Energy Transfer between Flavin and Cytochrome c

Energy transfer between photoexcited flavin and cytochrome c has been investigated in order to estimate intermolecular forces between flavin and cytochrome c. The quenching of the fluorescence of flavin by cytochrome c excited at 372 nm was found to be much greater than that excited at 465 nm. This dependence of the quenching on the exciting wavelength is considered to be due to the “prerelaxational” fast energy transfer. From the analysis of the quenching of the fluorescence of FMN and lumiflavin by cytochrome c excited at 465 nm, it was concluded that 1) the quenching is mainly controlled by resonance energy transfer, and 2) the heterogeneous dispersion state of molecules due to electrostatic forces makes the critical transfer distance, R ₀, of the resonance process longer than the real distance. For the quenching of the fluorescence of flavodoxin by cytochrome c, it was found that complex formation is a dominant process and is controlled to a great extent by electrostatic forces. Furthermore, fluorescence decay curves were measured by a single-photon counting method in order to estimate the dynamic processes of flavin fluorescence. The results also showed that the resonance process exists in the energy transfer between flavin and cytochrome c.     

Fluorescence quenching of flavins by reductive agents

The fluorescence behaviour of the flavins riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and lumiflavin in aqueous solution at pH 8 in the presence of the reducing agents β-mercaptoethanol (β-ME), dithiothreitol (DTT), and sodium nitrite (NaNO₂) is studied under aerobic conditions. The fluorescence quantum yields and fluorescence lifetimes are determined as a function of the reducing agent concentration. For all three reducing agents diffusion controlled dynamic fluorescence quenching is observed which is thought to be due to photo-induced reductive electron transfer. For DTT additionally static fluorescence quenching occurs.     

Photochemical degradation of flavins. IV. Studies of the anaerobic photolysis of riboflavin

Abstract A variety of substances which quench the fluorescence of riboflavin decrease the rate of anaerobic photobleaching (photolysis) of the flavin at concentrations which have little effect on the fluorescence. A semi-quantitative estimate of the yield of various products of photolysis using thin layer chromatography and autoradiography with C¹⁴-labelled riboflavin shows that at least ten radioactive products are formed. Whereas the yield of most of these is decreased by low concentrations of quenchers, such as phenol, some are decreased only at quencher concentrations high enough to decrease the fluorescence significantly. The effects of quenchers on the phosphorescence and ESR signal induced by light were also observed. It is concluded that lumichrome arises, in part, from degradation of the excited singlet state, but that 9-formylmethylflavin and other products arise through the triplet state. A major product of anaerobic photolysis is a substance moving just ahead of riboflavin on chromatograms and which, like 9-formylmethylflavin, is destroyed by treatment with sodium hydroxide. Effects of varying the nature of the solvent, the flavin structure and the pH are reported, and the kinetics of processes involved in the primary photochemical acts and in quenching are discussed.     

Photoinduced interaction between riboflavin and TiO(2) colloid

The adsorption of riboflavin on the surface of TiO(2) colloidal particles and the electron transfer process from its singlet excited state to the conduction band of TiO(2) were examined by absorption and fluorescence quenching measurements. The apparent association constants (K(app)) were determined. The quenching mechanism is discussed involving electron transfer from riboflavin to TiO(2).     

Short range photoinduced electron transfer in proteins: QM-MM simulations of tryptophan and flavin fluorescence quenching in proteins

Hybrid quantum mechanical-molecular mechanics (dynamics) were performed on flavin reductase (Fre) and flavodoxin reductase (Fdr), both from Escherichia coli. Each was complexed with riboflavin (Rbf) or flavin mononucleotide (FMN). During 50 ps trajectories, the relative energies of the fluorescing state (S₁) of the isoalloxazine ring and the lowest charge transfer state (CT) were assessed to aid prediction of fluorescence lifetimes that are shortened due to quenching by electron transfer from tyrosine. The simulations for the four cases display a wide range in CT–S₁ energy gap caused by the presence of phosphate, other charged and polar residues, water, and by intermolecular separation between donor and acceptor. This suggests that the Gibbs energy change (ΔG₀) and reorganization energy (λ) for the electron transfer may differ in different flavoproteins.     

Quenching of the fluorescence of riboflavin by the histidinates and alaninates of nickel,copper, and zinc

The formation of complexes of histidinates and alaninates of Ni(II), Cu(II), and Zn(II) with riboflavin (RF) in the ground state and the quenching of the fluorescence of RF by these compounds has been investigated. It has been found that the quenching of the fluorescence of RF by the Cu²⁺ and Ni²⁺ complexes is caused mainly by the nonemissive energy transfer from the donor (RF) to the metal ions. In the case of the Cu²⁺, Ni²⁺, and Zn²⁺ histidinates the formation of nonfluorescing unstable complexes (K_stab — 3–10) of the metal histidinates with RF in the ground state also contribute to the quenching. Free histidine and zinc histidinate quench the fluorescence of RF by the transfer of an electron to the excited molecule of the flavin with the formation of nonfluorescing reduced RF.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 21, No. 4, pp. 488–493, July–August, 1985.     

