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Analysis of Electron Flow in Nitric Oxide Synthase

一氧化氮合酶中的电子流分析

基本信息

批准号：
10045076
负责人：
NAKAZAWA Hiroe
金额：
$ 6.78万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Nitric oxide synthase (NOS) transfers electrons supplied with NADPH to oxygen on heme of cytochrome P450-type and oxidizes L-arginine to produce NO during enzyme turnover. But, if the activated oxygen cannot react with L-arginine, the oxygen is released from NOS as reactive oxygen species (ROS). This phenomenon which is called uncoupling reaction, was clarified in detail in each NOS isoform and dependency upon cofactor and substrates was determined. Generations of NO, superoxide (・O_2^-), and H_2O_2 were measured at 25℃ with the metHb formation from HbO_2, the reduction of succinyl cytochrome c, and the generation of Fe (SCN)_4 from Fe^<2+>, respectively. NADPH consumption was also measured to determine total electron flux. Under BH_4^- and arginine-saturated condition, NOS-I and NOS-II did not show significant uncoupling. However, in NOS-III almost a half of electrons remained unconsumed during NO synthesis. In all NOSs, the depletion of substrate, arginine led to uncoupling reaction, whose IC_<50> corresponded to the arginine affinity to each NOS isoform. Under uncoupling condition, major ROS generated from NOS was superoxide in NOS-I and NOS-III, whereas it was H_2O_2 for NOS-II.For NOS-I and NOS-II, the change in electron transfer rate by substrates or cofactors did not alter the ・O_2^-/H_2O_2 ratio, whereas, for NOS-III, the increase in electron transfer rate enhanced ・O_2^- generation. These differences of three NOS isoforms may have an impact on its role in the pathogenesis of NOS-related inflammation processes.

一氧化氮合酶在细胞色素P450型血红素上将NADPH提供的电子传递给氧，并在酶的代谢过程中氧化L-精氨酸生成NO。但是，如果活性氧不能与L精氨酸反应，氧就以活性氧种(ROS)的形式从一氧化氮合酶中释放出来。这种现象被称为解偶联反应，在每一种NOS亚型中都得到了详细的阐明，并确定了对辅因子和底物的依赖关系。在25℃下测定了NO、超氧化物(·O_2^-)和H_2O_2的生成，其中高压氧生成甲基Hb，琥珀酸基细胞色素c还原，Fe~(2+)和Gt~(2+)生成Fe(SCN)_4。NADPH消耗也被测量以确定总的电子通量。在BH4~+和精氨酸饱和条件下，NOS-I和NOS-II没有明显的解偶联现象。然而，在NOS-III中，几乎一半的电子在NO合成过程中仍未被消耗。在所有一氧化氮合酶中，底物精氨酸的耗竭导致解偶联反应，其IC50和gt；对应于精氨酸对各NOS亚型的亲和力。在去偶联条件下，NOS-I和NOS-III产生的主要ROS是超氧化物歧化，而NOS-II产生的主要ROS是H_2O_2。对于NOS-I和NOS-II，底物或辅助因子改变电子传递速率不会改变·O_2^-/H_2O_2比值，而对于NOS-III，电子传递速率的增加增加了·O_2^-的产生。这三种一氧化氮合酶亚型的差异可能影响其在一氧化氮合酶相关炎症过程的发病机制中的作用。