FLAVIN COMPOUNDS AS AGENTS FOR THE OXIDATION OF PLASTOCYANIN IN BLUE LIGHT

Abstract—Flavins, flavin nucleotides and selected flavoproteins have been compared in a reaction using blue light. in which plastocyanin is oxidized as the flavin is photoreduced. Per unit of light absorbed. flavin mononucleotide is more effective than flavin adenine dinucleotide, ribotlavin or lumiflavin. Of the flavoproteins tested, diaphorase from Clostridium kluyveri was most effective, but was less active than free flavin mononucleotide. The oxidation of plastocyanin requires aerobic conditions. and appears to be mediated by the production of singlet oxygen when the flavin is irradiated.     

THE FLAVIN SENSITISED PHOTOOXIDATION OF ASCORBIC ACID: A CONTINUOUS AND FLASH PHOTOLYSIS STUDY

Abstract —The mechanism of the photooxidation of ascorbic acid by flavin mononucleotide has been studied by steady state and flash photolysis techniques. The primary reaction consists of an electron abstraction from ascorbic acid by the flavin singlet and triplet states by a diffusional process and in the latter case also by the formation of a triplet flavin-ascorbic acid complex. Under anaerobic conditions, the secondary reactions consist primarily of a radical recombination and a 'dark'chemical back reaction, leading to the reformation of unchanged flavin and ascorbic acid. In the presence of oxygen, the 'back reactions'are prevented, resulting in the efficient photooxidation of ascorbic acid.     

EXCITED-STATE REACTIONS OF OXIDIZED FLAVIN DERIVATIVES

Abstract— The reactivity of flavin mononucleotide and of lumiflavin triplets was studied by flash and laser photolysis. The rate constants of the triplets with oxygen, with flavin ground-state molecules, and with Br^- ions were determined. Although in solution at room temperature, the protonated flavin triplet, ³F1H⁺, is not formed directly from its very short lived singlet state, a transient, which we think is this triplet, results from protonation of the neutral triplet. This conclusion is based on a comparison between the neutral and the protonated triplet spectra in a low-temperature glass. It is proposed that the protonated triplet can also be formed by sensitization via the phenanthrene triplet.     

STUDIES ON THE in vitro O2-DEPENDENT INACTIVATION OF NADH-GLUTAMATE SYNTHASE FROM Chlamydomonas reinhardii STIMULATED BY FLAVINS

The NADH-glutamate synthase (EC 1.4.1.14) complex from Chlamydomonas reinhardii may experience in vitro two kinds of 0₂-dependent inactivation stimulated by flavins.
A peroxide-mediated inactivation of enzyme, which affects the NADH- and MVt-glutamate synthase activities of the complex, but not the NADH-dehydrogenase activity, can be obtained by aerobic incubation of the enzyme with NAD(P)H plus flavin. This inactivation of enzyme seems to be due to a permanent modification of sulfhydryl groups near the active site for L-glutamine or 2-oxoglutarate. The addition of 10 mM dithioerythritol to inactive NADH-glutamate synthase produces a significant, but not complete, reactivation of the enzyme.
On the other hand, the NADH-glutamate synthase is highly susceptible to a photodynamic inactivation caused by singlet 0₂. Aerobic incubation of the active enzyme with flavin under illumination leads to the irreversible inactivation of all the activities associated with the enzymatic complex.     

PHOTOREGULATION OF NITRATE UTILIZATION IN GREEN ALGAE AND HIGHER PLANTS*

Abstract— Nitrate reductase from eukaryotes can be reversibly inactivated, blue light being an effective activating agent both in vitro and in vivo. Hydroxylamine proved to be a powerful inactivating agent of Ankistrodesmus braunii nitrate reductase. Irradiation with blue light of NH₂OH-inactivated nitrate reductase, specially in the presence of μM amounts of FAD, promoted the recovery of the enzyme activity. Similarly, photoexcited methylene blue reactivated spinach nitrate reductase. On the other hand, in vitro nitrate reductase is highly susceptible to photodynamic inactivation caused by singlet O₂. Aerobic incubation of the active spinach enzyme with either FMN or methylene blue under either blue or red light respectively led to its irreversible inactivation. Irradiation of frozen and thawed spinach leaf discs also promoted, in situ, an irreversible inactivation of nitrate reductase, provided that 6₂ was present in the incubation mixture. Thus, either in vitro or in situ, light can cause two quite different responses of nitrate reductase, its blue light-dependent photoactivation in a flavin sensitized reaction and its photodynamic inactivation in a singlet O₂-dependent process.     

