NADPH-cytochrome P450 oxidoreductase - Structural basis for hydride and electron transfer
NADPH-cytochrome P450 oxidoreductase - Structural basis for hydride and electron transfer
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DOI：
10.1074/jbc.m101731200
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发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Kim, JJP
中科院分区：
文献类型：
--
作者：
Hubbard, PA;Shen, AL;Kim, JJP
NADPH-细胞色素P450氧化还原酶催化电子从NADPH通过两个黄素辅因子转移到各种细胞色素P450。与NADP（+）复合的大鼠还原酶的晶体结构表明，烟酰胺与FAD的接触被芳香残基（Trp-677）阻断，该残基与黄素的异咯嗪环的表面堆叠。为了研究在催化循环期间烟酰胺、FAD和Trp-677之间的相互作用的性质，通过晶体学研究了三种突变蛋白。第一个突变体W 677 X去除了最后两个C-末端残基Trp-677和Ser-678;第二个突变体W 677 G保留了C-末端丝氨酸残基。第三突变体具有以下三个催化残基被取代：S457 A、C630 A和D 675 N。在W 677 X和W 677 G结构中，NADP（+）的烟酰胺部分位于FAD异咯嗪环上，两个环的平面之间倾斜约30度。这些结果，连同S457 A/C630 A/D 675 N结构，使我们能够提出一种机制，通过改变氢键和π-π之间的相互作用的异咯嗪环和烟酰胺环或色氨酸-677吲哚环的氢化物转移调节。突变体和野生型结构的叠加显示了酶的两个黄素结构域之间的显着流动性。这一点，连同在所有三个突变体结构的FMN结构域中观察到的高度无序，表明在催化过程中发生构象变化。
NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the reface of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of similar to 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.