Control of electron transfer in neuronal NO synthase
Control of electron transfer in neuronal NO synthase
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DOI：
10.1042/0300-5127:0290147
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发表时间：
2001-05-01
影响因子：
3.9
通讯作者：
Shimizu, T
中科院分区：
文献类型：
--
作者：
Daff, S;Noble, MA;Shimizu, T
一氧化氮合酶 (NOS) 是二聚体黄素细胞色素，由加氧酶结构域和细胞色素 P450 样 Cys 连接血红素组成，并与与细胞色素 P450 还原酶相关的二黄素还原酶结构域偶联。 NOS 催化精氨酸顺序单氧化为 N-羟基精氨酸，然后催化为瓜氨酸和 NO。组成型 NOS 亚型 (cNOS) 受钙调蛋白 (CaM) 调节，钙调素在游离 Ca2+ 浓度升高时结合，而诱导型亚型不可逆地结合 CaM。组成型和诱导型异构体之间的主要结构差异之一是 cNOS 的 FMN 结合域中插入了 40-50 个氨基酸。删除大鼠神经元 NOS (nNOS) 中的插入片段会产生一种突变酶，该酶在较低 Ca2+ 浓度下结合 CaM，并在没有 CaM 的情况下保留活性。为了解决 CaM 激活的作用机制，我们使用重组形式的还原酶结构域测定了在存在和不存在 CaM 的情况下大鼠 nNOS 的 FMN 和 FAD 辅因子的还原电位。结果表明，CaM 结合不会调节黄素的还原电位，但似乎主要通过大的结构重排来控制电子转移。我们还报道了嵌合酶的产生，其中 nNOS 和黄素细胞色素 P450 BM3（巨大芽孢杆菌 III）的还原酶结构域已交换。尽管其黄素氧化还原电位非常不同，但 BM3 还原酶结构域能够支持低水平的 CaM 依赖性 NO 合成，而 NOS 还原酶结构域不能有效替代细胞色素 P450 BM3。
The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca2+, whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca2+ concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. The results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement, We also report the creation of chimaeric enzymes in which the reductase domains of nNOS and flavocytochrome P450 BM3 (Bacillus megaterium III) have been exchanged. Despite its very different flavin redox potentials, the BM3 reductase domain was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.