Studies on FAD- and FMN-binding domains in NADPH-cytochrome P-450 reductase from rabbit liver microsomes.
Six sulfhydryl group were determined after complete denaturation of NADPH-cytochrome P-450 reductase; of these, about 5.2 in both the native holoenzyme and FMN-depleted enzyme are accessible to p-hydroxychloromercuribenzoate (pCMB), which may be differentiated as follows: four --SH groups are modified by low concentration of the reagent but are not essentially involved in the catalytic function; additional block of one --SH group at high concentrations of pCMB completely inhibited the reductase activity. The fluorescence quenching of the FAD in the FMN-depleted enzyme was removed after the fifth --SH group was reacted slowly with pCMB. Kinetic and fluorometric analysis indicated that this finally modified --SH group was assumed to be essential for the activity and significantly protected by either 1 mM NADP+ or 2'-AMP against attack by mercurial compounds. A strong negative ellipticity at around 450 nm is clearly decreased upon binding of pCMB to an essential --SH group, while the CD spectra in the near and far UV region show only minor differences during the modification of --SH groups. Removal of the FMN prosthetic group from the native holoprotein results in 1.25-fold greater tryptophan fluorescence with a slight red shift of the emission maximum from 332 to 336 nm, and FMN reconstitution reduces the protein fluorescence quantum yield to approximately that of the holoprotein. Oxidation of tryptophan indol rings of the FMN-depleted enzyme is associated with a loss of FMN binding ability to the protein which causes the inactivation of cytochrome c reductase activity, but ferricyanide reductase activity is not strongly affected by tryptophan modification.
D000818:Animals; D001665:Binding Sites; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011817:Rabbits; D013053:Spectrophotometry; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D005182:Flavin-Adenine Dinucleotide; D020245:p-Chloromercuribenzoic Acid; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase