Cytochrome b_5 reductase-cytochrome b_5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

摘要

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b_5 reductase-cyt b _5. In fungi, limited information is available for the cyt b _5 reductase-cyt b_5 system. Here we characterized the kinetic mechanism of (cyt b_5r)-cyt b_5 redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b _5r followed a "ping-pong" mechanism and could directly reduce cytochrome c. However, unlike other cyt b_5 reductases, Pc-cyt b_5r lacked the typical ferricyanide reduction activity, a standard for cyt b_5 reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b_5r-cyt b_5 complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b_5r-cyt b_5 complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b_5r-cyt b_5) in terms of supporting a P450 monooxygenase activity.