Interaction of Cyanoketone and Other Steroid Nitriles with Cytochrome Oxidase, Hemoglobin, and Cytochrome P-450*

Abstract

The inhibitory activity of cyanoketone (CNK; 2alpha-cyano-4,4,17alpha-trimethyl-17beta-hydroxy-5-androsten-3-one), was investigated for enzymes of the respiratory chain and cholesterol side chain cleavage (CSCC). In bovine corpus luteum mitochondria incubated with [26-14C]cholesterol, 500 micron CNK caused 90% inhibition of pregnenolone synthesis. Comparable results were obtained with adrenal and placental mitochondria. Addition of CNK to bovine corpus luteum mitochondria or to cytochrome P-450 purified from this source elicited a concentration-dependent, reverse type I difference spectrum with an absorption maximum at about 423 nm and a minimum at about 395 nm, confirming binding to oxidized cytochrome P-450. This spectral change resembles those of steroids which inhibit CSCC. In mitochondrial preparations, CNK induced a second peak at about 445 nm. This peak was similar to that elicited by the interaction of potassium cyanide with cytochrome a3 when the former is added to rabbit heart mitochondria which are devoid of P-450. Like cyanide, CNK block mitochondrial respiration at the cytochrome oxidase site, and induced spectral changes in human hemoglobin. Therefore, this peak at 445 nm probably represents the interaction of CNK with oxidized cytochrome a3. Several other steroid nitriles had little, if any, effect on CSCC activity, nor did they induce spectral changes with cytochrome oxidase or hemoglobin. It appears that the steroid configuration of CNK is responsible for the binding to P-450 and inhibition of CSCC, whereas the binding to cytochrome a3 and hemoglobin and the inhibitory effect on electron transfer are probably related to the cyano group of CNK.