Oxidation-reduction potentials of cytochromes P-450 and ferredoxin in the bovine adrenal : Their modification by substrates and inhibitors

Abstract

The midpoint reduction potentials of the haem iron in bovine adrenal cytochrome P-450 and its associated iron-sulphur protein, adrenal ferredoxin, have been measured, using EPR spectroscopy to monitor the high and low spin ferric haem iron and reduced adrenal ferredoxin signals as a function of potential, in mitochondrial and microsomal suspensions. In mitochondria the high spin (substrate-bound) cytochrome P-450 showed single-component one-electron plots under most conditions; at pH 6.65 cholesterol side-chain cleavage cytochrome P-450 ( P-450 scc) had a midpoint E m = −305 mV; at pH 8.0 11β-hydroxylase cytochrome P-450 ( P-450 11 β ) had E m = −335 mV. Low spin cytochrome P-450 showed more complex titration curves under all conditions, which could be most simply interpreted in terms of two one-electron components with midpoint potentials approx. −360 and −470 mV, with varying intensities. During treatments that caused substrate binding, only the −470 mV component was reduced in magnitude. On sonication and removal of adrenal ferredoxin, the −470 mV low spin component was converted to higher potential. The potentials could also be altered by the cytochrome P-450 inhibitors aminoglutethimide and metyrapone. In the microsomes, a high spin component of cytochrome P-450 ( E m ≈ −290 mV) was observed even at pH 8.0, suggesting the binding of an endogenous substrate, while the low spin P-450 showed a predominance of the −360 mV component. The midpoint potential of membrane-bound adrenal ferredoxin under these various conditions was found to be −248 mV ± 15 mV.