Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains

被引:75
作者
Gutierrez, A
Lian, LY
Wolf, CR
Scrutton, NS
Roberts, GCK
机构
[1] Univ Leicester, Dept Biochem, Leicester LE1 7RH, Leics, England
[2] Univ Leicester, Ctr Mechanisms Human Tox, Leicester LE1 9HN, Leics, England
[3] Univ Leicester, Biol NMR Ctr, Leicester LE1 9HN, Leics, England
[4] Univ Dundee, Ninewells Hosp & Med Sch, Biomed Res Ctr, Dundee DD1 9SY, Scotland
关键词
D O I
10.1021/bi001719m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (> 500 s(-1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization (similar to 200 s(-1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (similar to 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH, The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].
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页码:1964 / 1975
页数:12
相关论文
共 52 条
[1]   H-1,N-15 and C-13 NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase [J].
Barsukov, I ;
Modi, S ;
Lian, LY ;
Sze, KH ;
Paine, MJI ;
Wolf, CR ;
Roberts, GCK .
JOURNAL OF BIOMOLECULAR NMR, 1997, 10 (01) :63-75
[2]   ENHANCEMENT OF DOXORUBICIN TOXICITY FOLLOWING ACTIVATION BY NADPH CYTOCHROME P450 REDUCTASE [J].
BARTOSZEK, A ;
WOLF, CR .
BIOCHEMICAL PHARMACOLOGY, 1992, 43 (07) :1449-1457
[3]  
BATIE CJ, 1986, J BIOL CHEM, V261, P1214
[4]   LASER FLASH-PHOTOLYSIS STUDIES OF THE REDUCTION KINETICS OF NADPH-CYTOCHROME-P-450 REDUCTASE [J].
BHATTACHARYYA, AK ;
LIPKA, JJ ;
WASKELL, L ;
TOLLIN, G .
BIOCHEMISTRY, 1991, 30 (03) :759-765
[5]  
BLIGH HFJ, 1990, CANCER RES, V50, P7789
[6]   Reductive and oxidative half-reactions of morphinone reductase from Pseudomonas putida M10:: A kinetic and thermodynamic analysis [J].
Craig, DH ;
Moody, PCE ;
Bruce, NC ;
Scrutton, NS .
BIOCHEMISTRY, 1998, 37 (20) :7598-7607
[7]  
Deng Z, 1999, NAT STRUCT BIOL, V6, P847
[8]  
ENOCH HG, 1979, J BIOL CHEM, V254, P8976
[9]  
GELLER D, 1989, P NATL ACAD SCI USA, V90, P3491
[10]   NITRIC OXIDES SYNTHASES - PROPERTIES AND CATALYTIC MECHANISM [J].
GRIFFITH, OW ;
STUEHR, DJ .
ANNUAL REVIEW OF PHYSIOLOGY, 1995, 57 :707-736