Flavin-mediated photoreactions in artificial systems: a possible model for the blue-light photoreceptor pigment in living systems.

Abstract— The photoreduction of cytochrome c and the photostimulation of oxygen uptake were studied in solutions of flavin and cytochrome as a possible model system for similar photoreactions which have been observed in vivo. Light causes the photoreduction of the flavin. Under aerobic conditions the photoreduced flavin reacts with oxygen to form the superoxide anion which in turn can reduce cytochrome c. Dismutation of the superoxide anions forms hydrogen peroxide which mediates the dark oxidation of the photoreduced cytochrome. Superoxide formation and dismutation also account for the light-induced oxygen uptake. Action spectra confirm the role of flavin in the photoreduction of cytochrome c and the photostimulation of oxygen uptake. Under anaerobic conditions the photoreduced flavin reduces cytochrome c directly. In the presence of an electron donor only catalytic amounts of flavin are required. In the absence of an added electron donor flavin itself can act as the electron donor if substrate amounts are present. Azide inhibits all of these flavin-mediated photoresponses. Azide also inhibits the photoreduction of cytochrome b which occurs in the mycelium of Newospora.     

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.     

Light-induced absorbance changes in cell-free extracts of Neurospora crassa.

Abstract— The mycelium of Neurospora crassa was ground and extracted with buffer and separated into a soluble supernatant fraction and a particulate fraction by centrifugation. Both fractions were examined for light-induced absorbance changes. Irradiation of the supernatant fraction caused a reversible photooxidation of cytochrome c which was inhibited by sodium azide or by dialysis. The action spectrum for the photooxidation showed that the response was mediated by an endogenous flavin. The photooxidation of cytochrome c, lost by dialysis, could be restored by adding flavin mononucleotide. Irradiation of the particulate fraction with blue light caused a reversible photoreduction of cytochrome c and cytochrome oxidase and, in some samples, of cytochrome b as well. The supernatant fraction showed photooxidation of cytochrome c rather than the more usual photoreductive changes because of the presence of super-oxide dismutase activity.     

THE SUPEROXIDE ANION AS ELECTRON DONOR TO THE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN

Abstract— In isolated respiratory multienzyme complexes of beef heart mitochondria the b -type cytochromes can be photoreduced in presence of flavin via the superoxide anion. O^-₂ does not reduce cytochrome c ₁. In an anaerobic system, FMNH₂ formed by irradiation with blue light in presence of EDTA reduces cytochromes b and c ₁ The possible implication of O^-₂ in the electron transfer from flavin/flavoprotein to cytochrome b in blue light-controlled biological processes is discussed.     

PHOTOINACTIVATION OF SPINACH NITRATE REDUCTASE SENSITIZED BY FLAVIN MONONUCLEOTIDE. EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN

Abstract All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase plus catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.     

VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVEN BY TWO PHOTOREACTIONS. I. ETHYLENEDIAMINETETRAACETIC ACID AS AN ELECTRON DONOR via FLAVIN TRIPLET STATE IN THE INNER COMPARTMENT AND CYTOCHROME c AS AN ELECTRON ACCEPTOR FROM CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

Negatively charged vesicle suspensions containing chlorophyll a (chl) dissolved in the lipid bilayer, flavin mononucleotide (FMN) and/or ethylenediaminetetraacetic acid (EDTA) enclosed in the inner compartment as electron sources and oxidized cytochrome c (cyt c[ox]) in the outer compartment as an electron acceptor have been studied using laser flash photolysis and steady-state irradiation methods. Cytochrome c initially quenches the chl triplet state (³chl) generating the chlorophyll cation radical (chi⁺′) in the membrane. Reverse electron transfer from cyt c(red) to chl^+. subsequently occurs in a kinetically biphasic reaction, with rate constants of 430 pT 30 and 21.9 pT 1.7 s^?1 for the fast and slow phases, respectively. In the absence of FMN, reduction of chl⁺′ by EDTA in the inner compartment can be observed during steady-state irradiation but not in a laser flash photolysis experiment. This is due to a low reaction yield, which is probably limited by the repulsive electrostatic interaction between EDTA and the negatively charged membrane. When FMN was enclosed together with EDTA in the inner Compartment, the reaction yield of vectorial electron transfer across the bilayer from EDTA to cyt c(oX) was increased by a factor of six during steadystate white light irradiation. Laser flash photolysis and steady-state irradiation experiments using red and blue light excitation have demonstrated that the enhancement mechanism involves the formation of fully reduced FMN by blue light-sensitized photooxidation of EDTA via the flavin triplet state, occumng simultaneously with red lightsensitized electron transfer to cyt c via the chlorophyll triplet state.     