Spectrofluorimetric Studies of the Interaction Between Quinine Sulfate and Riboflavin

The molecular interaction between quinine sulfate [QS] and riboflavin [RF] was investigated by fluorescence and UV-VIS absorption spectroscopy in aqueous solutions. The fluorescence of QS was quenched by RF. The Stern-Volmer quenching equation was successfully applied and the corresponding thermodynamic parameters enthalpy change (??H), Gibbs energy change (??G) and entropy change (??S) were calculated at different temperatures (294, 301, 307 and 314 K) according to the van??t Hoff equation. This information showed that hydrogen bonds play a major role in stabilizing the complex. The value of the critical energy transfer distance (R ₀) was found to be 46.47 Å, less than 50 Å, which proves that an efficient resonance energy transfer takes place between QS (donor) and RF (acceptor). The oxidation and reduction potentials determined from cyclic voltammetry indicated that association of QS and RF occurs in the excited state by electron transfer. The fluorescence quenching method was successfully applied for the direct determination of riboflavin (vitamin B₂) from pharmaceutical samples.     

亚油酸与核黄素或FAD激发态之间电荷转移的直接证据

根据荧光显微镜方法,我们首次发现核黄素(维生素B2)主要分布在细胞核的膜上和核的内部,故核黄素光敏化与辐射化的靶位置主要集中在细胞核内;当核黄素的浓度较大时,细胞膜上也有药物的分布,即在高浓度时,细胞膜也是光敏化与辐射敏化的作用位点一。应用308nn激光光解时间分辨吸收方法,以亚油酸作为脂质的模型化合物,研究了亚油酸与核黄素和黄素腺嘌呤二核苷酸(FAD)的激发三重态之间的电荷转移过程,首次给出了电荷转移的直接证据。     

DYNAMICS OF EXCITED FLAVOPROTEINS—PICOSECOND LASER PHOTOLYSIS STUDIES

Abstract— Molecular mechanism of fluorescence quenching of flavins in flavodoxin from Desulfovibrio vulgaris , strain Miyazaki, and riboflavin binding protein from egg white has been investigated by means of picosecond laser photolysis technique. In the case of flavodoxin, a transient absorption band characteristic of the non-fluorescent exciplex formed by electron transfer from indole to excited flavins in model systems has been observed around 600 nm at the delay time of 33 ps from exciting ps pulse pulse width, 25 ps). In the case of riboflavin binding protein, the transient absorption spectra were different from those of flavin-indole exciplex and rather similar to the spectra of the model system of flavin-phenol. These results suggest that tryptophan residue exists near the isoalloxazine nucleus in flavodoxin, and in riboflavin binding protein, tyrosine residue exists near the flavin. Direct measurements of the ultrafast process of the electron transfer in flavoproteins as developed here could provide useful information for elucidating protein dynamics, associated with redox reaction, in the picosecond time region.     

Flavin-sensitized Photo-oxidation of Lysozyme and Serum Albumin

The excited state processes of riboflavin, flavin mononucleotide and flavin adenine dinucleotide in argon-saturated aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). UV–Vis absorption and fluorescence spectroscopy indicates that the noncovalent flavin-protein binding is relatively weak. Quenching of the flavin triplet state by BSA, observed by time-resolved photolysis, is less efficient than by lysozyme. Light-induced oxidation of the two proteins and reduction of the three flavins were studied. The quantum yields of the former and latter in the absence of oxygen are up to 0.1 and 0.04, respectively. The effects of pH and sensitizer and protein concentrations were examined in greater detail. The proposed reaction is electron transfer from the tryptophan moiety to the flavin triplet rather than excited singlet state.     

核黄素与牛血清白蛋白相互作用的荧光光谱研究

采用荧光猝灭光谱、同步荧光光谱研究了核黄素与牛血清白蛋白(BSA)相互作用的光谱行为。结果发现,在温度为293 K和310 K时核黄素与BSA的结合常数(Kb)分别为4.879×105L.mol-1和1.880×105L.mol-1,结合热力学方程计算得到了对应温度下的热力学参数。结果表明核黄素对BSA有较强的荧光猝灭作用,其荧光猝灭过程属于动态猝灭机制,二者主要靠疏水作用力结合。采用同步荧光光谱探讨了核黄素对BSA构象的影响。     

Oxygen uptake after electron transfer from amines, amino acids and ascorbic acid to triplet flavins in air-saturated aqueous solution

The photolysis of lumichrome, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) was studied in air-saturated aqueous solution at room temperature in the presence of appropriate electron donors: ascorbic acid, aromatic amino acids or amines, e.g. ethylenediaminetetraacetate (EDTA). The overall reaction is conversion of oxygen via the hydroperoxyl/superoxide radical into hydrogen peroxide. The quantum yield of oxygen uptake increases with the donor concentration, e.g. up to 0.3 for riboflavin, FMN or FAD in the presence of EDTA or ascorbic acid (0.3-10mM). The formation of H(2)O(2) is initiated by quenching of the acceptor triplet state by the electron donor and subsequent reaction of the semiquinone radical with oxygen. Specific properties of flavins are discussed including the radicals involved and the pH and concentration dependences. The quantum yield of photodegradation is low under air, but substantial under argon, where the major product absorbing in the visible spectral range is the corresponding hydroquinone.     

