P450SCC

Encoded by the CYP11A1 gene, P450scc consumes three equivalents of NADPH and molecular oxygen during the conversion of cholesterol to pregnenolone. Although the enzyme is named for the cleavage of the cholesterol side chain, this process consists of three discrete steps: (1) the 22-hydroxylation of cholesterol; (2) the 20-hydroxylation of 22(R)-hydroxycholesterol; and (3) the oxidative scission of the C20-C22 bond of 20(R), 22(R)-dihydroxycholesterol—the side-chain cleavage event. The enzyme will utilize free hydroxysterol intermediates as substrates for the side-chain cleavage reaction, a tool that is used experimentally because the hydroxysterols are much more soluble than cholesterol and because their access to P450scc is independent of StAR.¹³ In vivo, however, little of these free intermediates probably accumulate because their k_cat/K_m ratios are much higher than for cholesterol,⁴¹ and the high K_d for pregnenolone (about 3000 nM) drives product dissociation. This complex process is the rate-limiting step in steroidogenesis, with turnover numbers of only about 20 molecules of cholesterol per molecule P450scc per minute.⁴¹ P450scc will also cleave the side chain of other hydroxysterols (e.g., 7-dehydrocholesterol), and 20- and 22-hydroxylates vitamin D.⁴²

The single human gene for P450scc⁴³ encodes an mRNA of 2 kb.⁴⁴ A 39-amino-acid mitochondrial leader peptide that targets P450scc to the mitochondria is then proteolytically removed to yield a 482-amino-acid protein. Forms of P450scc targeted to the endoplasmic reticulum are inactive,⁴⁵ demonstrating that the mitochondrial environment is required for activity. Expression of P450scc is induced in the adrenal zona fasciculata/reticularis,⁴⁶ testis,⁴⁷ and ovary by cAMP; and in the zona glomerulosa by intracellular calcium/protein kinase C.^48,49 In contrast, placental P450scc expression is constitutive⁵⁰ and is caused at least in part by the LBP family of transcription factors.^35,51 Side-chain cleavage activity and pregnenolone biosynthesis have been demonstrated in the rat and human brain⁵²; and abundant P450scc expression is found in the rodent brain, especially in fetal life. Deletion of the gene for P450scc has been described in rabbits⁵³ and mice,⁵⁴ abrogating all steroidogenesis and thus proving that P450scc is the only enzyme that can convert cholesterol to pregnenolone. While homozygous mutations in P450scc are expected to be embryonic lethal by eliminating placental progesterone synthesis, a small number of patients has been described having P450scc mutations that typically retain partial enzymatic activity.^55,56

P450C17

For investigators studying the enzymology and genetics of the steroidogenic pathways, P450c17 is especially interesting. Clinical observations showed that adrenal 17α-hydroxylase activity (reflected by serum cortisol concentrations) was fairly constant throughout life, whereas adrenal 17,20-lyase activity (reflected by serum DHEA and DHEAS concentrations) was low in early childhood but rose abruptly during adrenarche at ages 8 to 10 years.^57,58 This dissociation between adrenal secretion of 17α-hydroxylase products (cortisol) and 17,20-lyase products (DHEA) suggested that distinct enzymes performed the two transformations, a hypothesis that was reinforced by the description of patients with putative isolated 17,20-lyase deficiency. Consequently, reports⁵⁹ that the 17α-hydroxylase and 17,20-lyase activities of neonatal pig testes copurified were initially received with great skepticism. This controversy of “one enzyme or two” persisted until the cDNA for bovine P450c17 was cloned and shown to confer both 17-hydroxylase and 17,20-lyase activities when expressed in nonsteroidogenic COS-1 cells.⁶⁰ The human genome has one gene for P450c17,⁶¹ which is expressed in the adrenals and gonads,⁶² and not two tissue-specific isozymes as had been thought. A single 2.1-kb mRNA species yields a 57-kD protein in these tissues, and mutations in this gene produce a spectrum of deficiencies in 17-hydroxysteroids and C₁₉ steroids.

