SPECTROSCOPIC AND KINETIC-STUDIES ON REACTION OF CYTOCHROME P450NOR WITH NITRIC-OXIDE - IMPLICATION FOR ITS NITRIC-OXIDE REDUCTION-MECHANISM
SPECTROSCOPIC AND KINETIC-STUDIES ON REACTION OF CYTOCHROME P450NOR WITH NITRIC-OXIDE - IMPLICATION FOR ITS NITRIC-OXIDE REDUCTION-MECHANISM
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DOI：
10.1074/jbc.270.4.1617
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发表时间：
1995-01-27
影响因子：
4.8
通讯作者：
SHOUN, H
中科院分区：
文献类型：
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作者：
SHIRO, Y;FUJII, M;SHOUN, H
从尖孢镰刀菌中提纯的细胞色素P450(P450nor)是一种独特的血红素酶，它直接从NADH(2NO+NADH+H+-&>；N2O+H2O+NAD(+))转移电子，催化一氧化氮还原为一氧化二氮。我们用停流快速扫描和低温光谱方法研究了P450nor与NO和NADH的反应。NO配体能与铁酶结合形成稳定的NO结合络合物P450nor(Fe3+NO)。用NADH还原P450 Nor(Fe3+NO)生成中间体，该中间体瞬间形成(tau=类似于100ms)，并自发分解为Fe3+态。该中间体的光学吸收光谱不同于P450nor(Fe3+NO)与Na2S2O4单电子还原或与P450nor(Fe2+)结合形成的P450nor(Fe2+NO)的光学吸收光谱。在这些观察的基础上，我们推测该中间体可能是P450nor(Fe3+NO)被NADH两电子还原的产物，正式为(Fe3+NO)(2-)络合物。我们测定了10℃下NO与P450 Nor(Fe~(3+))(2.6×10~(-7)M(-1)S(-1))结合、P450 Nor(Fe_8+NO)(0.9×10~(6)M(~(-1))S(-1))的NADH还原反应以及中间体(0.027×10~(-1)M~(-1))自发分解的速率常数。在这些动力学测量中，发现前两个过程足够快，而后一个过程非常慢，与我们通过监测NADH消耗测得的催化反应的快速周转率(10℃下的1200 S(-1))相比，我们认为在催化循环中，游离NO对中间体的分解相当快，导致了如此快的周转率。根据光谱和动力学证据，我们提出了P450 Nor还原NO的可能机理。
Cytochrome P450 purified from Fusarium oxysporum (P450nor) is a unique heme enzyme that catalyzes the reduction of nitric oxide to nitrous oxide with electrons directly transferred from NADH (2NO + NADH + H+ --> N2O + H2O + NAD(+)). We studied the reaction of P450nor with NO and NADH using stopped-flow rapid scan and low temperature spectroscopic methods. The NO ligand can bind to the ferric enzyme to form the stable NO bound complex, P450nor(Fe3+NO). Reduction of P450nor(Fe3+NO) with NADH yielded an intermediate, which transiently formed (tau = similar to 100 ms) and spontaneously decomposed to the Fe3+ state. The optical absorption spectrum of the intermediate was different from that of P450nor(Fe2+NO), which was formed by either a one-electron reduction of P450nor(Fe3+NO) with Na2S2O4 or NO binding to P450nor(Fe2+), On the basis of these observations, we suggested that the intermediate is presumably a two electron reduced product of P450nor(Fe3+NO) by NADH, formally the (Fe3+NO)(2-) complex. We determined the rate constants of these re actions at 10 degrees C for the NO binding to P450nor(Fe3+) (2.6 x 10(7) M(-1) s(-1)), the NADH reduction of P450nor(Fe8+NO) (0.9 x 10(6) M(-1) s(-1)), and the spontaneous decomposition of the intermediate (0.027 s(-1)). In these kinetic measurements, it was found that the former two processes are fast enough, while the latter is extremely slow, compared with the fast turnover of the catalytic reaction (1200 s(-1) at 10 degrees C), which we measured by monitoring the NADH consumption, Therefore, we suggested that in the catalytic cycle, decomposition of the intermediate is fairly accelerated by free NO, resulting in such a fast turnover, On the basis of several lines of the spectroscopic and the kinetic evidence, we proposed a possible mechanism of the NO reduction by P450nor.