pH modulation of ligand binding to adrenal mitochondrial cytochrome P-450scc.
Interactions of substrate (cholesterol), product (pregnenolone), and electron donor (adrenodoxin) with adrenal mitochondrial cytochrome P-450scc are strongly interdependent. The binding data are indicative of cooperative binding of three ligands (H+, cholesterol, and adrenodoxin) to cytochrome P-450scc. Cholesterol binding in 0.3% Tween 20 is enhanced by decreased pH in a manner quantitatively consistent with preferential binding to protonated cytochrome (Kd = 5 microM) as compared to deprotonated cytochrome (Kd congruent to 85 microM). Protonation of fully low spin P-450scc in the absence of cholesterol exhibits a pK = 6.5 and occurs without change of spin state (less than 5% high spin). Cholesterol binding increases the affinity for protonation (pK = 7.9). Adrenodoxin binding in absence of cholesterol is unaffected by protonation but is activated, in a temperature-dependent manner, by cholesterol (6-fold at 35 degrees C). The spin states of the respective complexes are: adrenodoxin-P-450 (15% high), cholesterol-P-450 (80% high) and ternary complex (95% high). The selective binding of substrate (cholesterol) to one of two low spin states of uncomplexed P-450scc, interconvertible by protonation, differs markedly from substrate binding to cytochrome P-450cam (Sligar, S. G., and Gunsalus, I. C. (1979) Biochemistry 18, 2290-2296). Pregnenolone binds competitively with cholesterol while reverting the spin state to a low spin complex. Kd for pregnenolone in absence of cholesterol (17 microM at 25 degrees C) is independent of pH but is competitively increased by adrenodoxin. Evidence is presented for an additional weak pregnenolone complex (Kd = 170 microM) in which both adrenodoxin and cholesterol are retained.
D000311:Adrenal Glands / Q000378:metabolism*; D000325:Adrenodoxin / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002784:Cholesterol / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008433:Mathematics; D008928:Mitochondria / Q000378:metabolism*; D011284:Pregnenolone / Q000378:metabolism
D008024:Ligands; D000325:Adrenodoxin; D011284:Pregnenolone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol