Cytochrome P450 Reductase

The class II P450 redox system exploits the FAD- and FMN-containing CPR enzyme, which favors NADPH as its coenzyme. Mammalian CPRs are membrane-associated enzymes, with N-terminal anchor regions retaining these proteins in cellular membranes, and with the catalytic domains facing intracellularly [45]. All membranous hepatic P450s are supported by CPR. Analysis of the amino acid sequence of CPR enzymes indicates that they have two major domains (FAD/ NADPH-binding and FMN-binding) that have evolved from the fusion of genes encoding NADP(H)-ferredoxin reductase-like and flavodoxin-like proteins [46]. Removal of the N-terminal anchor region of the protein (either by proteolysis or protein engineering) enables production of a soluble form of the microsomal CPR [47]. The atomic structure for solubilized rat CPR confirms this domain organization and also demonstrates that the FAD and FMN flavins are closely juxtaposed to enable efficient inter-cofactor electron transfer [48]. As with the P450BM-3 system (see Section 5.2.6), the component FAD/NADPH and FMN domains have been expressed and purified, and shown to have redox and other properties consistent with those in the intact enzyme [49].

In CPR, NADPH reduces the FAD cofactor by hydride (2-electron) transfer, and electrons are transferred to the P450 via the FMN cofactor. Thus, the FMN acts as a single electron shuttle between FAD and heme. Potentiometric studies are consistent with this model, with the FMN being the more positive potential flavin

[50]. Also, the relative potentials of the FMN oxidized/semiquinone and semiqui-none/hydroquinone couples are consistent with the hydroquinone FMN as the electron donor to the heme iron, and with a model in which the enzyme undergoes a 1-3-2-1 cycle, where the digits refer to the number of electrons on the CPR flavins [51].

CPR is considered to be in a single electron reduced state at the start of the cycle, with the FMN in a semiquinone state. Reduction by NADPH places a further two electrons on the enzyme, with the FMN then being reduced to hydroquinone and the FAD to its semiquinone state on electronic equilibration. Both flavosemiqui-nones (FAD and FMN) are the blue (neutral) form. The first FMN-to-heme electron transfer from FMN hydroquinone is followed by redistribution of electrons in the CPR to reform the FMN hydroquinone and oxidize the FAD to its quinone state. The second FMN-to-heme electron transfer restores the starting (FMN semiquinone) state [52]. Eukaryotic CPR enzymes are often purified in a single electron reduced (FMN semiquinone) form, referred to as the "air stable semiquinone" state [53].

Protein engineering studies on CPR (predominantly rabbit, human, and rat isoforms) have demonstrated important roles for a "catalytic triad" of residues (Cys629, Asp674, and Ser457 in the human enzyme) in CPR that are crucial to efficient binding and electron transfer from NADPH [54]. Other CPR-like proteins include methionine synthase reductase, the reductase domain of nitric oxide synthase and the human cancer-related enzyme novel reductase 1 (NR1) [55-57]. NR1 has the slowest rate of flavin (FAD) reduction of all these enzymes and this is the rate-limiting step in catalysis for the enzyme [58]. In NR1, two of the three catalytic triad residues are mutated to other amino acids (Ala549 corresponds to Cys629, and Glu594 to Asp674 in human CPR), and this is almost certainly the reason for its slow kinetics.

Other important mutations made to human CPR were the removal of the aromatic "lid" over the FAD cofactor in W676A/H variants. These effected substantial switches in coenzyme selectivity from NADPH to NADH, as discussed in Section 5.5 [59]. Several studies (modeling, chemical modification, and mutagenesis) of the interactions between microsomal CPR and its P450 partners point to the importance of acidic (Glu and Asp) residues in the FMN domain and basic residues on the P450 partners in electrostatic binding interactions [51, 60-64]. However, these data contrast with others demonstrating increased rates of electron transfer between CPR and P450s as ionic strength is increased, leading to disfavoring of ionic interactions [51, 65-67]. Plausible explanations include the possibility than alterations in the nature of interdomain (FAD/NADPH and FMN) interactions in CPR occur at elevated ionic strength so as to promote electron transfer in the systems, or that hydrophobic interactions that "fine tune" configurations that enhance interprotein electron transfer take place under such conditions. Much work clearly remains to be done to understand fully the nature of CPR interactions with its multiple P450 partners in cell membranes.

In the 1980s it became clear that CPR was not an exclusively eukaryotic enzyme, and the characterization of microbial P450-CPR fusion enzymes provided an important leap forward in the characterization of the P450 superfamily, as discussed in the section below.

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