Dynamics and Mechanisms of Ultrafast Fluorescence Quenching Reactions of Flavin Chromophores in Protein Nanospace

Abstract

We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), d-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp . NH) or tyrosine (Tyr . OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90−100 fs) could be attributed to the compact stacked arrangement, Trp . NH.....F.....Tyr . OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck−Condon) → Fl (fluorescence) state conversion in PNS within the time resolution of the apparatus. The ultrafast FC → Fl conversion may be a coherent process coupled with intra-chromophore high-frequency modes leading to formation of vibrationally nonrelaxed or only partially relaxed Fl state, from which barrierless ET seems to occur. Fluorescence dynamics of DAOB have indicated faster initial decay in both blue and red sides of the spectrum contrary to other flavoproteins which showed practically wavelength-independent fluorescence dynamics. This result of DAOB is similar to those of photoactive yellow protein and visual rhodopsin although their reaction mechanism (twisting) is different from DAOB (ET). We have proposed a possible mechanism for this fluorescence dynamics of DAOB on the basis of an extremely compact stacked configuration of F...benzoate-...Tyr . OH which seems to undergo moderate frequency intermolecular vibration coupled with intra-chromophore high-frequency modes of F in the course of ET from Tyr . OH to excited F.

Ultrafast real-time visualization of active site flexibility of flavoenzyme thymidylate synthase ThyX

“…Through its influence on the electronic coupling, the ET rate k critically depends on the edge-to-edge distance r between the flavin cofactor and the quenching residue as k = k 0 e −βr (3,27). Assuming ET between Tyr and excited FAD is near barrierless (3,11), and β = 1.36 Å −1 (28), we can convert the lifetime distribution (Fig. 3C) to a distribution of distances from which ET takes place (Fig.…”

“…Experimentally, we exploited the fact that photoexcited FAD (FAD*) can accept an electron from the aromatic residues Tyr and Trp on the femtosecond-to-nanosecond timescale, and showed that in the case of TmThyX from, Tyr-91 is the principal electron donor. It has been argued that the FAD*Tyr→FAD − Tyr°+ reaction occurs with a driving force in the range of 0.7-1 eV and in a near-activationless regime (3,11). Indeed, the rates of this electron transfer reaction were found to be temperature independent in flavoproteins with a much less heterogeneous fluorescence decay distribution than TmThyX (11, 13).…”

“…In particular, the fluorescence properties of intrinsic or external fluorophores can be exquisitely sensitive to the protein environment. In flavoproteins, interaction of the flavin cofactor with the protein environment has been shown to diminish the lifetime of the flavin fluorescence from the intrinsic nanosecond timescale to the femtoseconds-to-picoseconds timescale, due to quenching by photooxidation of nearby aromatic residues (3,(9)(10)(11)(12)(13)(14)(15). In the present study, we use ultrafast fluorescence spectroscopy to investigate conformational flexibility of the flavoenzyme thymidylate synthase ThyX.…”

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

“…Furthermore, the pattern of positive and negative absorbance changes shows distinctive differences: the intensive negative signal of the stimulated emission at 520 -600 nm observed for free riboflavin in aqueous solution is missing for HsDod A -bound riboflavin (2), and the positive signal does not reach that far into the long wavelength region above 700 nm. A discussed mechanism for the fast quenching of the excited state, on the basis of previous investigations on flavoproteins (9,18,19), is an ultrafast electron transfer from a tryptophan residue to the excited flavin. If such an electron transfer occurs, the spectrum should show the absorption characteristics of a cationic tryptophan radical, expected around 560 nm (38,39) and of an anionic flavosemiquinone around 510 nm (40 -42).…”

Theoretical Analyses of Photoinduced Electron Transfer from Aromatic Amino Acids to the Excited Flavins in Some Flavoproteins

“…Time-resolved fluorescence spectroscopy of flavins and flavoproteins has been reviewed by Berg and Visser (2001). However, a number of flavoproteins are practically non-fluorescent, but rather they emit fluorescence with very short lifetimes (sub-picoseconds) upon excitation with an ultra-short laser pulse (Mataga et al, 1998(Mataga et al, , 2000(Mataga et al, , 2002Tanaka et al 2007;Chosrowjan et al, 2007Chosrowjan et al, , 2008Chosrowjan et al, , 2010. In these flavoproteins tryptophan (Trp) and/or tyrosine (Tyr) residues always exist near the isoalloxazine ring (Iso).…”