Irreversible inhibition of cytochrome P450 by nitric oxide.
Nitric oxide (NO) modulates various metabolisms through interaction with thiol proteins and hemoproteins. Although NO interacts reversibly with iron moieties of heme proteins, including cytochrome P450 (P450), dynamic aspects of the formation, catalytic functions and fates of NO-P450 adducts remain to be elucidated. When incubated with NOC7, which spontaneously and stoichiometrically releases NO within 5 min, microsomal P450 rapidly formed nitrosyl-heme adducts as determined by the electron spin resonance method. The signal intensity for the complex increased with time, peaking at 30 min and decreasing to below detectable levels by 60 min of incubation. In contrast, the microsomal levels of low-spin ferric forms of P450 (g = 2.26) rapidly decreased during the initial 30 min but recovered time-dependently thereafter. Analysis by differential spectra (reduced form/CO-reduced form) revealed that on incubation with NOC7, the form of microsomal P450 also changed in a biphasic manner. To elucidate the mechanism for the decrease in the levels of P450, microsomal levels of P450 isozymes (CYPs) were determined by Western blot analysis using specific antibodies against CYP3A2 and CYP2C11, major isoforms found in male rat liver. Kinetic analysis revealed that no appreciable degradation of P450 proteins occurred during the incubation of microsomes with NOC7. The effect of NO on the catalytic activity of the enzymes was determined by using testosterone as substrate because hydroxylation of steroid hormones is one of the major functions of P450. When exposed to NO, the hydroxylation activity in microsomes rapidly decreased during the initial 10 min and then disappeared slowly. These results suggested that NO formed dissociable complexes with P450 isozymes and the catalytic functions of these isozymes were irreversibly inactivated after dissociation from their heme moiety.
Minamiyama
Y
Y
Department of Biochemistry, Osaka City University Medical School, Abeno-ku, Osaka 545, Japan. yukiko@msic.med.osaka-cu.ac.jp
Takemura
S
S
Imaoka
S
S
Funae
Y
Y
Tanimoto
Y
Y
Inoue
M
M
D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D006900:Hydroxylation; D007501:Iron / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008297:Male; D009569:Nitric Oxide / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds / Q000032:analysis; D013739:Testosterone / Q000378:metabolism
D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D007501:Iron