Couture M, Stuehr D J, Rousseau D L
Department of Physiology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2000 Feb 4;275(5):3201-5. doi: 10.1074/jbc.275.5.3201.
The mechanisms by which nitric-oxide synthases (NOSs) bind and activate oxygen at their P450-type heme active site in order to synthesize nitric oxide from the substrate L-arginine are mostly unknown. To obtain information concerning the structure and properties of the first oxygenated intermediate of the enzymatic cycle, we have used a rapid continuous flow mixer and resonance Raman spectroscopy to generate and identify the ferrous dioxygen complex of the oxygenase domain of nNOS (Fe(2+)O(2) nNOSoxy). We detect a line at 1135 cm(-1) in the resonance Raman spectrum of the intermediate formed from 0.6 to 3.0 ms after the rapid mixing of the ferrous enzyme with oxygen that is shifted to 1068 cm(-1) with (18)O(2). This line is assigned as the O-O stretching mode (nu(O-O)) of the oxygenated complex of nNOSoxy. Rapid mixing experiments performed with nNOSoxy saturated with L-arginine or N(omega)-hydroxy-L-arginine, in the presence or absence of (6R)-5,6, 7,8-tetrahydro-L-biopterin, reveal that the nu(O-O) line is insensitive to the presence of the substrate and the pterin. The optical spectrum of this ferrous dioxygen species, with a Soret band wavelength maximum at 430 nm, confirms the identification of the previously reported oxygenated complexes generated by stopped flow techniques.
一氧化氮合酶（NOSs）在其P450型血红素活性位点结合并激活氧气，从而从底物L-精氨酸合成一氧化氮的机制大多未知。为了获得有关酶促循环中首个氧合中间体的结构和性质的信息，我们使用了快速连续流动混合器和共振拉曼光谱来生成并鉴定神经元型一氧化氮合酶（nNOS）加氧酶结构域的亚铁双氧络合物（Fe(2+)O(2) nNOSoxy）。我们在亚铁酶与氧气快速混合后0.6至3.0毫秒形成的中间体的共振拉曼光谱中检测到一条位于1135 cm(-1) 的谱线，用 (18)O(2) 时该谱线移至1068 cm(-1)。这条谱线被指定为nNOSoxy氧合络合物的O - O伸缩模式（ν(O - O)）。在存在或不存在 (6R)-5,6,7,8-四氢-L-生物蝶呤的情况下，用饱和L-精氨酸或N(ω)-羟基-L-精氨酸的nNOSoxy进行的快速混合实验表明，ν(O - O) 谱线对底物和蝶呤的存在不敏感。这种亚铁双氧物种的光谱，其最大吸收波长在430 nm处的Soret带，证实了对先前报道的通过停流技术产生的氧合络合物的鉴定。
