The catalytic cycle of cytochrome P-450scc and intermediates in the conversion of cholesterol to pregnenolone

Abstract

Cytochrome P-45Oscc as isolated is a cholesterol-depleted low-spin haemoprotein; addition of cholesterol results in formation of a high-spin complex. Cytochrome P-45Oscc -cholesterol is a one-electron acceptor on titration with NADPH. Cytochrome P-45Oscc -cholesterol can be anaerobically reduced to the ferrous state which, on oxygenation, forms an oxygenated cytochrome P-45Oscc -cholesterol complex. This oxygenated complex in the absence of adrenodoxin autoxidises to ferric cytochrome P-45Oscc -cholesterol without oxidation of cholesterol. The decay of the oxygenated complex is first-order, k = 9.3 x s-' at 4 "C. The rate of autoxidation is influenced by pH, ionic strength and the chemical nature of bound sterol. The activation energy of autoxidation is 75 kJ mol-'. Addition of equimolar amounts of adrenodoxin to cytochrome P-45Oscc-cholesterol followed by stoichiometric reduction under anaerobic conditions and subsequent oxygenation, allows single catalytic turnover cycles of cytochrome P-45Oscc to be observed. This has led to detection of intermediates in the conversion of cholesterol to pregnenolone and a precursor/product sequence of cholesterol -+ 22-hydroxycholesterol -20,22-dihydroxycholesterol -+ pregnenolone has been established. Addition of oxidised adrenodoxin to oxygenated cytochrome P-45Oscc -cholesterol results in formation of 22-hydroxycholesterol.Cytochrome P-450 acts as the terminal oxidase of the electron-transport chain associated with the side-chain cleavage reaction of cholesterol to pregnenolone in adrenal cortex mitochondria [l]. In these mitochondria, the cytochrome P-450 (cytochrome P-45Oscc) associated with the side-chain cleavage reaction of cholesterol appears to be distinct from the cytochrome P-450 (cytochrome P-450,,,) associated with 1 1P-hydroxylation of deoxycorticosterone and these cytochromes have been partially resolved [2 -41.Cytochrome P-45OCA, is the terminal oxidase of the electron transport chain associated with hydroxylation of camphor to exo-5-hydroxy-camphor in Pseudomonas putida [5]. This cytochrome has been purified and crystallised [6] and a reaction cycle postulated [7, 81 for the catalytic turnover of the cytochrome to form a monohydroxylated product. However, the conversion of cholesterol to pregnenolone, catalysed by cytochrome P-45Oscc, appears to be a complex series of reactions which may involve stable intermediates. Trivial Names. Cholesterol, S-cholesten-'3B-ol; pregnenolone, 3B-hydroxy-5-pregnen-20-one; deoxycorticosterone, 21-hydroxy-4-pregnene-3

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

“…For CYP3A4, we obtained similar free energies of activation (Fig. 3) to those measured for CYP101 (16) and CYP11A1 (47,48). The mechanism of autoxidation in CYP3A4 is likely the same, but the rates are faster by 2-3 orders of magnitude both with and without substrate.…”

A pathway for the metabolism of vitamin D 3 : Unique hydroxylated metabolites formed during catalysis with cytochrome P450scc (CYP11A1)

“…We report here the role for a third mitochondrial P450 (CYP11A1) in the metabolism of D3. This P450 catalyzes the side-chain cleavage reaction for the conversion of cholesterol to pregnenolone and is of great interest because it represents a rate-limiting step in steroid hormone biosynthesis (1)(2)(3)(4)(5). During experiments designed to develop a simpler assay for measuring cholesterol metabolism we turned to the use of 7-DHC because one can monitor absorbance changes with the latter substrate rather than radioactive cholesterol.…”

“…The conversion of cholesterol to pregnenolone (P5) is reported to occur by forming in sequence the monohydroxylated product [22R-(OH)cholesterol] followed by the formation of the dihydroxylated intermediate [20␣,22R-(OH) 2 cholesterol] with subsequent carbon-carbon bond cleavage at the C20-C22 position to form the C19 steroid pregnenolone (1)(2)(3)(4)(5). It has been difficult to identify these hydroxylated intermediates of cholesterol metabolism during catalysis by CYP11A1 because they are not released from the active site of CYP11A1 during metabolism (1)(2)(3)(4)(5). Of interest is the recent report that the oxysterol 22R-(OH)cholesterol plays an important role as a ligand for the liver X receptor, a transcription factor (6).…”

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

“…The reaction involves removal of the side chain of cholesterol via production of enzyme-bound intermediates, 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol (Hume et al, 1984;Lambeth et al, 1982;Tuckey, 1990Tuckey, , 2005. Cytochrome P450scc can also metabolize vitamin D3 (D3) and vitamin D2 (D2), as well as their precursors 7-dehydrocholesterol and ergosterol (Guryev et al, 2003;Slominski et al, 2004Slominski et al, , 2005aSlominski et al, ,b, 2006Tuckey et al, 2008).…”