Flash photolysis studies on the CO complexes of ferrous cytochrome P-450scc and cytochrome P-45011 beta. Effects of steroid binding on the photochemical and ligand binding properties.
Upon irradiation by a light flash (100-J), the carbon monoxide complex of cytochrome P-450scc was fully photodissociated in both the presence and absence of cholesterol, while less than 20% of the CO complex was photodissociable with those of deoxycorticosterone-bound and -free forms of cytochrome P-45011 beta. When the quantum yield of the reaction was measured for each photodissociable portion, the values were 0.5 and 1.0 for the substrate-free and -bound forms of cytochrome P-450scc, and 0.03 and 0.8 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. Thus, CO complexes of these enzymes become more photosensitive upon binding with the specific substrates. Steroid binding also affected kinetic constants of reactions between the ferrous enzymes and CO. The rate constants for the CO recombination at 15 degrees C were 2.7 X 10(6) and 2.3 X 10(5) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-450scc, and were 7.0 X 10(5) and 5.4 X 10(3) M-1 s-1 for the substrate-free and -bound forms of cytochrome P-45011 beta, respectively. The rate constants for the CO dissociation also decreased upon the steroid bindings. The products of the enzyme reactions, pregnenolone and corticosterone, had similar effects on the kinetic constants. From these findings, we postulate that the binding of a steroid to the substrate site of each enzyme alters the bonding character of CO with the heme-iron, thereby affecting both photochemical and kinetic properties of the CO complex. The nature of the photoindissociable portion of the CO complex of cytochrome P-45011 beta is also discussed.
D000302:Adrenal Cortex / Q000378:metabolism; D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism*; D002417:Cattle; D002784:Cholesterol / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003900:Desoxycorticosterone / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D010782:Photolysis; D011485:Protein Binding
D008024:Ligands; D003900:Desoxycorticosterone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol