Photosensitization of singlet oxygen formation by pterins and flavins. Time-resolved studies of oxygen phosphorescence under laser excitation

To elucidate the biochemical roles of singlet molecular oxygen (1(O2)) in the light-dependent reactions photosensitized by biological blue-light photoreceptors, time-resolved measurements of photosensitized 1O2 phosphorescence (1270 nm) were performed in air-saturated aqueous ((D2)O) solutions of pterins (2-amino-4-hydroxy-6,7-dimethylpteridine (DMP) and 2-amino-4-hydroxy-6-tetrahydroxybutyl-(D-arabo)pteridine (TOP)) and flavins (riboflavin and flavin mononucleotide (FMN)) under excitation with nitrogen laser (337.1 nm) pulses. The 1(O2) quantum yields were found to be 0.16, 0.20, 0.50, and 0.50 for DMP, TOP, riboflavin, and FMN, respectively. The data indicate that pterins and flavins are rather efficient photosensitizers of 1(O2) production that might be important for their photobiological functions.     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

Microspectrophotometry ofEuglena gracilis

The paraflagellar bodies (PFBs) of isolated flagella ofEuglena gracilis were investigated microspectrophotometrically using a visible- and infrared-light microscope with image analyzer and microspectrophotometer. Flagella with attached PFBs were separated from the cell bodies by a short exposure to near-UV light. Fluorescence-emission spectra (excitation at 365 nm) of single PFBs had maxima near 470 and 520 nm, indicating the presence of pterins and flavins. No fluorescence was associated with the flagella themselves. Pterin- and flavin-like fluorescence emission was also found in blue-fluorescing vesicles distributed throughout the entire cell body ofEuglena. Their characterization by microfluorimetry was greatly aided by the use of chlorophyll-free mutants in which the signal-to-noise ratio was distinctly enhanced because of the lack of chlorophyll fluorescence. Our finding of flavin-like fluorescence associated with PFB strengthens similar earlier reports in the literature. The occurrence of pterin-like fluorescence in the PFB lends further support to our earlier proposal that pterins as well as flavins may function as photoreceptor pigments for near-UV and blue light.     

The role of mutants in the search for the photoreceptor for phototropism in higher plants

Early attempts to identify the chromophore of the photoreceptor for phototropism are reviewed. Carotenoids and flavins were the principal candidates, but studies with grass coleoptiles devoid of carotenoids suggest that at least in these organs carotenoids are most unlikely to play that role. The status of characterization of a gene for a putative photoreceptor protein is also reviewed. As the action spectrum for phototropism resembles the absorption spectrum of a flavoprotein, flavoproteins are attractive candidates at present, especially since the CRY1 photoreceptor in Arabidopsis thaliana that mediates blue light-dependent hypocotyl growth suppression has flavin adenine dinucleotide as one of its two chromophores. As the second chromophore appears to be pterin, pterins should not be ruled out as candidate chromophores for the photoreceptor for phototropism.     

Nonpigment components of the photochlorophyllide photoactive complex: studies of low-temperature blue-green fluorescence spectra

Fluorescence spectra in the blue-green region and excitation fluorescence spectra of green wheat leaves, etiolated wheat leaves and isolated inner etioplast membranes (prolamellar bodies and prothylakoids) were compared to specify the structure of the active protochlorophyllide pigment-protein complex of inner etioplast membranes. Three bands in the blue region at 420, 443 and 470 nm and a broader green band at 525 nm were found. Comparison of the emission and excitation spectra suggests that the main components responsible for the blue fluorescence of etioplast inner membranes are pyridine nucleotides and pterins. The green fluorescence (525 nm) excitation spectra of etiolated samples were identical to the excitation spectrum of flavin fluorescence. The fact confirms the suggestion that flavins are the constituents of the active protochlorophyllide-protein complex.     

Bioenergetic processes in guard cells related to stomatal function

The energy required for ion uptake in guard cells is provided by two important bioenergetic processes, namely respiration and photosynthesis. The blue light-sensitive plasma membrane redox system is considered as the third bioenergetic phenomenon, since it uses blue light to create a proton gradient across the membrane. The unique features of respiration and photosynthesis in guard cells and their role in stomatal function are emphasized. Evidence for and against the blue light-sensitive components on plasma membrane (ATPase/distinct redox chain) and the photoreceptors (flavins, carotenoids, pterins) in guard cells are presented. The information on ion channels and their response to various kinds of secondary messengers including G-proteins, phosphoinositides, diacylglycerol, calcium, cAMP and protein kinases are reviewed. A model is presented indicating the possible mechanism of perception and transduction by guard cells of external signals and their interaction with different bioenergetic components.     

