Consistent with the observation that mutations in the redox

Consistent with the observation that mutations in the redox-partner binding site of P450c17 that reverse charge from basic to acidic (R347H, R358Q) cause 17,20-lyase deficiency (Geller et al, 1997, Geller et al, 1999), at least one POR mutation that changes a residue in the FMN domain from neutral to basic (G539R) has also been described in a family with clinical and hormonal findings of selective 17,20 lyase deficiency (underdeveloped male genitalia and low C19 steroids) (Hershkovitz et al., 2008). Prior studies showed that POR G539R inhibited 17,20 lyase activity to a much greater degree than 17α-hydroxylase activity (Huang et al., 2005); this POR mutant also partially impairs the catalytic activities of CYP1A2 and CYP2C19 (Agrawal et al., 2008). As electron transfer from POR is needed for the 17α-hydroxylase reaction as well as the 17,20 lyase reaction, it is not clear why mutations that affect electron transfer should have such a selective effect on 17,20 lyase activity. By contrast, the P450c17 mutation E305G affects the active site and directly impairs the conversion of 17-Preg to DHEA, but does not impair 17α-hydroxylase activity when assayed in transfected yeast or mammalian ARCA Cy3 EGFP mRNA mg (Sherbet et al., 2003), although the affected patients had impaired cortisol responses to ACTH (Tiosano et al., 2008). Interestingly, this unusual mutant increased the very low capacity of P450c17 to convert 17OHP to androstenedione about 10-fold, partially ameliorating the loss of 17,20 lyase activity in the dominant Δ5 pathway; nevertheless, the 46,XY index patient had micropenis and hypospadias, consistent with the Δ5 pathway being the predominant pathway for human androgen biosynthesis (Auchus et al, 1998, Flück et al, 2003).
Cytochrome b5 is a small membrane-bound hemoprotein expressed in both the adrenals and gonads. Early studies showed that cytochrome b5 could augment 17,20 lyase activity (Onoda and Hall, 1982), and transfection experiments in non-steroidogenic cells showed that this action was only seen when POR is present (Lin et al., 1993). More quantitative experiments showed that cytochrome b5 can stimulate the rate of 17,20 lyase activity more than 10-fold (Auchus et al, 1998, Katagiri et al, 1995, Lee-Robichaud et al, 1995), but the Δ5 preference persists, and the rate of the lyase reaction never quite reaches the rate of the hydroxylase reactions. The adrenal expression is specific to the zona reticularis and may contribute to the genesis of adrenarche (Mapes et al, 1999, Suzuki et al, 2000). Cytochrome b5 can augment some activities of some other P450 enzymes, possibly by transferring the second electron from POR to the P450 during the P450 cycle (Bridges et al., 1998). However, although cytochrome b5 can receive electrons from POR, the redox potentials of cytochrome b5 and POR that have been reduced by one electron are unfavorable for electron transfer from cytochrome b5 to P450, and some of the actions of cytochrome b5 can be seen with apo-cytochrome b5 (Yamazaki et al., 1996), which lacks heme and hence cannot transfer electrons. The stimulation of the 17,20-lyase activity of human P450c17 by apo-cytochrome b5 is one of the major experimental bases for the conclusion that cytochrome b5 affects 17,20 lyase activity allosterically, promoting the interaction of P450c17 and POR, and not by electron transfer (Akhtar et al, 2005, Auchus et al, 1998, Lee-Robichaud et al, 1995). Functional and modeling studies suggest that cytochrome b5 promotes the interaction of P450c17 with POR (Pandey and Miller, 2005), but direct evidence for a heterotrimeric complex is lacking. The structure of cytochrome b5 shows that the C-terminal membrane-anchoring helix extends from the structural (core 2) domain, and the heme in the heme-ligand (core 1) domain extends toward the periphery, with the entire surface dominated by negatively charged residues. In apo-b5, which lacks heme, the core 1 domain has considerable flexibility, while the core 2 domain remains folded (Falzone et al., 1996). The C-terminal membrane-spanning helix is required to stimulate the 17,20-lyase activity of human P450c17, but the signal peptide is not (Lee-Robichaud et al., 1997). Genetic and biochemical studies show that basic residues in the redox-partner binding site of P450c17 (R347, R358, and perhaps R449 and K89) are important for the interaction of P450c17 with cytochrome b5 (Auchus, Miller, 1999, Geller et al, 1999, Lee-Robichaud et al, 1997) while E48 and E49 of cytochrome b5 are required for high 17,20-lyase activity (Naffin-Olivos and Auchus, 2006). However, the molecular details of how addition of cytochrome b5 to the P450c17/POR complex augments 17,20-lyase activity remain unclear. NMR studies indicate that cytochrome b5 and POR compete for binding to P450c17, and that binding of P450c17 to cytochrome b5 is stronger in the presence of pregnenolone than in the presence of 17-Preg; the weaker interaction between cytochrome b5 and P450c17 when 17-Preg is the substrate promotes 17,20 lyase activity by enhancing the interaction of P450c17 with POR (Estrada et al., 2013). Consistent with its role in facilitating electron transfer from POR to P450c17, mutations in cytochrome b5 can also cause 17,20 lyase deficiency (Giordano et al., 1994). Two mutations have been described in detail, W27X (Kok et al., 2010) and H44L (Idkowiak et al., 2012). The E48 and E49 residues required for the stimulation of 17,20 lyase activity (Naffin-Olivos and Auchus, 2006) are absent in the patient with W27X, but present in the patient with H44L. Thus much remains to be learned about how cytochrome b5 influences 17,20 lyase activity.