Photo-induced Benzyl Substitution of Flavins by Phenylacetate: a Possible Model for Flavoprotein Catalysis

Hemmerich, Peter; Massey, Vincent; Weber, Gisela

doi:10.1038/213728a0

Cited by 80 publications

(25 citation statements)

References 9 publications

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“…Azide also has been described as an inhibitor of electron transfer involving flavins in an in vitro system (12). Finally, the benzyl residue of the phenylacetic acid covalently binds to the flavin nucleus of an illuminated flavin forming'a phenyl derivative of the latter (7). We suggest that the specific inhibitory action of iodide, azide, and phenylacetate on phototropism is based upon their inhibition of a flavin, and that this flavin is the photoreceptor pigment itself.…”

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confidence: 97%

Specific Inhibition of Phototropism in Corn Seedlings

Schmidt¹,

Hart²,

Filner³

et al. 1977

Plant Physiol.

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Geotropism was used as a control for the specifficity of potential inhibitors of phototropism by the coleoptiles of corn (Zea mays) seedlings. The compounds tested fall into three categories showing: (a) no inhibition of either phototropism or geotropism (KCI); (b) nonspecic inhibition of both phototropism and geotropism (KCN); and (c) specific inhibition of phototropism (KI, NaN3, and phenylacetic acid).Simuitaneous irradiation of coleoptiles with phototropically inert g4ht in addition to the phototropicafly active blue light also results in an inhibition of phototropism. Since azide, iodide, and phenylacetic acid are known to interact with flavins while a simultaneous irradiation with a phototropicafly inert light may depopulate the first triplet state of flavins, these data support the hypothesis that the photoreceptor pigment for phototropism in corn is a flavin.The identification of the photoreceptor pigment controlling phototropism has in general not progressed beyond a description of action spectra for the physiological response. Although action spectra should provide, within limits, a reasonable representation of the absorption spectrum of the photoreceptor pigment, an action spectrum is insufficient for distinguishing between potential photoreceptor pigments with very similar absorption spectra (1,3,13). Moreover, the absorption spectrum for the photoreceptor pigment may depend strongly on its environment, but this environment is an unknown as long as the identity of the photoreceptor pigment itself is unknown.Because of these inadequacies of action spectra as the major criterion for the photoreceptor pigment, we have searched for other criteria to identify the photoreceptor pigment for phototropism. We report here the results of experiments on the effects of potential inhibitors on phototropism and geotropism, pursuing the observation of Hart and Filner (6) H20. The trays were placed in large closed black boxes to keep humidity constant and the boxes were kept in a constant temperature, controlled humidity, darkroom. Only that 20% of the seedlings with straight shoots were used. The seedlings were prepared for the experiments under dim (<1 ,w cm-2) 540 nm (10 nm half-bandwidth) safelight. Each seedling was placed in a 50-ml test tube with the root system in a bathing solution and with 3 cm of the shoot projecting through a hole in a cork stopper inserted in the opening of the test tube. The seedlings were incubated with the roots immersed in a solution of the potential inhibitor at pH 7 for 1 hr before application of the test stimulus (light or gravity). All experiments were conducted at22 + 1 C.To test for geotropism, seedlings were turned horizontal for 1 hr. The geotropic response was then permitted to develop in the vertical position for 4 hr. Phototropism was induced for 5 hr with 2 ,uw cm-2 blue (440 nm) light from a 40 w Unitron incandescent lamp through an interference filter with a 10 nm half-bandwidth. The phototropic response to this intensity of light probably falls into the ...

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confidence: 97%

Specific Inhibition of Phototropism in Corn Seedlings

Schmidt¹,

Hart²,

Filner³

et al. 1977

Plant Physiol.

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“…Final buffer concentrations were 10 mM with respect to both free acid and tetrabutylammonium salt, unless otherwise stated. Buffered flavin solutions were kept in the dark in order to prevent possible photochemical reactions such as the reductive photoalkylation observed with phenylacetate [28].…”

