Mitochondrial w-Hydroxylation of Cholesterol Side Chain*

The mitochondrial fraction of rat liver homogenate catalyzed the conversion of cholesterol into 5-cholestene-3P,25diol and 5-cholestene-30,26-diol in the presence of an NADPH-generating system and oxygen. 5-Cholestene3P,26-diol was the predominant product. 26-Hydroxylation of cholesterol was much more efficient with the NADPHgenerating system than with NADPH alone, and it was shown that the isocitrate in the generating system was responsible for most of the stimulation. Addition of Mg2+ increased both the NADPHand the isocitrate-dependent 26-hydroxylation, and at high concentrations of Mg2+ the 26hydroxylation in the presence of NADPH was about the same as in the presence of isocitrate. Addition of Ca2f increased NADPH-dependent but not isocitrate-dependent 26-hydroxylation. NADH and NADH-generating systems could not replace NADPH or the NADPH-generating system. However, NADH together with Mg2+ and ATP stimulated the reaction efficiently, probably due to energy-dependent intramitochondrial transhydrogenation. Citrate and isocitrate were found to stimulate mitochondrial 26-hydroxylation of cholesterol more efficiently than other citric acid cycle intermediates regardless of whether Mg2f was present or absent. Mitochondrial 26-hydroxylation was inhibited by p-hydroxymercuribenzoate. Rotenone, potassium cyanide, and dinitrophenol had little or no effect on isocitrate-dependent 26-hydroxylation. Hg2+, Zn*f, Cu2+, and Fe3f in 0.1 mM concentration inhibited the reaction strongly. Experiments with sonically disrupted mitochondria supported the contention that the mitochondrial 26-hydroxylase was mainly bound to the inner membranes. Experiments with ‘*02 and 2H20 showed that in 25as well as 26-hydroxylation of cholesterol the oxygen incorporated is derived from molecular oxygen.