Excited-State Dynamics of Oxyluciferin in Firefly Luciferase

Abstract

The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time-resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH-dependent emission to a single chemical species would be an oversimplification.

The Fluorescent Properties of pH‐Independent Cypridina Oxyluciferin Derivatives

“…Bioluminescence is a natural process in living organisms, which converts chemical energy into light emission . There are 700 genera can produce bioluminescence in the world . It′s worth mentioning that about 80% of bioluminescent organisms live in the ocean .…”

Luciferin Regeneration in Firefly Bioluminescence via Proton‐Transfer‐Facilitated Hydrolysis, Condensation and Chiral Inversion

“…Oxyluciferin is composed of six chemical forms (see molecular structures and abbreviations in Scheme S1), which can coexist and convert one another in solution. The excited‐state (S 1 ) of oxyluciferin, keto‐1, has been verified to be the main form of the light emitter of firefly bioluminescence,–, although a more complex situation may exist . Upon emitting light, the S 1 state, keto‐1, relaxes to the ground state (S 0 ), keto, which has been indicated from the crystallographic structure of luciferase (PDB: 2D1R) .…”

Theoretical Development of Near‐Infrared Bioluminescent Systems

“…So, the key to modify the light emission is to modify the fluorescence of the light emitter. The anionic keto form of oxyluciferin in its S 1 state has been verified to be the main form of the light emitter, although recent excited‐state dynamics indicates a more complex situation . The BL and fluorescence of the light emitter are conceptually different, but actually the same for firefly BL …”