Studies of the active site of cytochrome P-450 scc with a high-affinity spin-labeled inhibitor

Abstract

The intramolecular site of P-450scc for conversion of cholesterol to pregnenolone involves a substrate site, an active site, and a site for transmission of electrons. The substrate site was studied with a high-affinity, high-potency nitroxide spin-labeled inhibitor of cholesterol side-chain cleavage. This substance, 17 alpha-hydroxy-11-deoxycorticosterone nitroxide (SL-V), has an affinity comparable to that of the most active substrate inhibitors ever reported and 2-50 times greater than that of the natural substrate cholesterol. Competition experiments with cholesterol and its analogues confirmed that SL-V binds reversibly to the substrate site. Titration experiments showed a single binding site on the P-450 molecule. The substrate site is on the apoprotein and has little or no direct interaction with the heme. Spin-spin interactions between the Fe3+ and side-chain or A-ring spin-labeled groups could not be demonstrated, which is consistent with carbons 22 and 20 being closest to the heme iron. We postulate that substrate disrupts a histidine nitrogen coordination with the heme iron and induces conformational changes in the apoprotein. These changes lead to increased affinity for iron-sulfur protein.

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

“…Since, as discussed above, the doxyl ring is too far away from the heme to perturb the iron electronic environment by direct electromagnetic interaction, the spin probe may exert a constraint upon the heme via the protein structure. Interestingly, the binding of a side-chain spin-labelled deoxycorticosterone derivative, which is not a substrate for cytochrome P-450,,,, does not induce a change of the heme-iron ESR g-values [15]. In contrast, the orientation of the CNO molecule within the enzyme active site is more likely that of authentic cholesterol, due to the substrate nature of CNO.…”

“…Obviously the orientation of the CNO molecule within the cytochrome P-450,,, active site is very similar to that of authentic cholesterol. This is of particular interest, since previous investigations of the steroid binding site were undertaken mostly with P-450,,, inhibitors such as amino-cholesterols [5 -81, suicide substrates or recently with a spin-labeled deoxycorticosterone derivative [15]. CNO, on the contrary, may be used as a suitable spin-labelled substrate analogue for the study of the enzyme-substrate interaction in the course of the catalytic event.…”

“…Since we did not observe a flattening of the signal, we conclude that the nitroxide radical in CNO, and thus the terminal cholesterol side-chain carbons, must be farther away from the heme iron in cytochrome P-450,,, than 0.6 nm. On the other hand, Seeley et al [15], using space filling models, have calculated for a comparable compound a maximal Fe-NO distance of 1.0 nm. Under the assumption that the CNO C20 and C22 atoms are very close to the heme, the CNO spin probe would also be distant from the heme by maximally 1.0 nm.…”

“…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].…”

P450scc Engineering and Nanostructuring for Cholesterol Sensing

“…(2) The center of the cholesterol molecule should be as close as possible to the heme because experimental data show that, in the productive complex, the C22 atom of cholesterol located near the center of the molecule should be close to the heme iron. [42][43][44][45] Application of the first criterion left only 4 out of 12 possible regions for cholesterol contact. Those regions comprised the following residues: (1) Pro131, Leu132, Pro135, and the adjacent Ile130, Leu267, and Val424 (region 1); (2) Ile245, Phe246, Leu250, and the adjacent Leu184 (region 2); (3) Ile409, Leu414, Trp418, and the adjacent Phe390 (region 3); (4) Ile468, Phe469, Leu470, Val471, and the adjacent Leu164 (region 4).…”