Recruitment of governing elements for electron transfer in the nitric oxide synthase family
Recruitment of governing elements for electron transfer in the nitric oxide synthase family
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DOI：
10.1073/pnas.0506522102
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发表时间：
2005-11-01
影响因子：
11.1
通讯作者：
Masters, BSS
中科院分区：
文献类型：
--
作者：
Jáchymová, M;Martásek, P;Masters, BSS
至少有三个结构单元负责调节一氧化氮合酶（NOS）亚型中的电子转移的分子基础：钙调蛋白结合序列、C-末端延伸和还原酶结构域中的自动调节环。我们试图赋予的C末端的NOS细胞色素P450氧化还原酶（CYPOR），其中不包含这些调控元件的控制。这些C末端对CYPOR性质的影响揭示了NOS可能的进化起源，并解决了CYPOR新功能开发中新肽的招募问题。NOS的C末端调节黄素蛋白介导的电子转移到各种电子受体。将这些C末端中的任何一个添加到CYPOR中，都会抑制人工电子受体细胞色素c、2，6-二氯酚靛酚和铁氰化物的还原，而分子O-2的还原则增加。这表明在限速步骤的转变，表明NOS的C末端中断黄素单核苷酸（FMN）和黄素腺嘌呤二核苷酸（FAD）和/或电子受体之间的电子通量。通过添加NOS C末端调节CYPOR也得到黄素再氧化和荧光猝灭研究以及C末端延伸的抗体识别的支持。这些实验支持NOS酶的起源模块组成的血红素结构域和CYPOR或铁氧还蛋白-NADP（+）还原酶-和黄素氧还蛋白样亚结构域构成CYPOR，随后进一步招募更小的调节元件到黄素结合域。
At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O-2 was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.