PDF(Pubmed)
Abstract：
Nitrophorins are heme proteins used by blood feeding insects to deliver nitric oxide (NO) to a victim, leading to vasodilation and antiplatelet activity. Cimex lectularius (bedbug) nitrophorin (cNP) accomplishes this with a cysteine ligated ferric (Fe(III)) heme. In the acidic environment of the insect\'s salivary glands, NO binds tightly to cNP. During a blood meal, cNP-NO is delivered to the feeding site where dilution and increased pH lead to NO release. In a previous study, cNP was shown to not only bind heme, but to also nitrosate the proximal cysteine, leading to Cys-NO (SNO) formation. SNO formation requires oxidation of the proximal cysteine, which was proposed to be metal-assisted through accompanying reduction of ferric heme and formation of Fe(II)-NO. Here, we report the 1.6 Å crystal structure of cNP first chemically reduced and then exposed to NO, and show that Fe(II)-NO is formed but SNO is not, supporting a metal-assisted SNO formation mechanism. Crystallographic and spectroscopic studies of mutated cNP show that steric crowding of the proximal site inhibits SNO formation while a sterically relaxed proximal site enhances SNO formation, providing insight into specificity for this poorly understood modification. Experiments examining the pH dependence for NO implicate direct protonation of the proximal cysteine as the underlying mechanism. At lower pH, thiol heme ligation predominates, leading to a smaller trans effect and 60-fold enhanced NO affinity (Kd = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.
摘要：
Nitrophorins是血红素蛋白，用于喂养昆虫的血液将一氧化氮（NO）传递给受害者，导致血管舒张和抗血小板活性。Cimexlectularius（臭虫）硝化蛋白（cNP）通过半胱氨酸连接的三价铁（Fe（III））血红素来实现这一目标。在昆虫唾液腺的酸性环境中，NO与cNP紧密结合。在血餐期间，cNP-NO被递送到喂食部位,在那里稀释和增加的pH导致NO释放。在之前的研究中,表明cNP不仅结合血红素，还要对近端半胱氨酸进行硝化，导致Cys-NO(SNO)形成。SNO的形成需要近端半胱氨酸的氧化,建议通过伴随铁血红素的还原和Fe（II）-NO的形成来进行金属辅助。这里,我们报告了cNP首先化学还原然后暴露于NO的1.6μ晶体结构，并显示形成了Fe(II)-NO，但没有形成SNO，支持金属辅助SNO形成机制。突变的cNP的晶体学和光谱学研究表明，近端位置的空间拥挤抑制了SNO的形成，而空间松弛的近端位置增强了SNO的形成。提供对这种知之甚少的修饰的特异性的见解。检查NO的pH依赖性的实验暗示近端半胱氨酸的直接质子化是潜在机制。在较低的pH值，巯基血红素连接占主导地位，导致较小的反式效应和60倍增强的NO亲和力（Kd=70nM）。出乎意料的是,我们发现硫醇的形成会干扰SNO的形成，表明cNP-SNO不太可能在昆虫唾液腺中形成。
