Firefly luciferases emit yellow-green light and are pH-sensitive, changing the bioluminescence color to red in the presence of heavy metals, acidic pH and high temperatures. These pH and metal-sensitivities have been recently harnessed for intracellular pH indication and toxic metal biosensing. However, whereas the structure of the pH sensor and the metal binding site, which consists mainly of two salt bridges that close the active site (E311/R337 and H310/E354), has been identified, the specific role of residue H310 in pH and metal sensing is still under debate. The Amydetes vivianii firefly luciferase has one of the lowest pH sensitivities among the group of pH-sensitive firefly luciferases, displaying high bioluminescent activity and special spectral selectivity for cadmium and mercury, which makes it a promising analytical reagent. Using site-directed mutagenesis, we have investigated in detail the role of residue H310 on pH and metal sensitivity in this luciferase. Negatively charged residues at position 310 increase the pH sensitivity and metal sensitivity; H310G considerably increases the size of the cavity, severely impacting the activity, H310R closes the cavity, and H310F considerably decreases both pH and metal sensitivities. However, no substitution completely abolished pH and metal sensitivities. The results indicate that the presence of negatively charged and basic side chains at position 310 is important for pH sensitivity and metals coordination, but not essential, indicating that the remaining side chains of E311 and E354 may still coordinate some metals in this site. Furthermore, a metal binding site search predicted that H310 mutations decrease the affinity mainly for Zn, Ni and Hg but less for Cd, and revealed the possible existence of additional binding sites for Zn, Ni and Hg.

Graphical abstract

中文翻译：

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组氨酸 310 在 Amydetes vivianii 萤火虫荧光素酶 pH 和金属敏感性中的作用及其颜色调节特性的改进

萤火虫荧光素酶发出黄绿色光，并且对 pH 值敏感，在存在重金属、酸性 pH 值和高温的情况下，生物发光颜色会变为红色。这些 pH 和金属敏感性最近已被用于细胞内 pH 指示和有毒金属生物传感。然而，虽然 pH 传感器和金属结合位点的结构已被确定，主要由两个关闭活性位点的盐桥（E311/R337 和 H310/E354）组成，但残基 H310 在 pH 和金属传感仍处于争论之中。 Amydetes vivianii萤火虫荧光素酶是 pH 敏感性萤火虫荧光素酶中 pH 敏感性最低的酶之一，具有高生物发光活性以及对镉和汞的特殊光谱选择性，这使其成为一种很有前景的分析试剂。通过定点诱变，我们详细研究了残基 H310 对这种荧光素酶 pH 和金属敏感性的作用。 310位带负电的残基增加pH敏感性和金属敏感性； H310G 显着增加空腔的尺寸，严重影响活性，H310R 封闭空腔，H310F 显着降低 pH 值和金属敏感性。然而，没有任何替代品可以完全消除 pH 值和金属敏感性。结果表明，310位上带负电和碱性侧链的存在对于pH敏感性和金属配位很重要，但不是必需的，表明E311和E354的剩余侧链可能仍然在该位点配位一些金属。此外，金属结合位点搜索预测，H310 突变主要降低对 Zn、Ni 和 Hg 的亲和力，但对 Cd 的亲和力较小，并揭示了可能存在额外的 Zn、Ni 和 Hg 结合位点。

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