Human P450c17 17-hydroxylates both pregnenolone and progesterone with approximately equal efficiency,^63,64 but all other reactions show prominent differences between Δ⁴ and Δ⁵ substrates. The 17,20-lyase activity is about 50 times more efficient for the 17α-hydroxypregnenolone-to-DHEA reaction than for the 17α-hydroxyprogesterone-to-androstenedione reaction.^63,64 Although the rate of the lyase reaction can be increased more than 10-fold by the addition of a molar excess of cytochrome b₅,^63–65 the Δ⁵ preference persists, and the lyase rate never quite reaches the rate of the hydroxylase reactions. In addition, human P450c17 16α-hydroxylates progesterone but not pregnenolone⁶⁴; in the presence of cytochrome b₅, it diverts about 10% of pregnenolone metabolism to a Δ¹⁶ andiene product⁶³ that is also formed by this pathway in pigs and that acts as a pheromone in that species. Although experiments to study the chemistry of human P450c17 often require manipulations that could be considered nonphysiologic, the remarkable consistency for substrate preferences and kinetic constants observed for the modified, solubilized P450c17 expressed in Escherichia coli^63,65 and native P450c17 expressed in yeast microsomes,⁶⁴ or intact COS-1 cells,⁶⁶ or that obtained from human tissues and cells,^64,67 serve to verify these conclusions.

Given the diverse repertoire of reactions catalyzed by P450c17 in the classical pathways, it is not surprising that synthetic steroids such as dexamethasone⁶⁸ and the enantiomer of progesterone,⁶⁹ as well as planar drugs such as troglitazone,⁷⁰ also bind to and inhibit P450c17. In addition, the 5α-reduced C₂₁ steroids dihydroprogesterone (5α-pregnane-3,20-dione) and allopregnanolone (5α-pregnan-3α-ol-20-one) are excellent substrates for the 17α-hydroxylase activity of P450c17⁷¹ (Fig. 96-4A). Furthermore, 17α-hydroxylated allopregnanolone (5α-pregnane-3α,17α-diol-20-one; 17OH-Allo) is the most efficient substrate yet identified for the 17,20-lyase activity of human P450c17, and its cleavage to androsterone is minimally dependent on cytochrome b₅,⁷¹ unlike 17α-hydroxypregnenolone metabolism to DHEA.^63–65 The conversion of 17OH-Allo to androsterone by the 17,20-lyase activity of P450c17, first described in the testes of tammar wallaby pouch young,⁷² provides an alternative or “backdoor” pathway to DHT, by which DHT is produced without utilizing DHEA, androstenedione, and testosterone as intermediates⁷³ (see Fig. 96-4B). Consequently, the presence of 5α-reductases in steroidogenic cells does not preclude the production of C₁₉ steroids but rather paradoxically enhances the production of DHT by directing flux to 5α-reduced precursors of DHT.

FIGURE 96-4. Reactions catalyzed by human P450c17 and pathways to C₁₉ steroids. A, The four principal A/B-ring configurations of active endogenous steroids and their precursors: Δ⁵, Δ⁴, 5α, and 5α,3α (boxes and structures at bottom). Progesterone and 17α-hydroxyprogesterone can be 5α-reduced, and once the A-ring is saturated, these 5α-reduced steroids are substrates for reductive 3α-HSDs of the AKR1C family. Human P450c17 17α-hydroxylates all four classes of C₂₁ steroids, but the 17,20-lyase activity is robust only with 17α-hydroxypregnenolone and 5α-pregnane-3α,17α-diol-20-one (Δ⁵– and 5α,3α-pathways, respectively). B, Two pathways to DHT using the different 17,20-lyase activities of human P450c17. In the conventional or Δ⁵-pathway (solid arrows), the 17,20-lyase activity of P450c17 requires cytochrome b₅ to efficiently convert 17α-hydroxyprogesterone to DHEA, and testosterone is reduced in target tissues by 5α-reductase 2 (5αR2) to DHT. In the “backdoor” or 5α,3α-pathway (broken arrows), 5α-reduction by 5αR1 and 3α-reduction of C₂₁ steroids occurs in the steroidogenic tissue prior to the 17,20-lyase reaction. In the best characterized pathway, 5α-pregnane-3α,17α-diol-20-one is cleaved to androsterone without requiring cytochrome b₅ and reduced to androstanediol. Androstanediol is exported from the testis and metabolized to DHT by oxidative 3α-HSDs (Ox. 3α-HSD). Note that testosterone is not an intermediate in the backdoor pathway to DHT, that different isoforms of 5α-reductase appear to be involved in the two pathways, and that both reductive and oxidative 3αHSDs are required for the “backdoor” pathway. Structures of testosterone, DHT, and androstanediol are shown at bottom.

