Studies on FAD- and FMN-binding domains in NADPH-cytochrome P-450 reductase from rabbit liver microsomes.

Y Nisimoto,Y Shibata

doi:10.1016/s0021-9258(18)33543-9

Abstract

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.

Highlights

A strong negative ellipticity at around 450 nm is clearly decreased binding domain is concerned with this “SH group essential for the catalytic function.Using both the holoenzyme and FMN-depleted enzyme, our present studies show that a catalytically important “ S H group is in the active sitein which FAD-binding domain and NADPH-binding domain are probably closely located and interacted with each other
Studies on FMN and profteluinorescence quenching have provided a possibility for tryptophan-flavin interactions in the reductase.It is well known that flavodoxin is the upon binding ofpCMB to an essential ” S H group, flavoprotein which shows aclose association of FMN withone while the CD spectra in the near and far W region tryptophan side chain in thceoenzyme bindingsite [9].Similar show only minor differencesduring themodification of observations have been made with a variety of other flavodoxins (10, ll),and this appears btoe a generalproperty
In the recent demonstration holoprotein results in1.25-fold greater tryptophan flu- based onx-ray crystal structure determination, they indicated orescence with a slightred shift of the emission maxi- that a tryptophan side chain is in close proximity to the mum from 332 to 336 nm, and FMN reconstitution re- isoalloxazine ring of FMN in the flavodoxin [12, 13]

Summary

EXPERIMENTA LP ROCEDURES

CL-GB, The detergent-solubilized hepatic NADPH-cytochrome P450 reductase (EC 1.6.2.4), the flavoprotein component of a liver microsomal mixed function oxidase, contains 1 mol each. On the other Reductase-Detergent-solubilized NADPH-cytochrome P-450 reduchand, FMN, which is the high potential flavin, appears to participate as an electron carrier in the process of electron transfer from NADPH to cytochromPe-450 during themixed tase was prepared by the method of Iyanagi and Mason [18] with some modifications [19]. Preparation of FMN-reconstituted Enzyme-The solution of FMN-depleted enzyme (35 p~ as FAD) dissolved in 0.1 M potassium phosphate buffer, pH 7.6, containing 10%glycerol, was incubated with a 1.5-fold excess of FMN for 30 min a t 20 "C and was dialyzed against. The Oxidationof Tryptophan Residueswith a Low Concentration of Ozone-Oxidation ofindolrings of tryptophan residues in the "c reductase was carried out a t 3 in 0.1 M potassium phosphate buffer, PH 7.2, containing 10% glycerol. For the measurementsof fluorescence intensities of the enzyme-bound flavin and N'-formylkynurenine, the excitation wavelengths were setat 450 and 320 nm, witha fixed spectral bandwidth of 3 nm

RESULTS

Flavin contents"

DISCUSSION