Fluorescence flavins

Callis PR, Liu T (2006) Short range photoinduced electron transfer in proteins QM-MM simulations of tryptophan and flavin fluorescence quenching in proteins. Chem Phys 326 (l) 230-239... 

Vames AW, Dodson RB, Wehry EL (1972) Interactions of transition-metal ions with photoexcited states of flavins. Fluorescence quenching studies. J Am Chem Soc 94 946-950... 

S.A. Siano and R. Mutharasan, NADH and flavin fluorescence responses of starved yeast cultures to substrate additions, Biotechnol. Bioeng. 34, 660-670 (1989). 

The quantum yield of flavin fluorescence in proteins is very low in many cases, and the lifetimes are on the order of picoseconds. This is a result of the high electrophilicity of oxidized flavins, and their ability to quench fluorescence following electron transfer from the electron-rich groups of... 

Mutations in the active site of the enzyme can decrease the flavin-to-haem electron transfer rate to an extent that it is almost identical to the steady-state turnover rate of the enzyme (Noble et al., 1999). Also, problems with flavin fluorescence can be circumvented by studies of the haem domain of P450 BM3, and this has proven of great value for resonanee Raman characterisation of the haem site. One important finding from such studies has been that the active site of P450 BM3 is large enough to aeeommodate both a fatty acid and a large inhibitor molecule (metyrapone) simultaneously (Macdonald et al., 1996). 

The properties of the flavin are exploited for flavoenzyme characterization. Absorbance spectroscopy (under anaerobisis, as needed) can be used for binding studies, redox titrations, and rapid-reaction kinetics (Note the relevant articles in the See Also section). Other useful techniques include fluorescence spectroscopy (at equilibrium or time-resolved, although in most cases the flavin fluorescence is quenched in the holoen-zyme), EPR, and NMR. Several of these techniques have been developed early on (also) thanks to research on fiavoproteins. 

Flavoenzymes may be ideal model systems for the development of new tools to be made available for the study of enzymes in general. One example may be the use of cholesterol oxidase for single molecule enzymology, which exploits the flavin fluorescence changes during the catalytic cycle (29). 

Vissci A J.W.Gn 1984. Bnerics of slacking imetactions in flavin ademne rSnoefeoUde Crofu time-icsatved flavin fluorescence. Pho-tochem. PhotobM. 40 703-706... 

Visse A. J. W.G.. l984,Kindicsofsto(kingin(eiacUonsinflavin adenine (Emicleotide from dme iesolved flavin fluorescence, Pho todiem. Photobiol. 40 703-706. 

Riboflavin and the nucleotides FMN and FAD fluoresce, although at various pH values the relative intensities change sufficiently to permit this property to serve as the basis for a differential analysis of the flavin. Fluorescence disappears as groups dissociating at pH 1.7 and 10.2 are titrated with acid and alkali, respectively. It has been concluded... 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

Flavin mononucleotide (Na, 2H2O salt, FMN) [ 130-40-5JM 514.4, pKj 2.1 (PO4H2), pK2 6.5 (PO4H ), pKj 10.3 (CONH), fluorescence Xmax 530nm (870nm for reduced form). 

Latia luciferase is colorless and normally nonfluorescent. However, the luciferase fluoresces visibly in alkaline solutions. The fluorescence is most prominent in a KCN solution, showing an emission spectrum that is very close to the bioluminescence spectrum and also to the fluorescence emission of a flavin (FAD) except for the 370 nm... 

No information is available on the chemical nature of the luminophore, although the photoprotein must contain a chro-mophore to emit luminescence and fluorescence. Acid treatment of the protein, followed by extraction with organic solvents, did not yield coelenteramide or coelenteramine, indicating that this luminescence system is unrelated to coelenterazine. A flavin (FAD) was found in partially purified preparations of photoprotein, but not in highly purified preparations. 

Eley, M., et al. (1970). Bacterial bioluminescence. Comparisons of bioluminescence emission spectra, the fluorescence of luciferase reaction mixtures, and the fluorescence of flavin cations. Biochemistry 9 2902-2908. 

Macheroux, P., et al. (1987). Purification of the yellow fluorescent protein from Vibrio fischeri and identity of the flavin chromophore. Biochem. Biophys. Res. Commun. 146 101-106. 

Riboflavin is heat-stable in the absence of light, but extremely photosensitive. It has a high degree of natural fluorescence when excited by UV light. This property can be used for detection and determination. Two coenzymes (Fig. 2), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are derived from riboflavin. 

Fig. 21. Top The general Jablonski diagram for the flavin chromophore. The given wavelengths for absorption and luminescence represent crude average values derived from the actual spectra shown below. Due to the Franck-Condon principle the maxima of the peak positions generally do not represent so-called 0 — 0 transitions, but transitions between vibrational sublevels of the different electronically excited states (drawn schematically). Bottom Synopsis of spectra representing the different electronic transitions of the flavin nucleus. Differently substituted flavins show slightly modified spectra. Absorption (So- - S2, 345 nm S0 -> Si,450nm 1561) fluorescence (Sj — S0) 530 nm 156)) phosphorescence (Ty Sq, 605 nm 1051) triplet absorption (Tj ->Tn,...

A second approach with respect to anisotropic flavin (photo-)chemistry has been described by Trissl 18°) and Frehland and Trissl61). These authors anchored flavins in artificial lipid bilayers by means of C18-hydrocarbon chains at various positions of the chromophore. From fluorescence polarization analysis and model calculations they conclude, that the rotational relaxation time of the chromophore within the membrane is small compared to the fluorescence lifetime (about 2 ns74)). They further obtain the surprising result that the chromophore is localized within the water/lipid interface, with a tilt angle of about 30° (long axis of the chromophore against the normal of the membrane), irrespective of the position where the hydrocarbon chain is bound to the flavin nucleus. They estimate an upper limit of the microviscosity of the membrane of 1 Poise. 

The evidence for flavin involvement as deduced from action, absorption, and fluorescence spectra, as well as via non-spectroscopic methods, is then evaluated. It is concluded that all experimental results indirectly support the flavin hypothesis, but that direct proof will have to await the isolation and in vitro characterization of the chromophore. 

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