Fast Excited-State Deactivation in N(5)-Ethyl-4a-hydroxyflavin Pseudobase

Abstract

We present a study of the excited-state behavior of N(5)-ethyl-4a-hydroxyflavin (Et-FlOH), a model compound for bacterial bioluminescence. Using femtosecond pump-probe spectroscopy, we found that the Et-FlOH excited state exhibits multiexponential dynamics, with the dominant decay component having a 0.5 ps lifetime. Several possible mechanisms for fast excited-state decay in Et-FlOH were considered: (i) excited-state deprotonation of the -OH proton, (ii) thermal deactivation via (1)n,π* → (1)π,π* conical intersection, and (iii) excited-state release of OH(-) ion. These mechanisms were excluded based on transient absorption studies of two model compounds (N(5)-ethyl-4a-methoxyflavin, Et-FlOMe, and N(5)-ethyl-flavinium ion, Et-Fl(+)) and based on the results of time-dependent density functional theory (TD-DFT) calculations of Et-FlOH excited-states. Instead, we propose that the fast decay in Et-FlOH is caused by S(1) → S(0) internal conversion, initiated by the excited-state nitrogen planarization (sp(3) → sp(2) hybridization change at the N(5)-atom of Et-FlOH S(1) state) coupled with out-of-plane distortion of the pyrimidine moiety of flavin.

A Conical Intersection Controls the Deactivation of the Bacterial Luciferase Fluorophore

“…All three absorption bands were assigned, on the basis of TDDFT calculations in acetonitrile, to p,p* transitions with a degree of charge transfer character. [11] Similar bands were observed for other model 4a,5 flavin adducts whose energies were found to depend on the nature of substituents. [6,24] The room-temperature fluorescence of Et-FlOH displays a 496 nm l max (Figure 1 b) with a Stokes shift of more than 8000 cm À1 and weak intensity corresponding to a quantum yield of only 3 10 À3 .…”

“…The two emission bands are assigned to the vibronic progression of the Et-FlOH excited state, as the presence of multiple conformers was ruled out based on invariant variable-temperature absorption spectra of Et-FlOH in 2-MeTHF. Furthermore, the fluorescence lifetime is increased from a 500 fs dominant component at room temperature [11] to a nanosecond biexponential decay in the glass (Figure 1 c, lifetimes are t 1 = 2.8 ns and t 2 = 7.8 ns). Such a sudden increase in the fluorescence intensity upon confinement of a chromophore in a rigid molecular cavity has also been observed in other fluorophores.…”

“…[8,9] However, when the same cofactors are protein-bound, the confinement [6,7] leads to an enhanced fluorescence emission. [10][11][12][13][14][15] The exact geometry and electronic structure of the emitting conformer and the mechanism preventing the efficient internal conversion occurring in solution are presently unknown.A spectacular manifestation of the effects of protein confinement on flavin cofactors is seen in bacterial luciferases where a 4a,5 flavin adduct is held responsible for the bioluminescence of a vast group of marine eubacteria. In organisms such as Vibrio harveyi, the emission of the fluorophore may be strong enough to cause a blue luminescence of the microbial population that is so intense as to be observable from satellites.…”

“…Using femtosecond pump-probe spectroscopy, we found that, in solution, the first singlet excited (S 1 ) state of Et-FlOH decays to the ground (S 0 ) state with several time constants but with a dominant component that has a 500 fs lifetime. [11] The absorption spectrum of Et-FlOH in 2-methyl-tetrahydrofuran (2-MeTHF) exhibits three bands in the UV region with l max at 282, 308 and 347 nm (Figure 1 a). All three absorption bands were assigned, on the basis of TDDFT calculations in acetonitrile, to p,p* transitions with a degree of charge transfer character.…”

“…[6,24] The room-temperature fluorescence of Et-FlOH displays a 496 nm l max (Figure 1 b) with a Stokes shift of more than 8000 cm À1 and weak intensity corresponding to a quantum yield of only 3 10 À3 . [11] The large Stokes shift and low fluorescence quantum yield are both indicative of a significant change in geometry during the initial S 1 relaxation and following progression towards an effective radiationless decay channel. To gain a better understanding of this fast excited-state deactivation, we report a variable temperature fluorescence study of Et-FlOH.…”