Analysis of Electron Flow in Nitric Oxide Synthase

一氧化氮合酶 (NOS) 将 NADPH 提供的电子转移到细胞色素 P450 型血红素上的氧，并在酶周转过程中氧化 L-精氨酸以产生 NO。但是，如果活性氧不能与 L-精氨酸发生反应，氧就会以活性氧 (ROS) 的形式从 NOS 中释放出来。这种被称为解偶联反应的现象在每种 NOS 异构体中得到了详细阐明，并确定了其对辅因子和底物的依赖性。在25℃下，分别测量了HbO_2生成的metHb、琥珀酰细胞色素c的还原以及Fe^2生成Fe(SCN)_4的NO、超氧化物(·O_2^-)和H_2O_2的生成。还测量了 NADPH 消耗以确定总电子通量。在BH_4^-和精氨酸饱和条件下，NOS-I和NOS-II没有表现出明显的解偶联。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NOS中，底物精氨酸的消耗导致解偶联反应，其IC_<50>对应于精氨酸对每种NOS亚型的亲和力。在解偶联条件下，NOS产生的ROS在NOS-I和NOS-III中主要是超氧化物，而在NOS-II中主要是H_2O_2。对于NOS-I和NOS-II，底物或辅因子引起的电子转移速率的变化不会改变·O_2^-/H_2O_2比率，而对于NOS-III，电子转移速率的增加增强了·O_2^-的生成。三种 NOS 亚型的这些差异可能会影响其在 NOS 相关炎症过程发病机制中的作用。

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.