Fluorescence probing of the function-specific cysteines of rat microsomal NADPH-cytochrome P-450 reductase.
Titration of NADPH-cytochrome P-450 reductase with a fluorigenic maleimide suggests that approximately four cysteines are initially accessible and in close proximity to four tryptophans. Perturbation of the cysteines and/or tryptophans results in concomitant decreases in enzymic activity. These cysteines were correlated with functional components by binding studies and subsequent tryptic peptide mapping on the acid mobile phase-reverse phase HPLC. Adenine nucleotides and cytochrome c block labelling of the more hydrophilic peptides, while detergents facilitate labelling of the more hydrophobic peptides. The more hydrophobic peptides contain the microsomal binding site of cytochrome P-450. Removal of the prosthetic flavins exposes more cysteines in the more hydrophilic and hydrophobic regions of the peptide map, associating the former with FAD and the latter with FMN binding sites.
D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005453:Fluorescence; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D000227:Adenine Nucleotides; D003574:Cytochrome c Group; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; D003545:Cysteine