Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase.
A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface.
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006736:Horses; D007527:Isoenzymes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000032:analysis; D010634:Phenobarbital / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D007527:Isoenzymes; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide; D010634:Phenobarbital