Cytochrome b5, cytochrome c, and cytochrome P-450 interactions with NADPH-cytochrome P-450 reductase in phospholipid vesicles.
Upon incubation of detergent-solubilized NADPH-cytochrome P-450 reductase and either cytochrome b5 or cytochrome c in the presence of a water-soluble carbodiimide, a 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), covalently cross-linked complex was formed. The cross-linked derivative was a heterodimer consisting of one molecule each of flavoprotein and cytochrome, and it was purified to 90% or more homogeneity. The binary covalent complex between the flavoprotein and cytochrome b5 was exclusively observed following incubation of all three proteins including NADPH-cytochrome P-450 reductase, cytochrome b5, and cytochrome c in L-alpha-dimyristoylphosphatidylcholine vesicles, and no heterotrimer could be identified. The isolated reductase-cytochrome b5 complex was incapable of covalent binding with cytochrome c in the presence of EDC. No clear band for covalent complex formation between PB-1 and reductase was seen with the present EDC cross-linking technique. More than 90% of the cross-linked cytochrome c in the purified derivative was rapidly reduced upon addition of an NADPH-generating system, whereas approximately 80% of the cross-linked cytochrome b5 was rapidly reduced. These results showed that in the greater part of the complexes, the flavin-mediated pathway for reduction of cytochrome c or cytochrome b5 by pyridine nucleotide was intact. When reconstituted into phospholipid vesicles, the purified amphipathic derivative could hardly reduce exogenously added cytochrome c, cytochrome b5, or PB-1, indicating that the cross-linked cytochrome shields the single-electron-transferring interface of the flavoprotein. These results suggest that the covalent cross-linked derivative is a valid model of the noncovalent functional electron-transfer complex.
Nisimoto
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Y
Department of Biochemistry, Aichi Medical University, Japan.
Otsuka-Murakami
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H
D000818:Animals; D003432:Cross-Linking Reagents; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D003573:Cytochrome b Group / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D015786:Cytochromes b5; D004579:Electron Transport; D005022:Ethyldimethylaminopropyl Carbodiimide; D005486:Flavin Mononucleotide / Q000378:metabolism; D066298:In Vitro Techniques; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D051381:Rats
D003432:Cross-Linking Reagents; D003573:Cytochrome b Group; D003574:Cytochrome c Group; D008081:Liposomes; D005486:Flavin Mononucleotide; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide