Absorbance spectral change of the cytochrome P-450S21-phenylisocyanide complex upon binding of reduced NADPH-cytochrome-P-450 reductase.
Reduction of cytochrome P-450S21 (SF) (SF, substrate-free; purified from bovine adrenocortical microsomes) with sodium dithionite (Na2S2O4) in the presence of phenylisocyanide produced a ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex with Soret absorbance maxima at 429 and 456 nm. On the other hand, when a preformed ferric cytochrome P-450S21 (SF)-NADPH-cytochrome-P-450 reductase (Fp2) complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum of the ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex changed drastically, as characterized by an increase in absorbance intensity at 429 nm and a decrease at 456 nm. Similar spectral changes were observed by addition of reduced Fp2 to the preformed ferrous cytochrome P-450S21 (SF)-phenylisocyanide complex. Experiments to reduce a ferric cytochrome P-450S21 (SF)-phenylisocyanide complex with sodium dithionite in the presence of various amounts of Fp2 showed that; (1), the spectral change reached maxima for both absorption increase at 429 nm and decrease at 456 nm when cytochrome P-450S21 and Fp2 were previously mixed at the cytochrome P-450S21:Fp2 ratio of 1:5; (2), the spectral change was suppressed in 300 mM potassium phosphate buffer (pH 7.4). These results suggest that the absorbance spectral change is due to a conformational change around the heme moiety induced by association with reduced Fp2.
Iwamoto
Y
Y
Department of Biochemistry, Kagawa Medical School, Japan.
Tsubaki
M
M
Miura
S
S
Ichikawa
Y
Y
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004579:Electron Transport; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009570:Nitriles / Q000378:metabolism*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
D009570:Nitriles; D003577:Cytochrome P-450 Enzyme System; C019519:phenylisocyanide; D009251:NADPH-Ferrihemoprotein Reductase