Effects of Human Cytochrome b5on CYP3A4 Activity and Stabilityin Vivo

Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively. To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450. Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins,in vitro,which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase. There is strong evidence from such reconstitution experiments that, when cytochrome b5is included, the turnover of some substrates with certain P450s is increased. Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5to associate in anin vivo-like system, by coexpressing all three proteins together inEscherichia colifor the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5. The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes. The coexpression of cytochrome b5also resulted in the stabilization of the P450 during substrate turnover in wholeE. coli,with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5.