The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

Francisco Esteves; Diana Campelo; Bruno Costa Gomes; Philippe Urban; Sophie Bozonnet; Thomas Lautier; José Rueff; Gilles Truan; Michel Kranendonk

doi:10.3389/fphar.2020.00299

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners

Front Pharmacol. 2020 Mar 18:11:299. doi: 10.3389/fphar.2020.00299. eCollection 2020.

Authors

Francisco Esteves¹, Diana Campelo¹, Bruno Costa Gomes¹, Philippe Urban², Sophie Bozonnet², Thomas Lautier², José Rueff¹, Gilles Truan², Michel Kranendonk¹

Affiliations

¹ Centre for Toxicogenomics and Human Health (ToxOmics), Genetics, Oncology and Huma Toxicology, NOVA Medical School, Faculty of Medical Sciences, Universidade NOVA de Lisboa, Lisbon, Portugal.
² Centre National de la Recherche, Scientifique, Institut National de la Recherche Agronomique, Institut National des Sciences Appliqu es de Toulouse, Toulouse Biotechnology Institute, Universit de Toulouse, Toulouse, France.

Abstract

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b ₅ competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches - formerly indicated to be involved in CPR:CYP interactions - or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Keywords: FMN-domain; NADPH cytochrome P450 oxidoreductase (CPR); binding motifs; cytochrome P450 monooxygenases (CYP); electron transfer; protein–protein interaction; redox partner.