br Cytochrome b br Cytochrome P hydroxylase

Cytochrome b5
Cytochrome P450 17α-hydroxylase/17,20-lyase (P450 17A1) A number of P450s involved in steroid biosynthesis are multifunctional as they carry out two or more sequential hydroxylation reactions on the same substrate(s). P450 17A1 is a dual function endoplasmic reticulum enzyme encoded by the CYP17A1 gene consisting of eight exons, located on chromosome 10q24.3 [148]. The general reaction mechanism of P450 17A1 is believed to follow the consensus catalytic cycle (Fig. 4). This activity describes seven sequential ‘mechanistic’ steps [149] with an additional three unproductive shunts, termed ‘uncoupling pathways’ (Fig. 4). Reducing equivalents are provided to the P450 heme center by the nicotinamide nucleotide (NADPH or NADH) through the di-flavin- (FAD/FMN) containing reductase, CPR. The typical hydroxylation and oxygen GSK2578215A transfer reaction catalyzed by the P450s involves the consumption of one NAD(P)H molecule and one dioxygen molecule, with one oxygen atom inserted into the product and another oxygen reduced to oxide and incorporated into a water molecule. The ferric resting state of the P450 consists of a heme prosthetic group coordinated through an axial thiolate group provided by a cysteine residue and in the resting state by a water molecule occupying the sixth coordination position. The binding of the substrate (1) can partially or completely displace the water molecule, causing a concomitant shift from a low spin, six coordinate complex, to a high spin ferric state (2). This transition correlates with a change in the redox potential of the protein, shifting to more positive (∼130mV) values, and facilitates the transfer of electrons from the redox partner proteins. The binding of molecular oxygen to the ferrous protein (3) generates the ferrous oxygenated (oxy-ferrous) or (superoxide-ferric) state (4), which is a key intermediate in P450 catalytic cycle. The availability of a second electron to the ferric-oxy intermediate from the redox partner leads to a peroxo-ferric species (5a). Immediately following electron transfer, the “peroxo anion” state is formed. Protonation of the distal oxygen atom leads to the “hydroperoxo” anion (5b). Each of the peroxo species could undergo a non-productive release of peroxide (or hydrogen peroxide), regenerating the ferric form (2) of the enzyme. Alternatively, a second protonation of the distal oxygen cleaves the OO bond resulting in the release of a water molecule together with the generation of a higher valent metal-oxo species often termed “Compound I” (Cpd I) (6). The name Compound I is used due to the similarity with similar charged state in a number of heme-containing peroxidase enzymes. The P450 17A1 is a key enzyme in adrenal steroid hormone biosynthesis which in both hydroxylase and lyase activity occurs. In many mammalian species a hydroxylation process predominates in the adrenal zona fasciculate (ZF), where pregnenolone, a Δ5-steroid, and progesterone, a Δ4-steroid, are converted into the 17α-hydroxylase products as precursors of cortisol. However, in the adrenal zona reticularis (ZR) the dehydroepiandrosterone (DHEA) is produced, a precursor for androstenedione, testosterone and estrogens via 17,20-lyase activity in which the 21-carbon 17α-hydroxysteroids are cleaved to the 19-carbon 17-ketosteroids with the loss of acetic acid [150] (Fig. 5). It has been shown that the presence of cyt b5 in the adrenal zona reticularis, which acts as an allostoric regulator, activates the lyase reaction [151]. The absence of any P450 17A1 in the adrenal zona glomerulosa helps to direct the steroidogenic pathway toward the mineralocorticoid aldosterone production. Thus, P450 17A1 controls an important branch-point in steroidogenesis between glucocorticoid and sex hormone biosynthesis. In humans, progesterone is also converted to androstenedione; the Δ4 pathway, but the rate of conversion is only 3% of the Δ5 pathway [152] that converts 17-hydroxyprepnenolone to DHEA [153], [154].