LIGHT-DEPENDENT GENERATION OF SUPEROXIDE FROM HUMAN ERYTHROCYTES

Using the cytochrome c reduction method, we investigated light-dependent erythrocytic superoxide production. After 4 h light and dark exposure of erythrocytes from eight healthy human subjects, an average of 18.6% more superoxide was generated by erythrocytes exposed to light. Pretreatment of erythrocytes with the superoxide dismutase inhibitor N,N-diethyldithiocarbamate increased detection of superoxide while pretreatment with the anion channel blocker 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene decreased detection. These findings indicate that substantially more spontaneously generated superoxide is produced and escapes from normal erythrocytes at ambient oxygen tensions on exposure to light. This excess generation and escape of superoxide from erythrocytes exposed to light may result in tissue photosensitization, especially in the retina of the eye, where high oxygen tension, blood and chronic light exposure occur simultaneously.     

VECTORIAL ELECTRON TRANSPORT ACROSS LIPID VESICLE MEMBRANES DRIVE BY TWO PHOTOREACTIONS. II. PHOTOCHEMICAL REGENERATION OF REDUCED CYTOCHROME c BY FLAVIN TRIPLET STATE AND ETHYLENEDIAMINETETRAACETIC ACID IN THE INNER COMPARTMENT AND REDUCTION OF FERREDOXIN BY CHLOROPHYLL TRIPLET STATE IN THE OUTER COMPARTMENT

We have attempted to mimic natural photosynthesis with regard to the photogeneration of a powerful reductant, using a negatively charged lipid bilayer vesicle system incorporating two photoreactions sensitized by a flavin analog (flavin mononucleotide [FMN]) and chlorophyll (chl) in their respective triplet states. Ethylenediamine-tetraacetic acid (EDTA) in the inner aqueous compartment was used as a sacrificial electron donor to the FMN triplet, and ferredoxin in the outer aqueous compartment served as the final electron acceptor (mediated via triplet electron transfer chain in this multicomponent system to be elucidated. By itself, EDTA does not function as an effective donor to membran-bound oxidized chl (chl^+.), which is formed by electron transfer from triplet chl to the viologen follwed by transbilayer electron migration. This is a consequence of electrostatic repulsive interactions with the negatively charged membrane. This limitation is avoided when FMN is used as a photomediator between EDTA and chl^+.. The overall reaction is dramatically increased in rate by enclosing cytochrom c together with EDTA and FMN in the inner compartment. The rate constant of the key step in the reaction, i.e. elctron transfer from reduced cytochrome c, generated via photoreduction by the FMN/EDTA system, to chl^+. is increased 20-fold over that obtained with cytochrome c alone as the elctron donor. One of the important constraints that limited the net electron transfer across the bilayer to 50% of the added cytochrome, i.e. inhibition by oxidized cytochrome c formed in the inner compartment, is avoided by the inclusion of the second photoreaction in this system, thus allowing photoreduction of all of the added ferredoxin to be achieved. This system provides a model for a photochemical energy storage process that utilizes two photorections operating in series resulting in electron flow across a lipid bilayer membrane.     

PHOTOSENSITIZED PRODUCTION OF SUPEROXIDE ANION BY MONOCHROMATIC (290–405 nm) ULTRAVIOLET IRRADIATION OF NADH and NADPH COENZYMES

Abstract— The reduced pyridine coenzymes NADPH and NADH produced superoxide anion("CK") from ground state molecular oxygen when irradiated by ultraviolet (UV) radiation extending from 290 to 405 nm as detected by cytochrome c reduction. Superoxide dismutase (SOD), but not catalase or heat-inactivated SOD, decreased the amount of cytochrome c reduced, indicating that O₂⁻ was responsible for the reduction of cytochrome c. Decreased oxygen tension during irradiation also inhibited production of O₂⁻. Quantum yields for the production of the anion were in the region of 10⁻⁷ to 10⁻⁹ mol per photon. These data indicate that NADH and NADPH can act as type II photosensitizers of both far-and near-UV radiation, and that the deleterious biological effects of exposure to these radiations such as erythema and dermal carcinogenesis may be mediated at least in part through the generation of O₂⁻.     

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.     

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.     

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid-Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol . 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm² of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μ M as determined by the superoxide dismutase (SOD)-depen-dent increase in hydrogen peroxide formation and of 52 μ M by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.
Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N- dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H₂O₂ were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.     