Altered Pterin Patterns in Photoreceptor Mutants of Phycomyces blakesleeanus with Defective madl Gene

Action spectra of photogravitropic equilibrium were measured for the wild type of the lower fungus Phycomyces blakesleeanus and three photobehavioral mutants with defects in the madl gene. The action spectrum for the wild type NRRL1555 had major peaks at 383 and near 460 nm and subsidiary peaks at 365 and 422 nm. The action spectra of the mutants, L1 49 mad1712, L151 mad1714 and L153 mad1716 differed significantly from that of the wild type. One prominent feature of the three mutants was hat the near-UV peaks at 365 and 383 nm, which were not well resolved in the wild type, were of approximately equal height in the mutants and were separated by an extremely sharp valley at 378 nm. The steepness of this valley suggests interaction of multiple receptors. The second prominent feature of the mutants was their enhanced 422 nm peak. The gross changes of the photogravitropic action spectra associated with the madl genotype indicate that the respective gene product acts early in the photosensory transduction chain, very likely at the level of a complex photoreceptor system. Flavins and pterins, two pigment classes which were expected to function as chromophores of the near-UV/blue light photoreceptor system, were analyzed for stage I sporangiophores of the wild-type and the mutant strains by HPLC with fluorescence detection. In the wild-type strain NRRL1555, and also in the three madl mutants, flavins were found to be present at the following concentrations: riboflavin (2.9 × 10^?6 M), FMN (3.8 × 10^?6M) and FAD (1.3 × 10^?6 M). No significant effect of the madl mutations on the flavin content could be discerned. Among the pterins found in the wild type and the madl mutants were biopterin, 6,7-dimethylpterin, neopterin, pterin and xanthopterin. These pterins occurred in all strains in the micromolar range and none of them was significantly reduced in the mutants. However, biopterin, 6,7-dimethylpterin and xanthopterin occurred in some excess in one of the madl mutants. The most significant feature of the madl mutants was that they had almost completely lost one unidentified pterin with a retention time of 18 ? 20 min. Another two unidentified pterins were reduced about twofold in the mutants compared to the wild type. The results suggest an involvement of pterins in the photoreception of near-UV and blue light in Phycomyces.     

Blue light-dependent monovalent anion uptake

Blue light is one of the most important environmental signals regulating monovalent anion transport in plant cells. In the unicellular freshwater chlorophyte Monoraphidium braunii , blue light is essential for the activation of HCO₃⁻, NO₃⁻, NO₂² and Cl⁻ transport systems. These anions are taken up when blue light is present but the uptake ceases when this radiation is suppressed, indicating that blue light is a switch signal for the monovalent anion transport system(s) of this alga. Similar results have been obtained in other green algae and higher plants. The action spectra for the uptake of NO₃⁻ and Cl⁻ in M. braunii are very similar and resemble the absorption spectra of flavins or a combination of flavins and pterins. It is proposed that both anions share the same transport system(s). The uptake of monovalent anions consists of a cotransport with H⁺, thus producing alkalinization of the external medium. The time between the onset of blue light and the beginning of alkalinization can be as short as 2 s. Taken together, the results suggest that the photoreceptor mediating the blue light activation of monovalent anion uptake in this green alga is a plasma membrane-bound flavoprotein.     

Inhibition of hypocotyl elongation by ultraviolet-B radiation in de-etiolating tomato seedlings. I. The photoreceptor

Broad-band UV-B radiation inhibited hypocotyl elongation in etiolated tomato ( Lycopersicon esculentum Mill. cv. Alisa Craig) seedlings. This inhibition could be elicited by < 3 μmol m⁻² s⁻¹ of UV-B radiation provided against a background of white light (> 620 μmol m⁻² s⁻¹ between 320 and 800 nm), and was similar in wild-type and phytochrome-1-deficient aurea mutant seedlings. These observations suggest that the effect of UV-B radiation is not mediated by phytochrome. An activity spectrum obtained by delivering 1 μmol m⁻² s⁻¹ of monochromatic UV radiation against a while light background (63 μmol m⁻² s⁻¹ showed maximum effectiveness around 300 nm, which suggests that DNA or aromatic residues in proteins are not the chromophores mediating UV-B induced inhibition of elongation. Chemicals that affect the normal (photo)chemistry of flavins and possibly pterins (KI, NaN, and phenylacetic acid) largely abolished the inhibitor) effect of broad-hand UV-B radiation when applied to the root zone before irradiation. KI was effective at concentrations < 10⁻⁴ M , which have been shown in vitro to be effective in quenching the triplet excited stales of flavins but not fluorescence from pterine or singlet states of flavins. Elimination of blue light or reduction of UV-A, two sources of flavin excitation, promoted hypocotyl elongation, but did not affect the inhibition of elongation evened by UV-B. Kl applied after UV-B irradiation had no effect on the inhibition response. Taken together these findings suggest that the chromophore of the photoreceptor system invoked in UV-B perception by tomato seedlings during de-etiolation may be a flavin.     