Section: Solventmentioning

confidence: 99%

Oxidation Kinetics of 1, 5-Dihydroflavin by Oxygen in Non-aqueous Solvent

Favaudon

1977

Eur J Biochem

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The oxidation of reduced flavin anion by oxygen in aprotic dimethylformamide starts with the rapid preequilibrium formation of an anionic intermediate (~3 7 5 8.0 mM-l . cm-I). The preferred irreversible decay mode of this complex is the heterolytic rupture yielding oxidized flavin and hydrogen peroxide anion.The oxidation kinetics of neutral reduced flavin in unbuffered dimethylformamide are strictly dependent on the flavin concentration. In dilute solution, the reaction kinetics are identical to those observed with the anionic form, which requires proton eviction on formation of the flavin-oxygen complex. The whole preequilibrium step is extremely fast. Increasing the amount of neutral reduced flavin results in secondary kinetic effects corresponding to neutralization (pK, in dimethylformamide, pK* = 11.1) of the intermediate. The rate constant for the heterolytic splitting of the neutral complex is 2300-fold lower than that of the anionic form.Two sorts of pH-dependent coupled reactions between the peroxydihydroflavin adduct and uncomplexed reduced flavin appear in buffered dimethylformamide. A dismutation-type reaction yielding hydrogen peroxide and 2 mol of the flavin radical, forms the first mode. Its rate is maximum around the pK* of reduced flavin (10.15). Dismutation of the flavin radical results in autocatalytic kinetics. The second concerted mode (pK* = 10.4) allows simultaneous release of two oxidized flavin and water molecules; it has been characterized as a two-step process.The flavin-oxygen complex also forms in aqueous media, on the acidic side, with an estimated pK near neutrality. On the other hand, direct electron transfer without intermediate formation is the preferred path under strongly alkaline aqueous conditions, yielding flavin and hyperoxide radicals as the primary products.Oxygen activation in biological systems can be brought about by a variety of cofactors such as hemes or iron-sulfur clusters, pteridins and flavins. It is the fully reduced flavin which controls the reduction of oxygen through the biocatalytic turnover of numerous flavoproteins. These flavoenzymes can be separated into two classes, depending on whether the substrate undergoes reaction with the flavin-activated oxygen. Flavoprotein oxidases [l], on the one hand, simply utilize oxygen as the terminal acceptor of the two reducing equivalents transferred from the substrate to the flavin. In flavoprotein monooxygenases [2], also termAbbreviations. AC~RFI, tetraacetylriboflavin; 3MeAc4RF1, 3-methyl-tetraacetylnboflavin ; NMR, nuclear magnetic resonance; ESR, electron spin resonance; Fl,,dHZ, Fl,,dH-, FIH, Fif, FI,,, FIG, neutral and anionic forms of reduced, radical and oxidized flavins, respectively; pH*, pK*, pH and pK, values in dimethylformamide.ed mixed-function oxidases, one atom of the oxygen molecule is incorporated into the substrate while the other is reduced into water.First insights into the oxidation mechanism of fully reduced flavin by oxygen came from studies performed with free flavins in aqueous media [3 ...

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“…2). Hemmerich et al (12), studying details of the reaction, report that compounds similar to PAA are photodecarboxylated with the remaining benzyl residue attaching covalently to position N-5 or C-4a on the flavin nucleus. Such photoadducts can be stable in the presence of 02.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of Specific Inhibition of Phototropism by Phenylacetic Acid in Corn Seedling

Vierstra

Poff²

1981

Plant Physiol.

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Using geotropism as a control for phototropism, compounds similar to phenylacetic acid that photoreact with flavins and/or have auxin-like activty were examined for their ability to specifically inhibit phototropism in corn seedlings using geotropism as a control. Results using indole-3-acetic acid, napthalene-1-acetic acid, naphthalene-2-acetic acid, phenylacetic acid, and ,8-phenylpyruvic acid suggest that such compounds will specifically inhibit phototropism primarily because of their photoreactivity with flavins and not their auxin activity. For example, strong auxins, indole-3-acetic acid and naphthalene-1-acetic acid, affected both tropic responses at aDl concentrations tested whereas weak auxins, phenylacetic acid and naphthalene-2-acetic acid, exhibited specific inhibition. In addition, the in vivo concentration of phenylacetic acid required to induce specificity was well below that required to stimulate coleoptile growth. Estimates of the percentage of photoreceptor pigment inactivated by phenylacetic acid (>10%) suggest that phenylacetic acid could be used to photoaffinity label the flavoprotein involved in corn seedling phototropism.Blue light is known to control many metabolic and morphogenic responses in plants, such as phototropism in Phycomyces, A vena, and Zea mays (9), carotenoid synthesis in Neurospora (8), photophobic movements in Euglena (7), and changes in stomatal aperture in many plant species (13). The striking similarities between action spectra suggest a comon photoreceptor pigment for these responses. However, because these action spectra resemble the absorption spectra of two prominent plant pigments (flavins and carotenoids) and because of the lack of any assay specific for the photoreceptor pigment beyond the action spectrum, the identity of the chromophore responsible remains controversial. Indirect evidence, mainly from the photoresponses of "carotenoidless" mutants of Phycomyces (15), and Neurospora (17)

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Photo-induced Benzyl Substitution of Flavins by Phenylacetate: a Possible Model for Flavoprotein Catalysis

Cited by 80 publications

References 9 publications

Specific Inhibition of Phototropism in Corn Seedlings

Specific Inhibition of Phototropism in Corn Seedlings

Oxidation Kinetics of 1, 5-Dihydroflavin by Oxygen in Non-aqueous Solvent

Mechanism of Specific Inhibition of Phototropism by Phenylacetic Acid in Corn Seedling

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