A mismatch in enzyme-redox partnerships underlies divergent cytochrome P450 activities between human hepatocytes and microsomes.

NADPH-P450 oxidoreductase (POR) is the accepted redox-partner for drug-metabolising cytochrome P450s (CYPs) over NADH-cytochrome b5 reductase (Cytb5R). Accordingly, POR-centric NADPH-supplemented human liver microsomes (HLM) and recombinant CYP-POR systems complement human hepatocytes (HH) as drug-metabolism models. However, HH may exhibit inexplicably lower CYP activities relative to NADPH-HLM, particularly for CYP3A4-substrates. Here, we show the phenomenon can manifest as disparate CYP phenotyping in which NADPH-HLM and recombinant CYP-POR systems incorrectly identify CYP3A4 while HH accurately assign the main-metabolising CYP exemplified by CYP1A2 for savolitinib. Mechanistically, we serendipitously discover that HH CYP3A4-mediated midazolam-metabolism is increased by gefitinib and find that this can only be recapitulated in non-canonical Cytb5R-dependent NADH-HLM and recombinant CYP3A4-Cytb5R. We conclude that Cytb5R is important for HH-CYP3A4 and show HH-consistent CYP3A4-activities in NADH-HLM. Imaging NADH/NADPH in hepatocytes shows equivalent concentrations suggesting CYP redox-partnerships are cofactor-independent and likely influenced by protein-protein interactions as mimicking the dense-intracellular protein using albumin recapitulates HH savolitinib-metabolism in HLM.