Interaction of a spin‐labelled cholesterol derivative with the cytochrome P‐450_SCC active site

Abstract

The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450,,,. Upon incubation with the cytochrome P-450,,, electron transfer system, CNO is transformed to pregnenolone (Km = 33 fiM, V,,, = 0.32 min-I). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 pM). It binds tightly to cytochrome P-450,,, as evidenced by a reversed type I spectral absorbance change (& = 5.9 pM) which is paralleled by a greater hyperfine splitting of the roomtemperature CNO ESR spectrum due to an enhanced probe immobilization (& = 1.9 pM). This finding is in accord with a rotational correlation time of about s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450,,, displays the ESR g-values gx = 2.404/2.456, g, = 2.245 and g, = 1.916; these are different from those of cholesterol-liganded cytochrome P-450,,, and may thus serve as a marker for cytochrome P-450,,,.Our data indicate that the stereospecificity of the cytochrome P-450,,, side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (CZS to CZ7). The substrate binding site is however rather sensitive to a modification of the side chain. The doxy1 ring confers a stronger affinity of the substrate to the enzyme. Upon binding it becomes embedded in the protein matrix, and we estimate that its final position is 0.6 -1 .0 nm from the heme moiety.The adrenal cortex mitochondria1 cytochrome P-450,,, is the key enzyme in the corticosteroid biosynthesis [l]. It cleaves the side chain of cholesterol to form 4-methylpentanal and pregnenolone, which is the common precursor of steroid hormones [2]. The high specificity and the physiological importance of this enzyme have deserved much attention in recent years; in particular, much work was focused on the study of the active site and of the enzyme-substrate interaction by the use of cholesterol derivatives with a chemically modified side chain such as hydroperoxy sterols [3, 41, aminocholesterols [5 -71 and the so-called suicide substrates [S].Another approach was the use of nitroxide-spin-labelled molecules in the study of cytochrome P-450,,, and of cytochrome P-450 from other sources. This method allowed the rotational correlation time of cytochrome P-45OC,, and of liver microsomal P-450LM to be determined [9 -141. Furthermore, Seeley et al. [15], using various nitroxide compounds bearing the pregnane carbon skeleton, studied the positioning of these inhibitors within the cyctochrome P-450,,, substrate binding site. Similarly, from the interaction of spin-labelled isocyanides with varying alipathic chain lengths with cytochrome P-45OLM2, information about the architecture of the active site could be gained [16]. The information obtained with the nitroxide-spin-labeled cytochrome P-450 inhibitors Abbreviations. CNO, 25-doxyl-27-nor-chloresterol; Me&, trimethylsilyl.Enzymes. Cytochrome P-450,,,, cholesterol monooxygenase, side-chain-cleaving (EC 1 A4.15.6); glucose-6-pho...

The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium

“…We always started from the lower temperature, heated the solution stepwise and waited for 5 -10 min for temperature and spectral stabilization. The instrument response time was set to 'slow', which resulted in a noise level of AA = 0.5 x The high -low-spin equilibrium constant Kspin = [hs]/[ls] was then determined at each step once the spectral changes had stabilised as described [13]. It should be kept in mind, however, that all our spin-state determinations are indirect: we did not measure directly the spin state by EPR or magnetic susceptibility, but by its manifestation in the visible/ultraviolet absorption spectrum.…”

Interaction of Tryptophan Residues of Cytochrome P450scc with a Highly Specific Fluorescence Quencher, a Substrate Analogue, Compared to Acrylamide and Iodide

“…Cytochrome P-45Oscc was isolated from beef adrenal cortex as described (Lange et al, 1988). The enzyme was electrophoretically homogeneous, and the typical specific concentration was 10 nmol P-450 heme/mg protein.…”

“…CNO was synthesized by the method of Keana et al (1967) with 27-nor-5-cholestene-3P-ol-25-one as precursor, according to Maurin et al (1988). Immediately before use, its concentration was determined with respect to a standard nitroxide spin probe by double integration of its ESR spectrum in ethanol with a Bruker ECS 106 instrument (Lange et al, 1988). Acrylamide, sodium thiosulfate and sodium iodide were from Merck.…”

“…For quenching experiments with CNO, the enzyme was incubated with CNO at 37°C for 6 min, then thermostatically controlled at the desired temperature. This procedure was necessary to account for the slow binding of CNO (Lange et al, 1988). The CNO results were corrected for a small quenching effect of methanol.…”

“…We chose this compound because it is accepted by P45Oscc as a substrate. In a previous report, we have shown that it interacts strongly with the substrate-binding site; the spin probe becomes immobilized in the heme pocket, and its stoichiometrical binding is reflected by a heme iron spin state change (Lange et al, 1988). This doxy1 radical appears, therefore, to be an ideal candidate for a screening of tryptophan residues in the heme pocket.…”