Lowell D. Wilson

Studies on adrenal cortical cytochrome P-450: I. Effects of substrates and electron donors on its catalytic, spectral, and electron paramagnetic resonance properties

Abstract The optical, electronic, and catalytic properties of cytochrome P-450 of bovine adrenal cortical mitochondria have been studied. 11-Deoxycortisol and other substrates for 11β-hydroxylation induce a characteristic difference spectrum in aerobic mitochondria showing absorption minima at 575, 530–535, and 420 mμ. Selective reduction of the hydroxylating enzymes in the absence of steroid substrate produces a typical hemoprotein reduced-minus-oxidized difference spectrum with α, β, and Soret absorption maxima at 575, 530–535, and 420 mμ, respectively. The kinetics of reduction and oxidation of this cytochrome have been correlated with oxygen uptake and steroid hydroxylation. Equilibration of the reduced hemoprotein with increasing carbon monoxide/oxygen ratios results in a progressive conversion of its 420mμ absorption band to the intense 450mμ absorption maximum characteristic of cytochrome P-450-CO. Electron paramagnetic resonance studies of aerobic mitochondria show signals ascribed to mitochondrial cytochrome P-450 at g values 2.42 and 2.26 which can be intensified by 11-deoxycortisol.

Biosynthesis of adrenal corticosteroids: energy metabolism and the hydroxylases

Abstract Evidence for the existence of at least two different cytochrome P450s concerned with corticosteroid hydroxylation has been presented. Metyrapone, a competitive inhibitor of steroid 11β-hydroxylation, affects the ligand fields of the iron of these mixed function oxidases. This agent interferes with the binding of 11β-hydroxylation substrates at the same or interacting sites at low concentrations and with other substrates at high concentrations. Because of the parallel changes in optical properties of the oxygenase, oxygen uptake and oxidation of reduced pyridine nucleotides upon addition of steroid substrates, in addition to their competitive interaction with metyrapone, it seems likely that they also react with the ligand field of cytochrome P450 to alter its redox properties. This presumably increases its rate of oxidation of electron donors and its rate of reaction with oxygen and hydroxylation of substrate. Such a substrate initiated event could be the site of primary control in steroidogenesis and might activate energy metabolism in general by reducing the level of citrate and ATP, and thereby, producing the activation observed in phosphofructokinase. However, the failure to observe an affect of cholesterol on the above parameters even though its oxygenase has very similar properties as other steroid oxygenases, as judged by its action spectrum, argues against such a substrate mediated control mechanism as a general phenomenon for control of corticosteroid hydroxylations. Since subcellular adrenal preparations generally show a high rate of synthesis of corticosteroids from endogenous precursor (cholesterol), when fortified with suitable electron donors, it is likely that the failure to demonstrate cholesterol effects on the cytochrome P450 of these particles is because the oxygenase is already saturated with endogenous cholesterol. Under such circumstances, the rate limiting reations would, most logically, be concerned with the generation of reducing equivalents and not with the provision of corticoid intermediates. The possibility exists that in the isolation of the subcellular particles the cholesterol hydroxylating enzyme is altered in some manner or a compartment is destroyed that permits it to interact with its substrate, a process normally mediated by ACTH. In this case, the primary action of ACTH would be either on the activation or synthesis of a “permease” or a specific synthetase. However, ability of NADPH-generator systems to bypass the stimulatory effect of ACTH on corticoid secretion in certain in vitro studies (8.10) argues against this possibility and further supports the hypothesis that places the control of corticosteroidogenesis on a regulation of energy metabolism. Basically similar conclusions have been drawn by many other investigators (51), differing only in the site at which the control is placed. The studies reported here suggest that phosphofructokinase, but not phosphorylase, could be a regulatory site. Whether or not it is primary, secondary, or concurrent to regulation at other sites is not known.