Mitochondrial Cytochrome P450

Abstract

Abstract A novel method employing the direct reduction of cytochrome oxidase in the presence of antimycin A has provided the means to reveal the presence of cytochrome P450 in chick kidney mitochondria as characterized by the carbon monoxide difference spectrum. This mitochondrial pigment has been found to have properties distinctly different from that of the kidney microsomal cytochrome P450. The mitochondrial content of cytochrome P450 has been found to be about 0.20 nmole per mg of protein. Reconstitution studies with the solubilized cytochrome have indicated that this carbon monoxide-binding hemoprotein is involved in the activity of the renal 25-hydroxycholecalciferol-1α-hydroxylase. The reconstitution required an added flavoprotein and iron-sulfur protein, as well as NADPH and the solubilized hemoprotein. Kinetic experiments on the competitive inhibition of 25-hydroxycholecalciferol-1α-hydroxylase activity of intact mitochondria by metyrapone and aminoglutethimide have shown that the inhibition constants, Ki, for the drugs are 1.6 x 10-4 m and 1.1 x 10-3 m, respectively. The spectral dissociation constants, Ks, for metyrapone and aminoglutethimide as determined from difference spectra are 1.4 x 10-4 m and 4.5 x 10-4 m, respectively. The microsomal spectral dissociation constant for metyrapone is 3.3 x 10-5 m. The similarity between the mitochondrial Ki and Ks values for metyrapone clearly indicates that the extent of the observed mitochondrial 1α-hydroxylase inhibition is a function of the amount of metyrapone bound to cytochrome P450. The kinetic data and the reconstitution studies have substantiated the role of the kidney mitochondrial cytochrome P450 as a terminal oxidase in the 1α hydroxylation of 25-hydroxycholecalciferol.