Pufahl R A, Wishnok J S, Marletta M A
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy University of Michigan, Ann Arbor 48109-1065.
Biochemistry. 1995 Feb 14;34(6):1930-41. doi: 10.1021/bi00006a014.
The ability of murine macrophage nitric oxide synthase (NOS) to utilize peroxides in place of O2 and NADPH was investigated using hydrogen peroxide (H2O2), tert-butylhydroperoxide, and cumene hydroperoxide with both L-arginine and NG-hydroxy-L-arginine (L-NHA) as substrates. Of the three peroxides examined, only H2O2 was able to support product formation using L-NHA as a substrate. No product formation was observed from L-arginine with any peroxide tested. Therefore, the L-NHA/H2O2 reaction was examined in greater detail. The products of the reaction were citrulline and nitrite/nitrate (NO2-/NO3-) with a stoichiometry of approximately 0.75:1 (citrulline to NO2-/NO3-). Product formation was greater in the presence of oxygen. Both the Km and Vmax of the reaction, determined under aerobic conditions, were affected by (6R)-tetrahydro-L-biopterin (H4B). Chemiluminescence experiments failed to detect nitric oxide (.NO) as a reaction product. However, spectral spectral experiments with L-NHA and H2O2 under anaerobic conditions demonstrated the appearance of a ferrous heme-.NO complex with a Soret peak at 440 nm and a broad single alpha/beta peak at 578 nm, which is believed to arise from single electron transfer of a ferric-NO- (nitroxyl) complex. Preliminary experiments detected nitrous oxide (N2O) formation by gas chromatography under anaerobic conditions. Stable isotope labeling experiments with [18O]H2O2 conclusively established incorporation of label exclusively into the ureido position of citrulline. Based on these results, a mechanism of oxidation of L-NHA by H2O2 is proposed.
利用过氧化氢（H₂O₂）、叔丁基过氧化氢和异丙苯过氧化氢，以L-精氨酸和NG-羟基-L-精氨酸（L-NHA）作为底物，研究了小鼠巨噬细胞一氧化氮合酶（NOS）利用过氧化物代替O₂和NADPH的能力。在所检测的三种过氧化物中，只有H₂O₂能够以L-NHA作为底物支持产物生成。用所测试的任何一种过氧化物处理L-精氨酸均未观察到产物生成。因此，对L-NHA/H₂O₂反应进行了更详细的研究。该反应的产物是瓜氨酸和亚硝酸盐/硝酸盐（NO₂⁻/NO₃⁻），其化学计量比约为0.75:1（瓜氨酸与NO₂⁻/NO₃⁻）。在有氧条件下产物生成更多。在有氧条件下测定的该反应的Km和Vmax均受（6R）-四氢-L-生物蝶呤（H4B）影响。化学发光实验未能检测到一氧化氮（·NO）作为反应产物。然而，在厌氧条件下用L-NHA和H₂O₂进行的光谱实验表明，出现了一种亚铁血红素-NO复合物，其Soret峰在440 nm，α/β宽单峰在578 nm，据信这是由铁-NO-（硝酰基）复合物的单电子转移产生的。初步实验通过气相色谱在厌氧条件下检测到了一氧化二氮（N₂O）的生成。用[¹⁸O]H₂O₂进行的稳定同位素标记实验最终确定标记仅掺入瓜氨酸的脲基位置。基于这些结果，提出了H₂O₂氧化L-NHA的机制。
