Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States of America.
Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, United States of America.
J Inorg Biochem. 2023 Sep;246:112263. doi: 10.1016/j.jinorgbio.2023.112263. Epub 2023 Jun 1.
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 (K = 70 nM). Unexpectedly, we find that thiol formation interferes with SNO formation, suggesting cNP-SNO is unlikely to form in the insect salivary glands.
硝普蛋白是一类血红素蛋白，可被吸血昆虫用于向受害者输送一氧化氮（NO），从而导致血管舒张和抗血小板活性。臭虫（Cimex lectularius）硝普蛋白（cNP）通过一个半胱氨酸配位的三价铁（Fe(III)）血红素来实现这一功能。在昆虫唾液腺的酸性环境中，NO 与 cNP 紧密结合。在吸血过程中，cNP-NO 被输送到进食部位，在那里稀释和 pH 值升高会导致 NO 释放。在之前的研究中，cNP 不仅被证明可以结合血红素，还可以硝化为近端半胱氨酸，导致 Cys-NO（SNO）的形成。SNO 的形成需要氧化近端半胱氨酸，这被认为是通过伴随的三价铁血红素还原和 Fe(II)-NO 的形成而实现的金属辅助过程。在这里，我们报告了 cNP 的 1.6 Å 晶体结构，该结构首先进行了化学还原，然后暴露于 NO 下，并表明形成了 Fe(II)-NO，但没有形成 SNO，支持了一种金属辅助的 SNO 形成机制。对突变 cNP 的晶体学和光谱学研究表明，近端部位的空间位阻抑制了 SNO 的形成，而空间位阻较小的近端部位则增强了 SNO 的形成，为深入了解这种理解甚少的修饰提供了见解。研究 NO 对 pH 值依赖性的实验表明，近端半胱氨酸的直接质子化是潜在的机制。在较低的 pH 值下，硫醇血红素配位占主导地位，导致较小的反位效应和 60 倍增强的 NO 亲和力（K=70 nM）。出乎意料的是，我们发现硫醇形成会干扰 SNO 的形成，这表明在昆虫的唾液腺中不太可能形成 cNP-SNO。
