Kinetics of dithionite-dependent reduction of cytochrome P450 3A4: Heterogeneity of the enzyme caused by its oligomerization

被引:80
作者
Davydov, DR
Fernando, H
Baas, BJ
Sligar, SG
Halpert, JR
机构
[1] Univ Texas, Med Branch, Dept Pharmacol & Toxicol, Galveston, TX 77555 USA
[2] Univ Illinois, Dept Biochem & Chem, Beckman Inst Adv Sci & Technol, Urbana, IL 61801 USA
关键词
D O I
10.1021/bi0509346
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
To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (LIP) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO2center dot- anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.
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页码:13902 / 13913
页数:12
相关论文
共 76 条
[11]   SLOW (RESTING) FORMS OF MITOCHONDRIAL CYTOCHROME-C-OXIDASE CONSIST OF 2 KINETICALLY DISTINCT CONFORMATIONS OF THE BINUCLEAR CUB/A(3) CENTER - RELEVANCE TO THE MECHANISM OF PROTON TRANSLOCATION [J].
COOPER, CE ;
JUNEMANN, S ;
IOANNIDIS, N ;
WRIGGLESWORTH, JM .
BIOCHIMICA ET BIOPHYSICA ACTA, 1993, 1144 (02) :149-160
[12]  
CREUTZ C, 1974, J BIOL CHEM, V249, P6788
[13]   KINETICS AND MECHANISM OF ELECTRON-TRANSFER FROM DITHIONITE TO MICROSOMAL CYTOCHROME-B5 AND TO FORMS OF THE PROTEIN ASSOCIATED WITH CHARGED AND NEUTRAL VESICLES [J].
DAVIES, DM ;
LAWTHER, JM .
BIOCHEMICAL JOURNAL, 1989, 258 (02) :375-380
[14]  
DAVYDOV D R, 1982, Biokhimiya, V47, P1476
[15]  
Davydov D.R., 1997, HIGH PRESSURE RES BI, P111
[16]   HIGH-PRESSURE INDUCED INACTIVATION OF FERROUS CYTOCHROME-P-450 LM2 (IIB4) CO COMPLEX - EVIDENCE FOR THE PRESENCE OF 2 CONFORMERS IN THE OLIGOMER [J].
DAVYDOV, DR ;
KNYUSHKO, TV ;
HOA, GHB .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1992, 188 (01) :216-221
[17]   Stabilization of P4502B4 by its association with P450 1A2 revealed by high-pressure spectroscopy [J].
Davydov, DR ;
Petushkova, NA ;
Archakov, AI ;
Hoa, GHB .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2000, 276 (03) :1005-1012
[18]   KINETIC-STUDIES ON REDUCTION OF CYTOCHROME-P-450 AND CYTOCHROME-B5 BY DITHIONITE [J].
DAVYDOV, DR ;
KARYAKIN, AV ;
BINAS, B ;
KURGANOV, BI ;
ARCHAKOV, AI .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1985, 150 (01) :155-159
[19]   Interactions of cytochrome P450 2B4 with NADPH-cytochrome P450 reductase studied by fluorescent probe [J].
Davydov, DR ;
Knyushko, TV ;
Kanaeva, IP ;
Koen, YM ;
Samenkova, NF ;
Archakov, AI ;
Hoa, GHB .
BIOCHIMIE, 1996, 78 (8-9) :734-743
[20]   HIGH-PRESSURE-INDUCED TRANSITIONS IN MICROSOMAL CYTOCHROME-P450 2B4 IN SOLUTION - EVIDENCE FOR CONFORMATIONAL INHOMOGENEITY IN THE OLIGOMERS [J].
DAVYDOV, DR ;
DEPREZ, E ;
HOA, GHB ;
KNYUSHKO, TV ;
KUZNETSOVA, GP ;
KOEN, YM ;
ARCHAKOV, AI .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1995, 320 (02) :330-344