Revisiting the Origin of Bacterial Bioluminescence: QM/MM Study on Oxygenation Reaction of Reduced Flavin in Protein

Bacterial bioluminescence is initiated by the oxygenation reaction of reduced flavin mononucleotide in luciferase. This enzymatic oxygenation occurs in a wide range of biological processes including cellular redox metabolism, biocatalysis, biosynthesis and homeostasis. However, little is known about the mechanism of the enzymatic reaction between singlet reduced flavin and triplet oxygen. To explore the enigmatic oxygenation, for the first time, the reaction of reduced flavin anion with oxygen was studied in bacterial luciferase by a combined quantum mechanics and molecular mechanics method as well as molecular dynamics simulation. The calculated results demonstrate that the reaction proceeds via a proton-coupled electron transfer (PCET) pathway, and the essential αHis44 acts as a catalytic acid to provide the proton. The currently proposed PCET mechanism clearly describes the initial steps of bacterial bioluminescence, and could be suitable for the other flavin oxygenation reactions in enzymes.     

Enhancement of riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by urocanic acid

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.     

A STUDY OF THE PHOTODYNAMIC EFFICIENCIES OF SOME EYE LENS CONSTITUENTS

We have studied the photochemical quantum yields of singlet oxygen production (using the RNO bleaching method) and superoxide production (using the EPR-spin trapping method and the SOD-inhibitable ferricytochrome c reduction spectral assay) of kynurenine (Ky), N-formylkynurenine (NFK), 3-hydroxykynurenine (3HK), kynurenic acid (KUA), and the flavins, riboflavin (RF) and flavin mononucleotide (FMN). Such a study of the photodynamic efficiencies is important since these compounds appear endogenously in the eye. The singlet oxygen quantum yields of the flavins and KUA are high, while Ky and 3HK generate no detectable amounts of singlet oxygen. The superoxide quantum yields of the sensitizers are low compared to their singlet oxygen, and Ky and 3HK produce no detectable amounts of superoxide. The production of the superoxide radical is enhanced in the presence of electron donor molecules such as EDTA and NADH. These results suggest that the production of oxyradicals in the lens may be modulated by the presence of endogenous electron donor molecules such as the coenzymes NADH and NADPH, which are present in significant amounts in some lenses. They also suggest that Ky and 3HK, which are known to be present in aged lenses, might play a protective rather than a deleterious role in the eye.     

Visible radiation effects on flavocytochrome b2 in dilute aqueous solution: a steady-state and laser flash photolysis study

Irradiation of flavocytochrome b2 by visible radiation at 450 nm in dilute aqueous solution is found to have a devastating effect not only on its activity but also on the important flavin mononucleotide (FMN) constituents. The active site and the substrate binding site are also found to be largely modified on exposure to visible radiation. This has a telling effect on the constituent aromatic amino acids, tryptophan and tyrosine, and therefore justifies the role of FMN as a very potent photosensitizer. Partial unfolding of the irradiated enzyme molecule is also observed. Damage is much greater in deaerated conditions, which indicates that molecular oxygen plays a protecting role in this particular system. The inactivation is mediated through rapid electron transfer from tryptophan and tyrosine to excited flavin, forming flavin semiquinone and tryptophanyl and tyrosinyl radicals, which in turn cause permanent damage at the molecular level.     

PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN

Abstract— It has been demonstrated that singlet oxygen is the major oxidizing entity in the photo-dynamic inactivation of hen egg white lysozyme by eosin, using D₂O to enhance the solvent-induced decay lifetime, and azide ion as a specific scavenger. Two regimes of inactivation can be distinguished depending on whether the sensitizer is free or complexed to the enzyme. The kinetic analysis for free dye sensitization, based on photostationary measurements and inactivation quantum yields, indicates that at least 1 in 15 singlet oxygen interactions with lysozyme leads to loss of lytic activity. The direct attack of triplet eosin makes a lesser overall contribution in air-saturated solutions, where 1 in 4 reactions induces inactivation. Lysozyme binds 1 eosin molecule from pH 4 to 12, leading to almost total quenching of the tryptophyl residue fluorescence without inhibition of the enzymic activity. The inactivation quantum yields indicate that singlet oxygen generated from the bound dye is the inactivating agent, but the dominant attack takes place with the complexed fraction of lysozyme molecules. The tryptophyl residue loss is the same or smaller in changing from H₂O to D₂O despite the 5–10 times increase in quantum yield, indicating that singlet oxygen inactivates also by reacting with residues other than tryptophan. The photochemical and fluorescence results are consistent with the the identification of tryptophyl site 108 with the eosin binding site and a reaction target for singlet oxygen. In a re-examination of earlier work on eosin-sensitized photo-oxidation of I", it has been found that singlet oxygen is the oxidizing agent in aerobic solutions.     

Flavin-mediated photoinactivation of spinach leaf nitrate reductase involving superoxide radical and activating effect of hydrogen peroxide

Irradiation with white light of spinach leaf nitrate reductase (NR) in the presence of flavin mononucleotide (FMN) and ethylenediaminetetraacetic acid (EDTA) resulted in a gradual loss of the enzyme activity, measured with reduced methyl viologen as electron donor. Inactivation of NR was dependent on oxygen and was prevented by superoxide dismutase. On the contrary, the presence of catalase markedly enhanced the rate of inactivation. Nitrate showed a protective effect. NR previously inactivated by irradiation in the presence of FMN and EDTA was greatly reactivated by a short preincubation of the inactive enzyme with either ferricyanide or H₂O₂. These results suggest that spinach leaf NR is reversibly inactivated by photogenerated O₂ , while H₂O₂ has an activating effect.     

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.     

BLUE LIGHT INDUCED, REVERSIBLE IN ACTIVATION OF THE TONOPLAST-TYPE H+-ATPase FROM CORN COLEOPTILES IN THE PRESENCE OF FLAVINS

Abstract— Irradiation (λ_max 447 nm; 58.5 W m^-2) of a microsomal membrane fraction of corn coleoptiles for 5 min in the presence of the in vivo concentration of riboflavin inactivates the tonoplast-type H⁺-ATPase. This inhibition is O₂-dependent, is enhanced in D₂O and suppressed by NaN₃, indicating participation of singlet molecular oxygen in the inactivating mechanism. Besides singlet oxygen, the superoxide anion (O₂^-) is generated during irradiation, which obviously has no effect on the H⁺-pumping activity. However, in the presence of superoxide dismutase (SOD), O₂^- is transformed into H₂O₂ which causes an additional strong inhibition of H⁺. ATPase activity. This inhibition can be increased by ethylenediaminetetraacetic acid (EDTA), which is known to be an electron donor of the excited flavin molecule. In contrast, catalase prevents the H₂O₂-mediated photoinactivation of the H⁺ -ATPase. The light dependent inactivation of H⁺-transport does not occur if reduced glutathion (GSH) is added prior to or after irradiation. These results indicate that the blue light mediated inhibition of the H⁺-ATPase is mediated by singlet oxygen and H₂O₂ which oxidize essential SH-groups of the enzyme into disulfides. Reduction of the formed disulfides by GSH restores the activity of the enzyme.     

PHOTODYNAMIC INACTIVATION OF YEAST CELLS SENSITIZED BY HEMATOPORPHYRIN

Abstract— Yeast cells are inactivated by treatment with hematoporphyrin and light. The inactivation is mainly mediated by singlet oxygen. The quantum yield of singlet oxygen increases with increasing pH, while the efficiency of cellular inactivation decreases with increasing pH. Cells in the stationary phase are much more resistant to the treatment than cells in exponential growth. Membrane damage seems to be the main determining step in the photoinactivation.     

Photo-induced reduction of flavin mononucleotide in aqueous solutions

The photo-induced reduction of flavin mononucleotide (FMN) in aqueous solutions is studied by absorption spectra measurement under aerobic and anaerobic conditions. Samples without exogenous reducing agent and with the exogenous reducing agents ethylene-diamine-tetraacetic acid (EDTA) and dithiothreitol (DTT) are investigated. Under anaerobic conditions the photo-induced reduction with and without reducing agents is irreversible. Under aerobic conditions the photo-reduction without added reducing agent is small compared to the photo-degradation, and the photo-reduction of FMN by the reducing agents is reversible (re-oxidation in the dark). During photo-excitation of FMN the dissolved oxygen is consumed by singlet oxygen formation and subsequent chemical reaction. After light switch-off slow re-oxidation (slow absorption recovery) occurs due to air in-diffusion from surface. EDTA degradation by FMN excitation leads to oxygen scavenging. The quantum efficiencies of photo-reduction under aerobic and anaerobic conditions are determined. The re-oxidation of reduced FMN under aerobic conditions and due to air injection is investigated.     

