Abstract
To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Formation of the CYP17A1·FMN domain complex induced differential line broadening of the NMR signal for each protein. Alterations in the exchange dynamics generally occurred for residues near the surface of the flavin mononucleotide, including 87-90 (loop 1), and for key CYP17A1 active site residues. These interactions were modulated by the identity of the substrate in the buried CYP17A1 active site and by b5. The FMN domain outcompetes b5 for binding to CYP17A1 in the three-component system. These results and comparison with previous NMR studies of the CYP17A1·b5 complex suggest a model of CYP17A1 enzyme regulation.