FLAVIN PIGMENTS EMBEDDED IN LIPID MATRICES: A SPECTROSCOPIC AND PHOTOCHEMICAL INVESTIGATION

Abstract— –The spectroscopic properties of riboflavin tetrabutyrate have been studied in lecithin lipo-somes and several detergent micellar systems. The results indicate that the flavin chromophore is located close to the liposome-water interphase rather than in the hydrophobic region. By varying the number of flavins contained in each liposome and employing fluorescence spectroscopy and steady state photolysis techniques, the possible existence of two distinct environments for the flavin chromophore was demonstrated. Several basic photochemical properties of riboflavin tetrabutyrate in liposomes and micellar systems were investigated using laser flash and microsecond flash photolysis techniques.     

THE MECHANISM OF THE FLAVIN SENSITIZED PHOTODESTRUCTION OF INDOLEACETIC ACID*

Abstract— A detailed in vitro study was made of the flavin sensitized photoinactivation of indoleacetic acid, using primarily riboflavin as sensitizer. The dependence of the quantum yield on reactant concentrations, pH, presence of oxygen, viscosity, temperature, KI concentration, and solvent was determined. The involvement of a limiting dark reaction was demonstrated, using an intermittent light technique. The results are consistent with a mechanism involving a metastable state of riboflavin as the photochemically reactive species. The calculated rate constant for intersystem crossing to this state was found to be 2.5 times 10⁸/sec. Riboflavin, in the metastable state, is believed to oxidize indoleacetic acid to indolealdehyde, with subsequent recovery of riboflavin by autoxidation. The maximum quantum yield of the photoinactivation of IAA is 0.71, indicating a highly efficient process, approaching 100% when energy loss due to riboflavin fluorescence is taken into account. Both carotenoids and pure chlorophyll- a were found to be inactive as sensitizers.     

The salt effect on riboflavin-photosensitized substitution of the sulfo group for bromine in 1-bromo-2-hydroxynaphthalene

It was shown that the substitution reaction of the sulfo group for bromine in 1-bromo-2-hydroxynaphthalene photosensitized with riboflavin occurred via the riboflavin triplet state. Electron-donor anions quench the fluorescence of riboflavin. Halide (iodide and bromide) ions increase the quantum yield of the substitution reaction at low concentrations because of an increase in the spin-orbital coupling in the radical ion pair generated in riboflavin fluorescence quenching. As a result of the spin-orbital conversion, the triplet-state radical ion pair is formed, which separates into the riboflavin radical anion and the halogen atom. The halogen atom is reduced by the sulfite ions with the simultaneous formation of the sulfite radical anions, which participate in the propagation of the substitution reaction chain. All salts increase the quantum yield of the substitution reaction at high concentrations (>0.5 mol l^?1).     

Photophysical studies of some dyes in aqueous solution of triton X-100

The spectral (both absorption and fluorescence) and photoelectrochemical studies of a few selective dyes, namely, anionic erythrosin B, neutral riboflavin and cationic safranin O have been carried out in aqueous solution of triton X-100, a neutral surfactant. The results show that the ionic dyes, erythrosin B and safranin O form 1:1 electron donor-acceptor (EDA) or charge-transfer (CT) complexes with triton X-100 both in the ground and excited states, whereas neutral dye riboflavin in its excited state forms 1:1 complex with triton X-100. In these complexes, the dyes act as electron acceptors whereas triton X-100 acts as an electron donor. The fluorescence spectra of erythrosin B and safranin O in presence of triton X-100 show enhancement of fluorescence intensity with red and blue shifts respectively while riboflavin shows normal quenching of fluorescence. A good correlation has been found among photovoltage generation of the systems consisting of these dyes and triton X-100, spectral shift due to complex formation and thermodynamic properties of these complexes.     

Spectroscopic study of molecular associations between FMN and β-carbolines

The spectrophotometric and thermodynamic properties of molecular complexes of flavin mononucleotide (FMN) (riboflavin 5′-phosphate) with some β-carboline derivatives have been investigated in aqueous solution. The molecular associations have been examined by means of electronic absorption spectra, since in each a new charge-transfer band has been located, and also the variation of the fluorescence emission of FMN on the solutions has been observed. The formation constants for the molecular complexes were determined from absorption data using the Foster—Hammick—Wardley method. The quenching phenomenon observed in FMN fluorescence is related to the concentration of the β-carboline derivatives, allowing the calculation of the quenching constants for FMN-β-carboline complexes. Thermodynamic parameters have been determined from the values of association constants for the molecular complexes at various temperatures. The influence of substituents in the β-carboline molecule on the stability of the complexes formed was also investigated.     

Time-resolved fluorescence analysis of the mobile flavin cofactor in p -hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.     

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.