BLUE LIGHT PERCEPTION BY ENDOGENOUS REDOX COMPONENTS OF THE PLANT PLASMA MEMBRANE

b-Type cytochromes of the higher plant plasma membrane may be reduced by irradiation with actinic blue light (light-induced absorbance change). Although this reaction has been reported to depend on the presence of an exogenous oxygen-scavenging system, significant cytochrome reduction was obtained in bean hook (Phaseolus vulgaris L. cv. “Limburgse Vroege”) plasma membranes without any addition. An endogenous oxygen-consuming reaction is apparently sufficient to achieve a proper redox balance. A blue light-mediated absorbance change with absorbance minima at 450 and 475 nm precedes cytochrome b reduction and indicates the presence of a flavoprotein in the plasma membrane fraction. Cytochrome b reduction by blue light in the absence of an oxygen scavenger is highly sensitive to flavin photosensitizers. Glucose oxidase, which has previously been used to lower the oxygen concentration in membrane samples, was demonstrated to have a photosensitizing effect. Inhibitors of flavin photochemical reactions (KI and phenylacetic acid) were highly effective in preventing cytochrome b reduction. These results indicate that the blue light-mediated reaction probably involves an endogenous plasma membrane flavoprotein as the photoreceptor. As plasma membrane NADH-dependent oxidoreductases potentially are flavoproteins these experiments raise the question whether a plasma membrane cytochrome b and a flavin-enzyme may cooperate in blue light reactions. Evidence is also discussed, suggesting the possible involvement of oxygen radicals in the blue light-induced cytochrome b reduction.     

GENERATION OF SUPEROXIDE ANION WITH A PHOTOREDUCED PHENOXAZINE DERIVATIVE

Abstract. 1-Hydroxy-5-oxo-5H-pyrido [3,2 a ] phenoxazine-3-carboxylic acid was found to be reduced when it was irradiated with visible light in the presence of certain electron donors such as tetramethyl ethylenediamine and reduced glutathione. Upon illuminating this compound with either of the above electron donors under aerobic conditions, the coexisting nitroblue tetrazolium was reduced. The reduction was inhibited by superoxide dismutase, which indicated that superoxide anion was generated during the photoinduced reaction.     

Spectrophotometric assay of superoxide anion formed in Maillard reaction based on highly water-soluble tetrazolium salt.

A novel highly water-soluble tetrazolium salt, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt), can be reduced to water-soluble formazan with a superoxide anion. Here, the WST-1 assay was applied to detect the superoxide anion generated during the Maillard reaction. A sample solution containing carbonyl compounds such as glyceraldehyde and glycolaldehyde (5 mM) and Na-acetyl-L-lysine (10 mM) was incubated for 2 days at 37 degrees C. The detection of a superoxide anion generated in the sample was performed by the WST-1 assay, and the result was compared with the cytochrome c assay. The reduction of WST-1 was almost perfectly (86-96%) inhibited by the addition of the superoxide dismutase (SOD). On the contrary, the reduction of cytochrome c was slightly (20-25%) inhibited by the addition of SOD. A similar result was observed for the addition of 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). These results mean that the specificity of WST-1 to the superoxide anion was superior to that of cytochrome c. It was also possible to continuously monitor superoxide anion generation during the Maillard reaction by the coexistence of WST-1 in the reaction solution.     

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.     

Photodynamic Crosslinking of Proteins. III. Kinetics of the FMN- and Rose Bengal-sensitized Photooxidation and Intermolecular Crosslinking of Model Tyrosine-containing N-(2-Hydroxypropyl)methacrylamide Copolymers

