Abu-Soud H M, Gachhui R, Raushel F M, Stuehr D J
Department of Immunology, The Cleveland Clinic Research Institute, Cleveland, Ohio 44195, USA.
J Biol Chem. 1997 Jul 11;272(28):17349-53. doi: 10.1074/jbc.272.28.17349.
Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of L-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis, but its interactions with O2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods to study formation and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 degrees C. Mixing ferrous nNOSoxy with air-saturated buffer generated a transient species with absorption maxima at 427 and approximately 560 nm. This species decayed within 1 s to form ferric nNOSoxy. Its formation was first order with respect to O2, monophasic, and gave rate constants for kon = 9 x 10(5) M-1 s-1 and koff = 108 s-1 for an L-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of L-arginine and/or H4B did not greatly effect O2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O2 concentration and was either biphasic or monophasic depending on sample conditions. L-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s-1), while H4B accelerated its decay by a factor of 70 irrespective of L-arginine. The spectral and kinetic properties of the intermediate identify it as the FeIIO2 complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.
一氧化氮合酶（NOS）是一种血红素蛋白，可催化L-精氨酸氧化为一氧化氮（NO）和瓜氨酸。预计NOS血红素铁在催化过程中参与氧的活化，但其与O2的相互作用尚未明确。我们利用神经元型一氧化氮合酶含血红素的加氧酶结构域（nNOSoxy）和停流法研究了10℃下nNOSoxy氧合复合物的形成和自氧化分解。将亚铁型nNOSoxy与空气饱和缓冲液混合产生了一种瞬态物质，其吸收峰分别位于427和约560nm处。该物质在1秒内衰变形成高铁型nNOSoxy。其形成对O2呈一级反应，单相反应，对于L-精氨酸和四氢生物蝶呤（H4B）饱和的nNOSoxy，结合速率常数kon = 9×105 M-1 s-1，解离速率常数koff = 108 s-1。省略L-精氨酸和/或H4B对O2的结合和解离速率影响不大。氧合中间体的分解与O2浓度无关，根据样品条件，其分解反应可能是双相的或单相的。L-精氨酸可稳定氧合中间体（衰变速率 = 0.14 s-1），而H4B无论有无L-精氨酸，均可将其衰变加速70倍。中间体的光谱和动力学性质表明它是nNOSoxy的FeIIO2复合物。H4B使金属-氧物种不稳定的现象前所未见，这可能与该辅因子在NO合成中的作用有关。
