Copper‐mediated oxidation of imidazopyrazinones inhibits marine luciferase activity

Abstract

The development of bioluminescence‐based tools has seen steady growth in the field of chemical biology over the past few decades ranging in uses from reporter genes to assay development and targeted imaging. More recently, coelenterazine‐utilizing luciferases such as Gaussia, Renilla, and the engineered nano‐luciferases have been utilized due to their intense luminescence relative to firefly luciferin/luciferase. The emerging importance of these systems warrants investigations into the components that affect their light production. Previous work has reported that one marine luciferase, Gaussia, is potently inhibited by copper salt. The mechanism for inhibition was not elucidated but was hypothesized to occur via binding to the enzyme. In this study, we provide the first report of a group of nonhomologous marine luciferases also exhibiting marked decreases in light emission in the presence of copper (II). We investigate the mechanism of action behind this inhibition and demonstrate that the observed copper inhibition does not stem from a luciferase interaction but rather the chemical oxidation of imidazopyrazinone luciferins generating inert, dehydrated luciferins.

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

“…Notably, high concentrations of Cu 2+ led to bioluminescence quenching ( Figure S4a ), which is consistent with a previously documented mechanism involving the Cu 2+ -mediated oxidation of the luciferin. 36 However, these high Cu 2+ concentrations are unlikely to be physiologically relevant. 37…”

Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase

“…The results confirmed the system’s selectivity toward K + . Notably, high concentrations of Cu 2+ led to bioluminescence quenching (Figure S4a), which is consistent with a previously documented mechanism involving the Cu 2+ -mediated oxidation of the luciferin . However, these high Cu 2+ concentrations are unlikely to be physiologically relevant …”

“…Notably, high concentrations of Cu 2+ led to bioluminescence quenching (Figure S4a), which is consistent with a previously documented mechanism involving the Cu 2+ -mediated oxidation of the luciferin. 36 However, these high Cu 2+ concentrations are unlikely to be physiologically relevant. 37 Moreover, we conducted additional investigations into the potassiorin concentration dependency of BRIPO bioluminescence in the presence or absence of 150 mM K + (Figure S4b).…”