The backdoor pathway enables production of C₁₉ steroids in the presence of abundant 3β-HSD activity, despite the poor 17,20-lyase activity of human P450c17 for 17α-hydroxyprogesterone, by using 17OH-Allo as the substrate for the 17,20-lyase reaction. The presence of 5α-reductase activity is a key requirement for the backdoor pathway. The best-studied example of 5α-reduction in a human steroidogenic tissue is the production of 5α-dihydroprogesterone in human corpus luteum by the type 1 enzyme.⁷⁴ Human enzymes catalyze all of the other reactions required to complete this alternate route to DHT, and good evidence documents production of 5α-reduced androgens by the fetal adrenal, at least in some pathologic states. Consequently, it is possible that the backdoor pathway is the principal route to DHT in pathologic states in which 17α-hydroxyprogesterone accumulates, including 21-hydroxylase deficiency and P450 oxidoreductase deficiency (see the following). Androgen production by the backdoor pathway may explain why newborn girls with 21- and 11-hydroxlase deficiencies can be severely virilized, while those with 3β-HSD2 deficiency, whose adrenals cannot make 17α-hydroxyprogesterone, are minimally virilized.⁷⁵ The fractional contributions of the conventional and backdoor pathways to DHT production during human sexual differentiation (at 8 to 12 weeks of gestation) and the expression of 5α-reductase in fetal adrenal and gonad tissues,⁷⁶ however, are only beginning to be determined.

The chemistry of P450c17-mediated hydroxylations is believed to proceed via the common iron oxene species and “oxygen rebound” mechanism proposed for prototypical P450 hydroxylations.⁷⁷ The mechanism of the 17,20-lyase reaction involving a carbon-carbon bond cleavage, however, is not known despite considerable study. The failure of hydrogen peroxide alone to support catalysis (as has been shown for some other P450-mediated deacylation reactions) and computer modeling studies suggest that the same heme-oxygen complex might participate in both hydroxylations and the 17,20-lyase reaction,⁷⁸ but no conclusive evidence to exclude proposed mechanisms exist.

One consequence of the Δ⁵ preference of the human enzyme for the 17,20-lyase reaction is that most human C₁₉ and C₁₈ steroids derive from DHEA as an intermediate.⁶⁷ This Δ⁵ preference allows for the phenomenon of adrenarche to occur in humans, an event that only takes place in large primates.^79,80 However, Δ⁵-lyase activity is not sufficient for adrenarche to occur, because some monkeys (e.g., rhesus macaques) produce high amounts of DHEA throughout life, but most mammals (e.g., cattle, dogs, cats, etc.) never produce much DHEA.⁷⁹ The biochemistry of P450c17, with its differential regulation of the 17α-hydroxylase and 17,20-lyase activities, provides clues to the genesis of this enigmatic process of adrenarche. P450c17 is a phosphoprotein, and phosphorylation selectively enhances the 17,20-lyase activity.^81,82 It appears likely that the regulation of P450c17 phosphorylation, which is a dynamic balance between phosphorylation and dephosphorylation, plays an important role in adrenarche and pathologic hyperandrogenic states such as polycystic ovary syndrome.⁸³ Whereas the kinase(s) responsible for P450c17 phosphorylation remain unknown, it is now apparent that the kinase activity is counterbalanced by protein phosphatase 2A, which in turn is regulated by cAMP via phosphoprotein SET.⁸⁴ Cytochrome b₅ also augments 17,20-lyase activity,^64,82 and high expression of b₅ in the zona reticularis of monkeys⁸⁵ and humans⁸⁶ suggests that the developmentally regulated expression of b₅ might be a key event. The transcriptional regulation of cytochrome b₅ in the adrenal is similar to that of P450c17,⁸⁷ but mechanisms enabling zone-specific expression have not been elucidated. Finally, limiting steroid flux to the Δ⁵ pathway by lowering 3β-HSD activity in the zona reticularis (where most DHEA derives) potentiates the effect of increased 17,20-lyase activity.^86,88

