Conformational change of the heme moiety of the ferrous cytochrome P-450scc-phenyl isocyanide complex upon binding of reduced adrenodoxin.
Reduction of cytochrome P-450scc(SF) (SF, substrate free) purified from bovine adrenocortical mitochondria with sodium dithionite (Na2S2O4) or with beta-NADPH mediated by catalytic amounts of adrenodoxin and adrenodoxin reductase in the presence of phenyl isocyanide produced a ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex with Soret absorbance maximum at 455 nm having a shoulder at 425 nm. On the other hand, when a preformed cytochrome P-450scc(SF)-adrenodoxin complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum showed drastic changes, i.e., an increase in intensity at 425 nm and a concomitant decrease in intensity at 455 nm. Similar spectral changes could be produced by addition of the same amount of reduced adrenodoxin afterward to the ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex. Titration experiments with adrenodoxin showed that (1) a 1:1 stoichiometric saturation of the spectral change was obtained for both the absorbance increase at 425 nm and the absorbance decrease at 455 nm, (2) there was no spectral change in the presence of 0.35 M NaCl, and (3) there was no spectral change for cytochrome P-450scc(SF) whose Lys residue(s) essential to the interaction with adrenodoxin had been covalently modified with PLP. These results suggest that ternary complex formation of ferrous cytochrome P-450scc(SF)-phenyl isocyanide with reduced adrenodoxin caused a conformational change around the ferrous heme moiety. By analysis of temperature and pH dependencies of the spectral change of the ternary complex, it was suggested that this conformational change may reflect the essential step for electron transfer from reduced adrenodoxin to the ferrous-dioxygen complex of cytochrome P-450scc.
Tsubaki
M
M
Department of Biochemistry, Kagawa Medical School, Japan.
Hiwatashi
A
A
Ichikawa
Y
Y
D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System; D004579:Electron Transport / Q000187:drug effects; D005296:Ferrous Compounds / Q000493:pharmacokinetics*; D006418:Heme; D008928:Mitochondria; D009570:Nitriles / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000187:drug effects
D005296:Ferrous Compounds; D009570:Nitriles; D000325:Adrenodoxin; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C019519:phenylisocyanide