Hydrogen-bonding modulation of excited-state properties of flavins in a model of aqueous confined environment

The singlet and triplet excited states properties of lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in reversed micelles (RM) of sodium docusate (AOT) in n-hexane solutions were evaluated as a function of the water to surfactant molar ratio, w(0) = [H(2)O]/[AOT], by both steady-state and time-resolved absorption and fluorescence spectroscopy. The results indicated that hydrogen-bonding interactions between the isoalloxazine ring of the flavins with the water molecules of the micellar interior play a crucial role on the modulation of the excited state properties of the flavins. Fluorescence dynamic experiments in the RM, allowed the calculation of similar values for both the internal rotational time of the flavins (θ(i)) and the hydrogen-bonding relaxation time (τ(HB)), e.g.≈ 7 and 1.5 ns at w(0) = 1 and 20, respectively. In turn, the triplet state lifetimes of the flavins were also enlarged in RM solutions at low w(0), without modifications of their quantum yields. A hydrogen bonding relaxation model is proposed to explain the singlet excited state properties of the flavins, while the changes of the triplet state decays of the flavins were related with the global composition and strength of the hydrogen bonding network inside of the RM.     

7-Substituted pterins: formation during phenylalanine hydroxylation in the absence of dehydratase

Previously we described a new form of human hyperphenylalaninemia characterized by the formation of 7-substituted pterins. We present evidence strongly suggesting that the 7-substituted pterins are formed by rearrangement of 6-substituted pterins. This rearrangement occurs during the phenylalanine hydroxylase reaction cycle which normally involves the enzymes phenylalanine hydroxylase, pterin-4a-OH-dehydratase, and q-dihydropterin reductase, specifically in the absence of dehydratase activity. We conclude that formation of 7-substituted pterins in humans is a consequence of an absence of dehydratase activity, which might result from a genetic defect. A chemical mechanism for this rearrangement is presented. Our results also suggest that tetrahydroneopterin can be a cofactor for the phenylalanine hydroxylase system in vivo.     

Flavin-photosensitized reactions of retinal and stilbene

1. Retinol and stilbene are both isomerized when they are illuminated anaerobically in the presence of flavins. 2. Triplet quenchers (e.g. oxygen, potassium iodide and paramagnetic ions) inhibit the reaction more efficiently than they quench flavin fluorescence. At 77 degrees C in a diethyl ether-isopentane-ethanol (5:5:2) glass retinol quenches flavin phosphorescence, but not its fluorescence. 3. For the stilbene reaction cis/trans photostationary-state mixtures are obtained with different flavins and these are linearly related to the phosphorescence transition energies of the flavins used. 4. The reaction involves the triplet state of flavin and a scheme for the reaction is suggested. 5. The dependence of the rate of reaction on substrate concentration is explicable in terms of this scheme. 6. The photobleaching of rhodopsin sensitized by flavin is also demonstrated.     

Crystal structure of NAD(P)H:flavin oxidoreductase from Escherichia coli.

Flavin reductases use flavins as substrates and are distinct from flavoenzymes which have tightly bound flavins. The reduced flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced flavins produced by flavin reductase. The crystal structure of E. coli flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboflavin in a substrate complex.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Inhibition of monocyte luminol-dependent chemiluminescence by tetrahydrobiopterin, and the free radical oxidation of tetrahydrobiopterin, dihydrobiopterin and dihydroneopterin

Luminol-dependent chemiluminescence of normal human monocytes activated by zymosan is demonstrated to be inhibited by tetrahydrobiopterin in a concentration-dependent manner. The reduced pterins tetrahydrobiopterin, dihydrobiopterin, and dihydroneopterin are all shown to be readily oxidized by the hydroxyl radical. The susceptibility of reduced pterins to free radical attack may explain the inhibition of chemiluminescence observed and an additional role of reduced pterins as free radical scavengers in tissues is considered.     