The initial description of 17α-hydroxylase deficiency was a case in which both 17α-hydroxylase and 17,20-lyase products were absent.⁸⁹ When the gene for human P450c17 was cloned,⁶¹ patients with 17α-hydroxylase deficiency were found to harbor mutations in the CYP17A1 gene, and more than 40 mutations scattered throughout the CYP17A1 gene have been characterized,⁹⁰ with mutations W406R and R362C being the most common and accounting for the high prevalence of 17α-hydroxylase deficiency in Brazil.⁹¹ The identification of CYP17A1 mutations causing apparent isolated 17,20-lyase deficiency is fraught with difficulty,⁹² but in the past decade, five cases of isolated 17,20-lyase deficiency caused by mutations in arginines 347 and 358 have been confirmed.^93,94 Computer modeling studies demonstrate that R347H and R358Q neutralize positive charges in the redox-partner binding site.^78,93 Biochemical studies confirm that mutations R347H and R358Q impair interactions of P450c17 with its electron donor POR and with cytochrome b₅.⁹⁵ Therefore, these cases of isolated 17,20-lyase deficiency are not caused by an inability of the mutant enzymes to bind the intermediate 17α-hydroxypregnenolone but rather are caused by subtle disturbances in interactions with redox partners.^93,95 In contrast, mutation E305G has been shown to cause 17,20-lyase deficiency by selectively disrupting binding of 17α-hydroxypregnenolone and DHEA synthesis despite enhanced conversion of 17α-hydroxyprogesterone to androstenedione.⁹⁶ This unusual variant of isolated 17,20-lyase deficiency provides further genetic evidence that the flux of androgens derived from conversion of 17α-hydroxyprogesterone to androstenedione in the minor Δ⁴ pathway is not sufficient to form normal male external genitalia. One of the first patients reported to have isolated 17,20 lyase deficiency was recently found to have a homozygous mutation in P450 oxidoreductase (G539R), further emphasizing the crucial role of efficient electron transfer in the 17,20 lyase reaction.⁹⁷

P450C21

Microsomal P450c21 performs the 21-hydroxylation of the Δ⁴ steroids 17α-hydroxyprogesterone and progesterone, an essential step in the biosynthesis of both mineralocorticoids and glucocorticoids (see Fig. 96-2). The human P450c21 protein is found only in the adrenal glands; the extra-adrenal 21-hydroxylase activity found in other organs such as the liver and the aorta⁹⁸ is not catalyzed by P450c21⁹⁹ but appears to be catalyzed by CYP2C9, CYP3A4, and possibly CYP2C19 and other enzymes as well.^100,101

The locus containing the CYP21 genes is among the most complex in the human genome and explains why 21-hydroxylase deficiency (affecting 1 of 14,000 live births) is one of the most common autosomal-recessive diseases. The CYP21A2 gene and the CYP21A1 pseudogene lie on chromosomal locus 6p21.1 in the midst of the human leukocyte antigen (HLA) locus. Because the HLA locus is highly recombinogenic, exchange between the CYP21A1 and CYP21A2 loci is common. Thus 85% of cases of 21-hydroxylase deficiency derive from micro- or macrogene conversion events where some or all of the CYP21A1 pseudogene replaces the corresponding area of the CYP21A2 gene, thus reducing the expression of the encoded P450c21 protein and/or impairing its activity.¹⁰² In addition, at least eight additional genes lie in this locus (Fig. 96-5), including the liver-specific C4A and C4B genes; the adrenal-specific “ZA” and “ZB” genes; and the ubiquitously expressed tenascin X or TNXB gene,¹⁰³ the disruption of which is one cause of Ehlers-Danlos syndrome.¹⁰⁴ Occasionally, a patient with 21-hydroxylase deficiency and Ehlers-Danlos syndrome will have a contiguous gene syndrome with tenascin X deficiency as well.¹⁰⁵

