A Conical Intersection Controls the Deactivation of the Bacterial Luciferase Fluorophore

Abstract

The photophysics of flavins is highly dependent on their environment. For example, 4a-hydroxy flavins display weak fluorescence in solution, but exhibit strong fluorescence when bound to a protein. To understand this behavior, we performed temperature-dependent fluorescent studies on an N(5)-alkylated 4a-hydroxy flavin: the putative bacterial luciferase fluorophore. We find an increase in fluorescence quantum yield upon reaching the glass transition temperature of the solvent. We then employ multiconfigurational quantum chemical methods to map the excited-state deactivation path of the system. The result reveals a shallow but barrierless excited state deactivation path that leads to a conical intersection displaying an orthogonal out-of-plane distortion of the terminal pyrimidine ring. The intersection structure readily explains the observed spectroscopic behavior in terms of an excited-state barrier imposed by the rigid glass cavity.

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase

“…In addition, the 13 C chemical shifts of the C(4a) atoms (Table ) strongly indicate that the alloxazines possess a somewhat lesser degree of sp 3 hybridization (downfield shift) than the isoalloxazines. The fluorescence property of N(5)‐ethyl‐C(4a)‐hydroxy‐4a,5‐dihydrolumiflavin (I in Table ) in glassy and fluid states has been investigated by computational and experimental methods . Interestingly, in the ground state a bond length of 1.34 Å has been found for the N(10)‐C(10a) bond , in close agreement with the X‐ray diffraction structure.…”

“…In the past two decades, there have been many attempts to justify this intersection and crossing of the potential energy surfaces (PES) of the reactants and excited state . In a sensitized process on the other hand, the immediate product is a “hot” S 0 vibrational state of the HEI, which on subsequent diffusion enters into a collision complex with an acceptor, wherein its decomposition deposits exothermicity into the excited state of the acceptor .…”

“…In spite of the computational challenge of the many‐body target consisting of a substrate within the protein binding site, there has been some progress in getting useful results on bioluminescence systems . The firefly and coelenterazine reactions have been the subject of most studies, recently one concluding that there was an unsuspected nonemissive diradical excited state in the coelenterazine light pathway.…”

“…Fewer computational efforts directed to bacterial bioluminescence have been made. One report showed why the fluorescence of the 4a‐hydroxyflavin model under rigid conditions was due to inhibition of an out of plane distortion of the pyrimidine ring, being relieved under fluid conditions that would provide a pathway quenching its fluorescence . Another study came to the conclusion that there was no support for CIEEL or a dioxirane mechanism in bacterial bioluminescence; instead, the chemical excitation was a direct chemiluminescence via CTIL to produce the 4a‐hydroxyflavin as the primary excited state and “proving” for the first time that the 4a‐hydroxyflavin S 1 state was the origin of the bioluminescence .…”

Synthesis and electronic properties of π-extended flavins

“…[1][2][3] They play an important role in light, oxygen, and voltage-sensitive (LOV) domains sensitive for blue light excitation. 13,15,16 The local environment, 17 hydrogen bonding, 18,19 π-π stacking 20 or metal coordination 21,22 to the chromophore and the type and position of substituents 23 determine the redox potentials and the photophysical properties of flavin derivatives. 13,15,16 The local environment, 17 hydrogen bonding, 18,19 π-π stacking 20 or metal coordination 21,22 to the chromophore and the type and position of substituents 23 determine the redox potentials and the photophysical properties of flavin derivatives.…”