PDF(Pubmed)
Abstract：
The sole known heme enzyme of the parasitic protist Giardia intestinalis is a flavohemoglobin (gFlHb) that acts as a nitric oxide dioxygenase (NOD) and protects the organism from the free radical nitric oxide. To learn more about the properties of this enzyme, we measured its nitric oxide dioxygenase, NADH oxidase, and cytochrome c reductase activities and compared these to the activities of the E. coli flavohemoglobin (Hmp). The turnover number for the NOD activity of gFlHb (23 s-1) is about two-thirds of that of Hmp (34 s-1) at pH 6.5 and 37 °C. The two enzymes differ in their sensitivity towards molecules that act as heme ligands. For both gFlHb and Hmp, inhibition with miconazole, a large imidazole ligand, is adequately described by simple competitive inhibition, with KI = 10 μM and 0.27 μM for gFlHb and Hmp, respectively. Inhibition plots with the small ligand imidazole were biphasic, which is consistent with previous experiments with carbon monoxide as a probe that show that the active site of flavohemoglobins exists in two conformations. Interestingly, the largest difference is observed with nitrite, which, like imidazole, also shows a biphasic inhibition plot; however, nitrite inhibits gFlHb at sub-millimolar concentrations while Hmp is not significantly affected. NADH oxidase activity measured under aerobic conditions in the absence of nitric oxide for Hmp was more than twice the activity of gFlHb. The addition of 1 mM hydrogen peroxide in these assays stimulated the NADH oxidase activity of gFlHb but not Hmp. Both enzymes had nearly identical cytochrome c reductase activities but the extent of the contribution of indirect reduction by flavohemoglobin-generated superoxide was much lower with gFlHb (4% SOD-inhibited) than with Hmp (17% SOD-inhibited). Although the active sites of the two enzymes share the same highly conserved residues that are important for catalysis, differences in the distal ligand binding site may account for these differences in activity and sensitivity towards NOD inhibitors. The differences observed in the NADH oxidase and cytochrome c reductase assays suggest that gFlHb may have evolved to protect the protist, which lacks both superoxide dismutase and catalase, from the damaging effects of superoxide by minimizing its production and from peroxide by actively reducing it.
摘要：
寄生原生贾第鞭毛虫肠的唯一已知血红素酶是黄素血红蛋白（gFlHb），可作为一氧化氮双加氧酶（NOD）并保护生物体免受自由基一氧化氮的侵害。要了解更多关于这种酶的特性，我们测量了它的一氧化氮双加氧酶，NADH氧化酶,和细胞色素C还原酶活性，并将其与大肠杆菌黄素血红蛋白（Hmp）的活性进行比较。在pH6.5和37°C下，gFlHb（23s-1）的NOD活性的周转数约为Hmp（34s-1）的三分之二。这两种酶对充当血红素配体的分子的敏感性不同。对于gFlHb和Hmp，用咪康唑抑制，一个大的咪唑配体，通过简单的竞争抑制来充分描述，对于gFlHb和Hmp，KI=10μM和0.27μM，分别。小配体咪唑的抑制图是双相的，这与先前以一氧化碳为探针的实验一致，该实验表明黄素血红蛋白的活性位点存在两种构象。有趣的是,最大的差异是亚硝酸盐，which,像咪唑，还显示了双相抑制图；然而，亚硝酸盐在亚毫摩尔浓度下抑制gFlHb，而Hmp不受显着影响。在不存在一氧化氮的情况下，在有氧条件下对Hmp测得的NADH氧化酶活性是gFlHb活性的两倍以上。在这些测定中加入1mM过氧化氢刺激gFlHb的NADH氧化酶活性,但不刺激Hmp。两种酶的细胞色素c还原酶活性几乎相同，但gFlHb（4％SOD抑制）的黄素血红蛋白生成的超氧化物间接还原的贡献程度远低于Hmp（17％SOD抑制）。尽管两种酶的活性位点共享相同的高度保守的残基，这些残基对催化很重要，远端配体结合位点的差异可能是NOD抑制剂活性和敏感性差异的原因.在NADH氧化酶和细胞色素c还原酶测定中观察到的差异表明gFlHb可能已经进化来保护原生生物，缺乏超氧化物歧化酶和过氧化氢酶，通过最大程度地减少超氧化物的产生来避免超氧化物的破坏作用，并通过积极地减少超氧化物。