FIGURE 96-5. Genetic map of the human leukocyte antigen (HLA) locus containing the genes for P450c21. The top line shows the p21.1 region of chromosome 6, with the telomere to the left and the centromere to the right. Most HLA genes are found in the class I and class II regions; the class III region containing the CYP21 genes lies between these two. The second line shows the scale (in kb) for the diagram immediately below, showing (from left to right) the genes for complement factor C2, properdin factor Bf, and the RD and G11/RP genes of unknown function; arrows indicate transcriptional orientation. The bottom line shows the 21-hydroxylase locus on an expanded scale, including the C4A and C4B genes for the fourth component of complement, the “CYP21A” pseudogene (CYP21A1, 21A), and the active “CYP21B” gene (CYP21A2, 21B) that encodes P450c21. XA, YA, and YB are adrenal-specific transcripts that lack open reading frames. The XB gene encodes the extracellular matrix protein tenascin-X; XB-S encodes a truncated adrenal-specific form of the tenascin-X protein whose function is unknown. ZA and ZB are adrenal-specific transcripts that arise within the C4 genes and have open reading frames, but it is not known if they are translated into protein; however, the promoter elements of these transcripts are essential components of the CYP21A1 and CYP21A2 promoters. The arrows indicate transcriptional orientation. The vertical dotted lines designate the boundaries of the genetic duplication event that led to the presence of A and B regions.

Much less is known about the enzymology of P450c21 than of P450c17, but the available evidence suggests that unlike P450c17, P450c21 is not very sensitive to the abundance of POR or cytochrome b₅. It is clear that genotype consistently predicts phenotype in very severe and very mild cases of 21-hydroxylase deficiency. In contrast, patients with P450c21 variants (e.g., the common Pro30Leu and Val281Leu mutations and less common mutations Arg339His and Pro453Ser), which have 20% to 50% of wild-type activity,^102,106 can have various phenotypes, implying additional factors that can modify the clinical manifestations of 21-hydroxylase deficiency.

P450C11β AND P450C11AS

The classical descriptions of distinct deficiencies in 11β-hydroxylase, 18-hydroxylase (also called corticosterone methyl oxidase I, or CMOI), and 18-oxidase (CMOII) suggested that three enzymes executed these three respective transformations.^107,108 Analogous to the scenario for P450c17, a single enzyme¹⁰⁹ and corresponding gene¹¹⁰ were found in bovine adrenals that possessed all three activities. In contrast, humans have two genes named CYP11B1 and CYP11B2¹¹¹ that encode the mitochondrial enzymes 11β-hydroxylase (P450c11β) and aldosterone synthase (P450c11AS), respectively, and rats but not mice have three functional CYP11B genes.¹¹² Although P450c11β and P450c11AS both possess 11β-hydroxylase activities, P450c11AS also performs the two oxygenations at C₁₈ required for aldosterone biosynthesis.^113,114 Mutations in CYP11B1 cause 11β-hydroxylase deficiency,¹¹⁵ whereas defects in CYP11B2 cause either CMOI or CMOII deficiencies.¹¹⁶ Severe defects can impair all P450c11AS activities, leading to the clinical phenotype of CMOI deficiency,¹¹³ whereas P450c11β provides 11β-hydroxylase activity in the zona fasciculata. Fortuitous site-directed mutagenesis experiments of nature have found amino acid substitutions such as Arg181Trp plus Val386Ala, which mainly impair 18-oxidase activity and lead to CMOII deficiency.¹¹⁷

The coding regions of the CYP11B1 and CYP11B2 genes share 93% amino acid identity and the same exonic gene structure found in all mitochondrial P450 genes.¹¹⁸ Despite the sequence similarities of these tandem genes, located within 40 kb on chromosome 8q24.3, the expression of P450c11AS is restricted to the adrenal zona glomerulosa, whereas P450c11β is found in the zona fasciculata and zona reticularis. The regulation of P450c11β is driven mainly by cAMP in response to ACTH, whereas P450c11AS expression derives from potassium and angiotensin II activation of the protein kinase C pathway.¹¹⁹ Thus, under normal circumstances, 18-hydroxylase and 18-oxidase activities are restricted to the zona glomerulosa, where 17-hydroxylase activity is low, limiting the repertoire of steroids that can undergo 18-oxygenation.

