Barber M J, Kay C J
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612, USA.
Arch Biochem Biophys. 1996 Feb 15;326(2):227-32. doi: 10.1006/abbi.1996.0069.
Assimilatory NADH:nitrate reductase catalyzes the transfer of reducing equivalents from NADH to molecular oxygen. Initial rate studies performed under conditions of optimal pH (8.0) and constant ionic strength (mu = 0.2) revealed that the maximal rate of activity with molecular oxygen was 0.5% (0.44 mumol NADH consumed/min/nmol heme) with a Km for O2 of 586 microM. NADH:molecular oxygen reductase activity exhibited a pH optimum of 9.2, was inhibited by cyanide, and was unaffected by changes in ionic strength or the presence of phosphate ions. Spectroscopic studies indicated NADH:molecular oxygen reductase activity resulted in the production of the superoxide radical, detected as the formation of adrenochrome from epinephrine and by the formation of adrenochrome from epinephrine and by the reduction of nitroblue tetrazolium, both of which could be inhibited by the addition of superoxide dismutase and were unaffected by the addition of catalase. Direct observation of superoxide production using spin-trapping in combination with EPR spectroscopy resulted in the detection of the spin adduct 5.5-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH). The formation of this spin adduct was abolished either in the absence of nitrate reductase, NADH, or DMPO or the the addition of superoxide dismutase or nitrate and was greatly reduced by the presence of cyanide. Inclusion of catalase or ethanol had no effect on the formation of the spin adduct. These results indicate that nitrate reductase can utilize molecular oxygen as an electron acceptor and that the product, O2.(-), is primarily generated via the Mopterin center.
同化型NADH：硝酸还原酶催化还原当量从NADH转移至分子氧。在最佳pH（8.0）和恒定离子强度（μ = 0.2）条件下进行的初始速率研究表明，以分子氧为底物时的最大活性速率为0.5%（0.44 μmol NADH消耗/分钟/ nmol血红素），O2的Km值为586 μM。NADH：分子氧还原酶活性的最适pH为9.2，受氰化物抑制，不受离子强度变化或磷酸根离子存在的影响。光谱研究表明，NADH：分子氧还原酶活性导致超氧阴离子自由基的产生，可通过肾上腺素生成肾上腺色素以及硝基蓝四氮唑还原进行检测，添加超氧化物歧化酶可抑制这两种反应，而过氧化氢酶的添加则无影响。使用自旋捕获结合电子顺磁共振光谱直接观察超氧阴离子的产生，检测到自旋加合物5,5-二甲基-5-羟基-1-吡咯烷基氧基（DMPO-OH）。在无硝酸还原酶、NADH或DMPO的情况下，或添加超氧化物歧化酶或硝酸盐时，这种自旋加合物的形成被消除，而氰化物的存在则使其大大减少。过氧化氢酶或乙醇的加入对自旋加合物的形成没有影响。这些结果表明，硝酸还原酶可以利用分子氧作为电子受体，并且产物O2(-)主要通过蝶呤中心产生。
