Role of Histidine 310 in Amydetes vivianii firefly luciferase pH and metal sensitivities and improvement of its color tuning properties.

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.