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

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Abstract
The cholesterol analogue 25-doxyl-27-nor-cholesterol (CNO), was found to be a substrate for cytochrome P-450scc. Upon incubation with the cytochrome P-450scc electron transfer system, CNO is transformed to pregnenolone (Km= 33 μM, Vmax= 0.32 min−1). The pregnenolone formation from endogenous cholesterol is strongly inhibited by CNO (50% at 5 μM). It binds tightly to cytochrome P-450scc as evidenced by a reversed type I spectral absorbance change (Kd= 5.9 μM) which is paralleled by a greater hyperfine splitting of the room-temperature CNO ESR spectrum due to an enhanced probe immobilization (Kd= 1.9 μM). This finding is in accord with a rotational correlation time of about 10−7 s, which is close to the tumbling rate of the protein. At 110 K the CNO-bound cytochrome P-450ssc displays the ESR g-values gx= 2.404/2.456, g,y= 2.245 and gz= 1.916; these are different from those of cholesterol-liganded cytochrome P-450scc and may thus serve as a marker for cytochrome P-450ssc. Our data indicate that the stereospecificity of the cytochrome P-450scc, side-chain-cleaving activity is not dependent on the nature of the cholesterol side-chain termination (C25 to C27). The substrate binding site is however rather sensitive to a modification of the side chain. The doxyl 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.