Department of Chemistry and Biochemistry , Florida Atlantic University , Boca Raton , Florida 33431 , United States.
Department of Biomedical Sciences , University of Copenhagen , Copenhagen N DK-2200 , Denmark.
Biochemistry. 2019 Feb 19;58(7):974-986. doi: 10.1021/acs.biochem.8b01231. Epub 2019 Jan 23.
The heme enzyme indoleamine 2,3-dioxygenase-1 (IDO1) catalyzes the first reaction of l-tryptophan oxidation along the kynurenine pathway. IDO1 is a central immunoregulatory enzyme with important implications for inflammation, infectious disease, autoimmune disorders, and cancer. Here we demonstrate that IDO1 is a mammalian nitrite reductase capable of chemically reducing nitrite to nitric oxide (NO) under hypoxia. Ultraviolet-visible absorption and resonance Raman spectroscopy showed that incubation of dithionite-reduced, ferrous-IDO1 protein (Fe-IDO1) with nitrite under anaerobic conditions resulted in the time-dependent formation of an Fe-nitrosyl IDO1 species, which was inhibited by substrate l-tryptophan, dependent on the concentration of nitrite or IDO1, and independent of the concentration of the reductant, dithionite. The bimolecular rate constant for IDO1 nitrite reductase activity was determined as 5.4 M s (pH 7.4, 23 °C), which was comparable to that measured for myoglobin (3.6 M s; pH 7.4, 23 °C), an efficient and biologically important mammalian heme-based nitrite reductase. IDO1 nitrite reductase activity was pH-dependent but differed with myoglobin in that it showed a reduced proton dependency at pH >7. Electron paramagnetic resonance studies measuring NO production showed that the conventional IDO1 dioxygenase reducing cofactors, ascorbate and methylene blue, enhanced IDO1's nitrite reductase activity and the time- and IDO1 concentration-dependent release of NO in a manner inhibited by l-tryptophan or the IDO inhibitor 1-methyl-l-tryptophan. These data identify IDO1 as an efficient mammalian nitrite reductase that is capable of generating NO under anaerobic conditions. IDO1's nitrite reductase activity may have important implications for the enzyme's biological actions when expressed within hypoxic tissues.
血红素酶吲哚胺 2,3-双加氧酶-1（IDO1）催化 l-色氨酸氧化沿犬尿氨酸途径的第一步反应。IDO1 是一种重要的免疫调节酶，对炎症、传染病、自身免疫性疾病和癌症具有重要意义。在这里，我们证明 IDO1 是一种哺乳动物亚硝酸盐还原酶，能够在缺氧条件下将亚硝酸盐化学还原为一氧化氮（NO）。紫外可见吸收和共振拉曼光谱显示，在厌氧条件下，将二硫代苏糖醇还原的亚铁-IDO1 蛋白（Fe-IDO1）与亚硝酸盐孵育导致时间依赖性形成 Fe-亚硝基 IDO1 物种，该物种被底物 l-色氨酸抑制，依赖于亚硝酸盐或 IDO1 的浓度，而与还原剂二硫代苏糖醇的浓度无关。IDO1 亚硝酸盐还原酶活性的双分子速率常数确定为 5.4 M s（pH 7.4，23°C），与肌红蛋白（3.6 M s；pH 7.4，23°C）相当，肌红蛋白是一种有效的和重要的生物哺乳动物血红素基亚硝酸盐还原酶。IDO1 亚硝酸盐还原酶活性依赖于 pH，但与肌红蛋白不同，其在 pH >7 时显示出降低的质子依赖性。测量 NO 产生的电子顺磁共振研究表明，常规 IDO1 加氧酶还原辅因子抗坏血酸和亚甲蓝增强了 IDO1 的亚硝酸盐还原酶活性，并以被 l-色氨酸或 IDO1 抑制剂 1-甲基-l-色氨酸抑制的方式时间和 IDO1 浓度依赖性地释放 NO。这些数据将 IDO1 鉴定为一种有效的哺乳动物亚硝酸盐还原酶，能够在缺氧条件下产生 NO。IDO1 的亚硝酸盐还原酶活性可能对其在缺氧组织中表达时的酶的生物学作用具有重要意义。