Although the organization of two highly homologous, adjacent CYP11B1 and CYP11B2 genes on chromosome 8 is reminiscent of the genetics of the CYP21A1 and CYP21A2, gene conversion in the CYP11B locus occurs rarely.¹²⁰ Instead, a clinical entity called glucocorticoid remediable aldosteronism (GRA) arises when an unequal crossing over of the CYP11B1 and CYP11B2 genes creates a third, hybrid gene in which the ACTH-regulated promoter of CYP11B1 drives expression of a chimeric protein with aldosterone synthase activity.^121,122 As a result, 18-hydroxylase and 18-oxidase activities are ectopically expressed in the zona fasciculata, leading to elevated renin-independent production of aldosterone, as well as 18-oxygenated metabolites of cortisol. The expression of this gene is suppressed by blunting ACTH production with glucocorticoids such as dexamethasone, which is used for diagnosis and treatment.¹²³ The prevalence of GRA varies from nil to as high 2% of referred patients with hypertension.¹²⁴

The genetics of GRA has assisted in the precise identification of residues in P450c11AS that enable 18-oxygenase activities. Residues 288, 296, 301, 302, 325, and perhaps most importantly, 320 are critical for 18-oxygenase activities.^125,126 Therefore, crossovers 3′ to codon 320 do not enable aldosterone synthase activity. These key residues lie in or near the I-helix, which contains the catalytically important threonine residue implicated in oxygen activation for almost all P450s; thus, these mutations would be expected to alter active site geometry.

P450ARO (AROMATASE)

The oxidative demethylation of C₁₉ steroids, mainly androstenedione and testosterone, consumes three equivalents of molecular oxygen and NADPH, yielding formic acid and C₁₈ steroids with an aromatic A-ring, hence the common name for this enzyme, aromatase. As is the case for P450scc, each subsequent oxygenation proceeds with greater efficiency, aiding in the completion of this transformation that is essential for estrogen biosynthesis in all animals.¹²⁷ The mechanism of this aromatization must account for the incorporation of the final oxygen atom from molecular oxygen into the formic acid byproduct. The weight of evidence favors a hydroxylation at C2 of 19-oxo-androstenedione, followed by an enzyme-assisted rearrangement and tautomerization of the intermediate dienone to the phenolic A-ring.¹²⁸

P450aro is expressed in steroidogenic tissues (ovarian granuloma cells, placenta), in brain, and in nonsteroidogenic tissues, especially fat and bone.¹²⁷ The CYP19A1 gene for P450aro spans over 75 kb¹²⁹ and contains five different transcriptional start sites¹³⁰ with individual promoters that permit the tissue-specific regulation of expression in diverse tissues. P450aro is a glycoprotein, but glycosylation per se does not appear to affect activity.

Studies of patients with aromatase deficiency confirm that biologically significant estrogen synthesis derives entirely from this enzyme,^131,132 although dietary phytoestrogens can provide some estrogen action in mice with targeted deletion of the aromatase gene.¹³³ Although very few cases of aromatase deficiency have been described, they are highly informative “knockouts of nature” that illustrate principles of fetoplacental steroidogenesis. In fetuses homozygous for aromatase deficiency, the principal manifestation results from its deficiency in the placenta,¹³¹ because ovarian steroidogenesis is quiescent during fetal life.¹³⁴ The fetal adrenal makes large amounts of C₁₉ steroids, principally DHEA-S, much of which is 16α-hydroxylated in the fetal liver before undergoing metabolism via steroid sulfatases, 3β-HSD1, aromatase, and 17β-HSD1 in the placenta to produce estriol, the characteristic estrogen of pregnancy. Although huge amounts of estriol and estradiol are produced by the fetoplacental unit, estrogens are not needed for fetal development, the maintenance of pregnancy, or the onset of parturition; all of these processes proceed normally in fetuses lacking StAR, P450c17, or aromatase, or even in fetuses wholly lacking adrenal glands because of mutations in SF-1 or DAX-1.¹³⁵ However, in the absence of placental aromatase activity, androgenic C₁₉ steroids derived from the fetal adrenal are passed into the maternal circulation, causing marked virilization of the mother.¹³¹

Furthermore, in pregnancies in which the mother has poorly treated 21-hydroxylase deficiency, maternal testosterone values can exceed 300 ng/dL (a midpubertal value for males), yet the fetus is not virilized¹³⁶ because the maternal testosterone is efficiently metabolized to estradiol by placental aromatase. Thus, placental aromatase is a key enzyme in protecting the fetus and mother from unwanted androgen exposure. After birth, individuals with aromatase deficiency grow normally and continue linear growth after completion of puberty, with males producing normal amounts of testosterone. However, when treated with estrogens, aromatase-deficient subjects fuse their epiphyses and cease linear growth.¹³⁷ These observations provide powerful evidence that bony maturation and epiphyseal fusion in children is mediated by estrogens, not androgens, even in males. These observations have led to the experimental use of aromatase inhibitors in various disorders of accelerated bone maturation.

