Nitrophorins (NP) 1-7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

中文翻译：

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硝化铁蛋白7中Glu27对配体结合机理的静电调节。

氮素（NP）1-7是在吸血昆虫Rhodnius prolixus唾液中发现的NO携带的血红素蛋白。异构体NP7显示出特殊的特性，例如异常高的等电点，结合带负电的膜的能力以及对NO亲和力的强pH敏感性。NP7的独特特征是在第27位存在Glu，在其他NP中被Val占据。Glu27似乎对调节血红素性质很重要，但仍未探索其对pH依赖性NO释放机制的影响，该影响由AB环的构象变化协助。在这里，为了深入了解Glu27的功能，我们使用CO作为模型，研究了Glu27→Val和Glu27→Gln突变对配体结合动力学的影响。结果表明，突变后Glu27负电荷的an灭会降低配体结合率的pH敏感性，而该过程又取决于Asp32的电离作用。我们提出，Glu27在Asp32上施加贯穿空间的静电作用，该作用将后一个氨基酸的pKa移至更多的酸性值，从而降低了在打开状态和关闭状态之间转换的pH敏感性。