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

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. Copyright © 1988, Wiley Blackwell. All rights reserved