ADRENODOXIN (FERREDOXIN)

Adrenodoxin (Adx), also known as ferredoxin, is encoded by a gene on chromosome 11q22 that spans over 30 kb. Adx is a small (14 kD), soluble, Fe₂S₂ electron shuttle protein that resides either free in the mitochondrial matrix or is loosely bound to the inner mitochondrial membrane.¹³⁸ Adx is expressed in many tissues, and its expression in steroidogenic tissues is induced by cAMP in parallel with P450scc.¹³⁹

Bovine Adx consists of two domains,¹⁴⁰ a core region and an interaction domain. The core region contains residues 1-55 and 91-end (bovine numbering), including the four cysteines whose sulfur atoms tether the Fe₂S₂ cluster to the protein. Residues 56 to 90 form the interaction domain, which is a hairpin containing a helix at its periphery that includes acidic residues critical for the interaction of Adx with P450scc¹⁴¹ (specifically, aspartates 72, 76, and 79, plus glutamate 73). The Fe₂S₂ cluster lies in a protuberance in the molecule at the junction of its two domains. The charged residues of Adx cluster in the interaction domain, giving the molecule a highly negatively charged surface above the Fe₂S₂ cluster (see Fig. 96-3A). This description of the Adx molecule concurs with earlier studies that showed that overlapping sets of negative charges on Adx drive Adx interactions with positive charges on both P450scc and adrenodoxin reductase (AdR).¹⁴² Because a preponderance of the evidence favors a model in which the same surface of Adx shuttles between AdR and the P450 to transport electrons,^142,143 a model of how Adx interacts with AdR would approximate how mitochondrial P450s interact with Adx.

ADRENODOXIN (FERREDOXIN) REDUCTASE

Like Adx, adrenodoxin reductase (AdR) is widely expressed in human tissues, but its expression is two orders of magnitude higher in steroidogenic tissues.¹⁴⁴ The primary RNA transcript from the 11-kb AdR gene¹⁴⁵ on chromosome 17q24-q25¹⁴⁶ is alternatively spliced, generating two mRNA species that differ by only 18 bp,¹⁴⁷ but only the protein encoded by the shorter mRNA is active in steroidogenesis.¹⁴⁸ Unlike most steroidogenic genes, the promoter for AdR contains six copies of GGGCGGG sequences,¹⁴⁵ which is the canonical binding site for the transcription factor SP-1 typically found in “housekeeping genes.” Accordingly, cAMP does not regulate transcription of the AdR gene, as is the case for Adx and P450scc,¹⁴⁴ implying that AdR plays additional roles in human physiology beyond steroidogenesis. Given their essential roles in the conversion of cholesterol to pregnenolone, no null mutations in AdR or Adx have been described in humans, and impairment of the Drosophila AdR homologue dare causes developmental arrest and degeneration of the adult nervous system owing to the loss of ecdysteroid production.¹⁴⁹

Bovine AdR also consists of two domains, each comprising a β-sheet core surrounded by α-helices.¹⁵⁰ The NADP(H)-binding domain is a compact region composed of residues 106 to 331 (bovine numbering), whereas the more open FAD domain, formed by the remaining amino- and carboxy-terminal residues, binds the dinucleotide portion of FAD across a Rossman fold, with the redox-active flavin isoalloxazine ring abutting the NADP(H) domain. By analogy to related structures, including glutathione and thioredoxin reductases, the nicotinamide ring of NADPH is modeled to lie adjacent to the flavin ring in a position to transfer its two electrons to the FAD. Intramolecular electron transfer occurs in the cleft formed by the angled apposition of these two domains. Within this cleft, basic residues abound, including arginines 240 and 244, which are important for interactions with Adx.^142,151 Hypothetical docking of the two structures suggests that the negative surface of Adx fits elegantly into the positive surface of AdR, even with NADP(H) bound.¹⁵⁰ Basic residues are also critical for the interaction of P450scc with the negative surface charges on Adx,¹⁴³ so that AdR-Adx docking is expected to share some key features with the mitochondrial P450-Adx interaction.