On the properties of fluorescing compounds in guard and epidermal cells of Allium cepa L.

Onion guard cells, in contrast to those of Vicia and Pisum, do not require an alkaline treatment in order to fluoresce. Fluorescing compounds of Allium cepa L. were characterized using in-vivo microspectrophotometry; furthermore, invitro chemical analysis for epidermal tissue, intact guard and epidermal cells, and isolated guard-cell protoplasts was performed. The emission intensity (_max 520 nm) decreased when intact onion guard cells were excited with 436 nm light, but increased (_max 470 nm) when excited at 365 nm. This photodecomposition at 436 nm is typical of flavins or flavoproteins whereas an increase in fluorescence intensity with excitation at 365 nm may be explained by the presence of other substances. The presence of flavins could not be unambiguously confirmed from these results. Indeed, the absorption spectra of the vacuolar area of guard cells did not show the peak at 445 nm which is characteristic for flavins. Furthermore, there was no decrease of absorption at the excitation wavelengths of 440 and 330 nm. Since spectral data indicate the presence at high amounts of flavonoids in guard and epidermal cells, this may reduce the sensitivity for the detection of flavins in guard cells. Using thin-layer chromatography and high-performance liquid chromatography together with hydrolytic procedures, flavonol glycosides with kaempferol and quercetin as aglycones substituted with sulphate and glucuronate were identified. Further studies on guard-cell metabolism should consider the presence of flavonoids in stomata of onion and other plants.Abbreviations GCP guard-cell protoplast - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Chloragosomes of lumbricidae as specific electron acceptors. In vitro investigations.

Measurements of the redox potential values in buffered salt solutions containing body wall homogenate, body wall homogenate with isolated chloragosomes and in both solutions enriched with NAD have shown that chloragosomes are specific electron acceptors which prevent the rapid decrease of the redox potential under anaerobic conditions. The substances responsible for the electron-acceptor activity are most probably identical with the flavins, carotenes and metalloporphyrins present in chloragosomes as shown by the visible absorption spectra of the extracts. The results support the assumption that chloragosomes may play an important role in the metabolism under hypoxic and anoxic conditions.     

Identification of FAD, FMN, and riboflavin in the retina by microextraction and high-performance liquid chromatography

The presence of flavins in the retina has been known for some time. However, the small size of the tissue has made it difficult to quantify the levels of the individual flavins, riboflavin (RB), FMN, and FAD without pooling large numbers of retinas. A procedure to extract and quantitate RB, FMN, and FAD in retinal tissue from as few as four rat retinas has been developed. The procedure resolves these three classes of flavins and provides a recovery near 100%. For the analysis, HPLC using a reverse-phase column with cyclohexyl functional groups was coupled to a fluorescence detector. The microextraction-HPLC procedure was reproducible for the quantitative analysis of flavins in the retina and equally applicable for analysis of flavins in liver and plasma.     

Determination of the stereoconfiguration of natural pterins by chiral high-performance liquid chromatography.

The separation of D- and L-enantiomers of 6-(polyhydroxypropyl)pterins was obtained by ligand-exchange chromatography using a reversed-phase column at 12 degrees C with a mobile phase containing D-phenylalanine as the chiral modifier and Cu(II) as the metal ion. This allowed the determination of the stereoconfiguration of natural pterins from very small amounts of biological sample containing pterins in the picomole range (nanogram range). Fluorescence detection was used both to increase the sensitivity and to confirm the identification by on-line fluorescence spectroscopy and comparison with reference compounds. The stereoconfiguration of optically active pterins present in a bacterium (Escherichia coli), in a ciliate protozoan (Tetrahymena pyriformis), in an amoeba (Dictyostelium discoideum), and in mammals (human urine) was obtained and compared to earlier determinations. Incidental findings resulting from the application of this method were that human urinary monapterin and the major pterin of T. pyriformis were identified as a D-monapterin, which, until now, was not known as a natural pterin.     

Chemical reduction of pterins to dihydropterins as substrates for enzymatic reactions

Dihydropterins are important intermediates in various metabolic pathways, including the biosynthesis of tetrahydrofolate and tetrahydromethanopterin, a key coenzyme in the one-carbon metabolism of methanogenic Archaea. Some procedures for the reduction of pterins to dihydropterins may produce undesirable tetrahydropterin contaminants. This work describes a procedure for the rapid reduction of pterins to dihydropterins while minimizing tetrahydropterin production that may be particularly useful in producing substrates for enzyme reactions when the dihydropterin substrate cannot be purchased commercially.