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 excitedstate barrier imposed by the rigid glass cavity.Oxidized flavin cofactors like flavin mononucleotide (FMN) contain a planar, stiff flavin moiety that displays intense fluorescence in solutions (with a lifetime of ca. 5 ns).[1] Such a fluorescence is quenched when the cofactor is proteinbound due to fast excited-state electron transfer from the flavin to neighboring aromatic amino acid residues (such as tyrosine or tryptophan). This behavior of oxidized flavins has been exploited to follow the conformational changes of flavoproteins over the course of enzymatic reactions. [2][3][4][5] In contrast, the 1,5 and 4a,5 flavin adducts (see Scheme 1 a) such as those found in reduced (FMNH 2 ) [6] and 4a-hydroxylated (FMNHOH) cofactors, respectively, [7] display a weak fluorescence in solution. [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.[16] Moreover, such flavin adducts are frequent intermediates in enzymatic catalysis by flavinbased monooxygenases, which activate molecular oxygen and catalyze the insertion of atomic oxygen into a number of substrates (e.g. in hydroxylations, sulfoxidations, epoxidations and Baeyer-Villiger oxidations).[17] A specific type of such monooxygenase activity is responsible for the bacterial luciferase fluorescence, [18][19][20][21][22] where the oxidation of aldehydes to acids via molecular oxygen would generate the 4a-hydroxy flavin byproduct as the putative light-emitting intermediate (FMNHOH in Sche...

Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence—A Theoretical Study

“…theoretically studied the deformation of a molecule analogous to LFOH; this deformation leads to a barrierless but nonradiative deactivation path from the S 1 state to S 0 state and subsequently results in the low quantum yield of fluorescence. Similarly, the structural rearrangement of S 1 –LFOH from S 0 –LFOH causes weak fluorescence in solutions . According to the current calculations, for S 1 ‐LFOH, its HOMO on benzene and pyrazine rings is nearly orthogonal to its LUMO on the pyrimidine ring (Supporting Information, Figure S3).…”

“…Under vacuum, LFOH in its S 1 state shows out‐of‐plane deformation of terminal pyrimidine (the C 9a ‐N 10 ‐C 10a ‐N 1 dihedral angle is 81.0°, Figure d). Gozem et al . theoretically studied the deformation of a molecule analogous to LFOH; this deformation leads to a barrierless but nonradiative deactivation path from the S 1 state to S 0 state and subsequently results in the low quantum yield of fluorescence.…”

“…[a] LF in water at pH 6 . [b] Stable LFOH analogues of 5‐Et‐LFOH in a 2‐methyl‐tetrahydrofuran solution . [c] FMN in the vibrio harveyi luciferase at pH 7, and the 445 nm band is resolved into two distinct peaks at 434 and 458 nm .…”

Fluorescence Properties of Flavin Semiquinone Radicals in Nitronate Monooxygenase

“…The lack of a single protein system capable of thermodynamically stabilizing both the anionic and neutral forms of flavin semiquinone is a fundamental problem that prevents any direct comparison of the photophysical properties of the flavin semiquinone in different ionized states due to the effects exerted by the surrounding protein environment . Glucose oxidase is the only known case in which both neutral and anionic flavin semiquinones can be transiently formed by photoreduction .…”

“…The fluorescence processes of flavin in the oxidized state typically yield excitation at 450 nm and emission at 525–535 nm, thus suggesting that small traces of oxidized flavin might have been present in the cryptochrome sample . Alternatively, a more‐dynamic protein environment surrounding the flavin anionic semiquinone in NMO than in type 1 cryptochrome might explain the lack of fluorescence emission in NMO …”