Side chain hydroxylation of C27-steroids and vitamin D3 by a cytochrome P-450 enzyme system isolated from human liver mitochondria

The present study was undertaken to obtain information on the involvement of cytochrome P-450 in the 26-hydroxylation of bile acid intermediates and in the 25- hydroxylation of vitamin D3 in human liver mitochondria. Cytochrome P-450 was solubilized from human liver mito- chondria and purified two times to a specific content of 0.125 nmol per mg protein. Furthermore, a ferredoxin was isolated from the mitochondria and partly purified. This iron-sulfur protein had properties similar to bovine adrenal ferredoxin. A mitochondrial NADPH-ferredoxin reductase was also isolated and purified to homogeneity. This enzyme was a flavoprotein with properties very similar to the bovine adrenal NADPH-ferredoxin reductase. The cytochrome P-450 preparation catalyzed 26-hydroxylation of CZ7- steroids and 25-hydroxylation of vitamin D3 when re- constructed with NADPH, the ferredoxin and the ferre- doxin reductase. With different substrates the following turnover numbers (nmol product X nmol P-450" X min") were found: cholesterol, 8; 5-cholestene-3P,7a-diol, 10; 7a-hydroxy-4-cholesten-3-one, 23; 7a, 12a-dihydroxy-4- cholesten-3-one, 27; 5P-cholestane-3a,7a-diol, 28; 5p- cholestane-3a,7a,l2a-triol, 41; and vitamin D3, 0.16. The hydroxylation reactions were inhibited by CO and metyra- ' pone. The human liver mitochondrial ferredoxin and ferredoxin reductase could be replaced by adrenal ferre- doxin and adrenal ferredoxin reductase without reduction of activity, but they could not be replaced by microsomal NADPH-cytochrome P-450 reductase.a It is concluded that human liver mitochondria contain cytochrome P-450 involved in the oxidation of the side chain of (&steroids and vitamin D,.-Oftebro, H., K. Saarem, I. Bjorkhem, and J. I. Pedersen. Side chain hydroxylation of <&-steroids and vitamin D3 by a cytochrome P-4.50 enzyme system isolated from human liver mitocl1ondria.J. L+id RPA. 198 1. 22: 1254- 1264.