the structural diversity and functional plasticity of CPRs and demonstrate that the broader redox environment can strongly influence CYP–CPR efficiency, extending the complexity of electron transfer beyond the canonical CYP–CPR pairing. Key points • CPRs from ascomycetes and basidiomycetes fungi exhibit distinct structural features. • The CPR from Mycosarcoma (Ustilago) maydis is structurally more similar to mammalian CPRs. • CYP-CPR activity is strongly influenced by reaction environment and cytochrome b5 and extends beyond the canonical protein pairing.