Mass spectrometry footprinting reveals microsomal CYP2A6 structural changes induced by interaction with its reductase...

NADPH cytochrome P450 reductase is the required redox partner for the majority of human cytochrome P450 enzymes, which are critically important for phase I drug metabolism of a wide variety of substrates. It is well understood that cytochrome P450 reductase supports P450 catalysis when its flavin mononucleotide (FMN)-containing domain (FMND) binds to the proximal side of P450 enzymes to deliver electrons to the P450 heme. Herein, we describe mass spectrometry-based footprinting approaches to compare the surface labeling of CYP2A6 and that of an artificial fusion protein composed of the reductase FMND linked to the N-terminus of CYP2A6 (FMND/CYP2A6). Three complementary footprinting approaches were used: hydrogen-deuterium exchange, benzoyl fluoride labeling, and fast photochemical oxidation of proteins (FPOP). Although the different labeling approaches target different amino acids and occur over varying reaction timescales, their outcomes generally agree. These experiments did not detect differential protection on the proximal P450 face where FMND is expected to bind. Instead, they consistently demonstrated increased exposure of CYP2A6 surface residues, indicative of structural changes in CYP2A6 in the presence of the FMND. Overall, the reduced protection is consistent with the FMN domain causing long-range allosteric modulation of the CYP2A6 structure. This structural evidence is consistent with increasing functional evidence that the reductase is an allosteric modulator of P450 enzymes in addition to its role in electron transfer. SIGNIFICANCE STATEMENT: Both established and new mass-spectrometry footprinting methods support structural changes in the CYP2A6 structure upon interaction with the FMN-containing domain of its reductase. This evidence supports the idea that the reductase is an allosteric modulator of P450 enzymes, in addition to its established role in electron transfer.