REACTION OF PHTHALATE DIOXYGENASE REDUCTASE WITH NADH AND NAD - KINETIC AND SPECTRAL CHARACTERIZATION OF INTERMEDIATES

被引:48
作者
GASSNER, G [1 ]
WANG, LH [1 ]
BATIE, C [1 ]
BALLOU, DP [1 ]
机构
[1] UNIV MICHIGAN,DEPT BIOL CHEM,ANN ARBOR,MI 48109
关键词
D O I
10.1021/bi00206a022
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
Phthalate dioxygenase reductase (PDR) is an electron transferase that contains FMN, which accepts a hydride from NADH, and a [2Fe-2S] center, which transfers electrons to phthalate dioxygenase. The reduction of PDR by NADH has been studied by stopped-flow spectroscopy. Data from studies using both protio- and deuterio-NADH were analyzed by nonlinear curve fitting and numerical simulation techniques. The results of these analyses indicate that the reductive half-reaction of PDR consists of five distinct kinetic phases: (a) NADH binds to form a primary Michaelis complex (MC-1) (K-d = 50 mu M). (b) The enzyme undergoes a structural change (116 +/- 5 s(-1)) resulting in a charge-transfer complex (CT-1). (c) The next phase in the reaction shows a deuterium isotope effect of 7.0 when (4R)-[H-2]NADH (NADD) is substituted for NADH, identifying this step as the one involving hydride transfer. The rate of hydride transfer from NADH to FMN is 70 s(-1), and this process results in a charge-transfer intermediate between the flavin hydroquinone anion and NAD (CT*). (d) Internal electron transfer from the flavin to the iron-sulfur center, which is only 35 +/- 4 s(-1), then results in an intermediate consisting of a reduced [2Fe-2S] center and a neutral flavin semiquinone (SQ). It is surprising that this rate is so slow, since the shortest interatomic distance between these centers is only 4.7 Angstrom [Correll, C. C., et al. (1992) Science 258, 1604-1610]. The 2-electron-reduced form of PDR (SQ in Figure 1) binds weakly to the reaction product, NAD (K-d = 3.7 mM), but forms a tight complex with NADH (K-d = 10 mu M). (e) TWO molecules of the reduced iron-sulfur flavin semiquinone (SQ) form of PDR then undergo a relatively slow second-order disproportionation reaction, resulting in one molecule of 3-electron-reduced PDR and one molecule of 1-electron-reduced PDR. The latter reacts rapidly with excess NADH to form a 3-electron-reduced PDR.
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页码:12184 / 12193
页数:10
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