Characterization of the FAD binding domain of cytochrome P450 reductase.
Characterization of the FAD binding domain of cytochrome P450 reductase.
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细胞色素 P450 还原酶 FAD 结合域的表征。
DOI：
10.1006/abbi.1996.0012
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Strobel,HW
中科院分区：
文献类型：
--
作者：
Hodgson,AV;Strobel,HW
细胞色素P450还原酶FAD和FMN在溶液中的氧化还原电位相当，但与还原酶结合时相差138 mV。每个黄素与其黄素结合区的相互作用赋予每个黄素独特的电子转移能力。为了确定FAD结合域的黄素结合特性和活性，我们在pTrcHis中表达了包含FAD和NADPH结合域的大鼠肝还原酶基因的三个片段(1161、1244和1556bp)。含有1161和1556碱基载体的细胞FAD结合片段稳定，分别结合0.66和0.71摩尔FAD/摩尔酶。这两个片段都还原了铁氰化物(分别为54%和104%的FMN还原酶/摩尔结合的黄素)，并参与了3-AcPyADP的转氢反应(分别为41%和65%的FMN还原酶/摩尔结合黄素)。纯化非FAD片段，并与8-氨基-FAD和8-氯-FAD重组，以确定结合效率。
The redox potentials of FAD and FMN of Cytochrome P450 reductase (reductase) are equivalent in solution but differ by 138 mV when bound to reductase. The interaction of each flavin with its flavin binding domain confers the unique electron transferring abilities to each flavin. In order to determine flavin binding properties and activity of the FAD binding domain, we have expressed in pTrcHis three fragments (1161, 1244, and 1556 bp) of rat liver reductase cDNA encompassing the proposed FAD and NADPH binding domain. The FAD binding fragments from cells harboring the 1161- and 1556-bp-containing vectors were stable and bound 0.66 and 0.71 mol FAD/mol enzyme, respectively. Both fragments reduce ferricyanide (54 and 104% of FMN-less reductase/mol bound flavin, respectively) and participate in the transhydrogenation reaction of 3-AcPyADP (41 and 65% of FMN-less reductase/mol bound flavin, respectively). FAD-less fragments were purified and reconstituted with 8-amino-FAD and 8-chloro-FAD to determine binding efficiencies.