As part of a study on the role of Tyr residues in the photosensitized intermolecular crosslinking of proteins, we have surveyed the kinetics of the rose bengal- and flavin mononucleotide (FMN)-sensitized photooxidation and crosslinking of a water-soluble N-(2-hydroxypropyl)methacrylamide copolymer with attached 6-carbon side chains terminating in tyrosinamide groups (thus the -OH group of the Tyr is free, but both the amino and carboxyl groups are blocked, simulating the situation of a nonterminal Tyr in a protein). The intermolecular photodynamic crosslinking of the Tyr copolymer can result only from the formation of Tyr-Tyr (dityrosine) bonds, because the copolymer itself is not photooxidizable. Rose bengal, primarily a Type II (singlet oxygen) sensitizer, sensitized the rapid photooxidation of the Tyr residue in the Tyr copolymer only at high pH, where the Tyr phenolic group is ionized; crosslinking did not occur with rose bengal under any of the reaction conditions used. In contrast, FMN, which can sensitize by both Type I (free radical) and Type II processes, sensitized the photooxidation of the Tyr copolymer over the pH range 4-9.5. Also, significant photocrosslinking occurred, but only from pH 4 to 8, with a maximum rate at pH 6. Crosslinking required the presence of oxygen. Studies with inhibitors, D2O as solvent, catalase and superoxide dismutase indicated that the photooxidation and photocrosslinking of the Tyr copolymer with FMN at pH 6 were not mediated by singlet oxygen, superoxide or hydrogen peroxide. It appears that crosslinking involves the abstraction of an H atom from the Tyr phenolic group to give Tyr and FMN radicals. The Tyr radical in one Tyr copolymer can then react with a Tyr radical in another Tyr copolymer to give an intermolecular dityrosine crosslink.     

Production of O2- in photolyzed water demonstrated through the use of superoxide dismutase

Abstract— The photolysis of water has been studied using ferricytochrome c as the detector of reducing radicals and ferrocytochrome c as the detector of oxidizing radicals. Mannitol was used as a scavenger of hydroxyl radicals and superoxide dismutase was used to expose the specific involvement of superoxide radicals. Aerobic photolysis caused a reduction of ferricytochrome c, which was inhibited by superoxide dismutase and was enhanced by mannitol. Aerobic photolysis also caused the oxidation of ferrocytochrome c, which was inhibited by mannitol and augmented by superoxide dismutase. The presence of superoxide dismutase also eliminated the effects of mannitol on the aerobic oxidation of ferrocytochrome c. Photolysis in the absence of oxygen also caused the reduction of ferricytochrome c and the oxidation of ferrocytochrome c, but under these anaerobic conditions neither mannitol nor superoxide dismutase exerted significant effects. An explanation of these observations is offered in terms of the reactivities of H^., OH^. and O^-2 radicals.     

BIOLUMINESCENCE FROM THE REACTION OF FMN, H2O2 AND LONG CHAIN ALDEHYDE WITH BACTERIAL LUCIFERASE

Abstract— The bioluminescent oxidation of reduced flavin mononucleotide by bacterial luciferase involves a long-lived flavoenzyme intermediate whose chromophore has been postulated to be the 4a-sub-stituted peroxy anion of reduced flavin. Reaction of long chain aldehyde with this intermediate results in light emission and formation of the corresponding acid. These experiments show that the typical aldehyde-dependent, luciferase-catalyzed bioluminescence can also be obtained starting with FMN and H₂O₂ instead of FMNH₂ and O₂. We postulate that the 4a-peroxy anion intermediate is formed directly by attack of H₂O₂ on FMN. The latter may be bound to luciferase. An enzyme bound intermediate is formed which by kinetic analysis, flavin specificity for luminescence, aldehyde dependence, and bioluminescent emission spectrum appears to be identical with the species generated by reaction of FMNH, and O₂ with luciferase. The quantum yield of the H₂O₂-_- and FMN-initiated biolumlnescence is low but can be enhanced by certain metal ions, which also stimulate a chemiluminescent reaction of oxidized flavin with H₂O₂. The peak of this chemiluminescence. however, appears to be at a shorter wavelength than that (490 nm) of the bioluminescence.     

PHOTOACTIVATION OF NITRATE REDUCTASE FROM NEUROSPORA CRASSA

Abstract— The photoactivation of nitrate reductase from Neurospora crassa was studied in partially purified extracts. The inactive enzyme [inactivated by reduction in the presence of potassium cyanide] could be reactivated by chemical oxidation with ferricyanide or by irradiation with blue light. The enzyme contains a short electron transfer chain consisting of flavin adenine dinucleotide, cytochrome b ₅₅₇ and molybdenum which normally transfers electrons from reduced pyridine nucleotide to nitrate. This overall activity, which was negligible in the inactive enzyme, was restored to approximately 70% of the ferricyanide control by irradiation. However, nitrate reduction using reduced methylviologen as reducing power, which was also negligible in the inactive enzyme, was photoactivated to 100%. The diaphorase activity of the enzyme mediated by the flavin adenine dinucleotide, which was fully active in the inactivated enzyme, was inhibited approximately 30% by the irradiation treatment. The action spectrum for photoactivation showed that a flavin was the photoreceptor chromophore. Photoactivation occurs only in the presence of oxygen.