KINETICS and THERMODYNAMICS OF THE AUTOXIDATION OF REDUCED FLAVIN MONONUCLEOTIDE (FMN)

Abstract— Pulse radiolysis of flavin mononucleotide (FMN) solutions produce flavin semiquinone radicals. The equilibrium constant of radical formation was determined in the pH range2–7 as a function of flavin concentration. Several complex constants as well as the kinetics of equilibration were measured in this pH regime. The rate constant of the autoxidation of the free flavin mononucleotide semiquinone radical was determined to be (1 ± 0.5) × 10⁴ M ⁻¹ s⁻¹. It is shown that between pH 2 and 7 the direct reaction of the dihydroflavin with oxygen is negligibly slow compared to the rate of autocatalysis. The autoxidation of dihydroflavin is discussed in relation to electron transfer theory.     

Ab initio quantum chemical investigation of the first steps of the photocycle of phototropin: a model study

Phototropin is a blue light-activated photoreceptor that plays a dominant role in the phototropism of plants. The protein contains two subunits that bind flavin mononucleotide (FMN), which are responsible for the initial steps of the light-induced reaction. It has been proposed that the photoexcited flavin molecule adds a cysteine residue of the protein backbone, thus activating autophosphorylation of the enzyme. In this study, the electronic properties of several FMN-related compounds in different charge and spin states are characterized by means of ab initio quantum mechanical calculations. The model compounds serve as idealized model chromophores for phototropism. Reaction energies are estimated for simple model reactions, roughly representing the addition of a cysteine residue to the flavin molecule. Excitation energies were calculated with the help of time-dependent density functional theory. On the basis of these calculations we propose the following mechanism for the addition reaction: (1) after photoexcitation of FMN out of the singlet ground state S0, excited singlet state(s) are populated; these relax to the lowest excited singlet state S1, and subsequently by intersystem crossing FMN in the lowest triplet state, T1 is formed; (2) the triplet easily removes the neutral hydrogen atom from the H-S group of the cysteine residue; and (3) the resulting thio radical is added.     

EXTENSIVE CROSSLINKING BETWEEN SUBUNITS OF OLIGOMERIC PROTEINS INDUCED BY FUROCOUMARINS PLUS UV-A IRRADIATION

Abstract— Exposure of oligomeric proteins to UV-A irradiation in the presence of various furocoumarins results in extensive crosslinking of the enzyme subunits. The mechanism by which these covalent bridges are formed does not involve the furocoumarin moiety as the crosslinking agent, but depends upon oxidative processes, mainly involving singlet oxygen and radical mediated processes not involving oxygen. The formation of crosslinks is not per se responsible for the inactivation of enzymes since in glutamate dehydrogenase the two reactions proceed at the same rate, in catalase it is not accompanied by enzyme inactivation and in alcohol dehydrogenase the inactivation rate is higher than polymerization.     

FLAVIN-MEDIATED PRODUCTION OF HYDROGEN PEROXIDE IN PHOTOELECTROCHEMICAL CELLS

A photoelectrochemical cell for hydrogen peroxide production using flavin photosystems is described. The anodic solution, which is kept free of oxygen by passing an Ar stream through it, contains the photoreceptor (flavin mononucleotide or lumiflavin) and the electron donor (EDTA, semicarbazide or hydroxylamine), while the peroxide is formed at the electrode of the cathodic solution, whose oxygen content is increased by bubbling with pure oxygen. Among several electrode metals that have been tested (Ag, Pt and Hg), Hg is the most efficient. The pH of the anodic and cathodic solutions should be adjusted to 14 and 1, respectively, for optimum results.     

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting Pt^IV and Ru^II complexes into potentially toxic Pt^II or Ru^II?OH₂ species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate Pt^IV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes Pt^IV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of Pt^IV chemotherapeutic agents.