Flash photolysis of flavins. IV. Some properties of the lumiflavin triplet state

Vaish, Shiv P.; Tollin, Gordon

doi:10.1007/bf01515980

Cited by 75 publications

(61 citation statements)

References 14 publications

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“…This D,D reaction corresponds to the photoelectron transfer, which has been observed earlier by Vaish and Tollin [5,6] with the 'binary' system flavin/ phenol, the latter being a 'le--only' donor substrate The rate of radical decay is then determined by the unstable substrate radical, and not by the much more slower dismutation of the flavosemiquinone.…”

Section: Flash Photolysis Of Flavin In Aqueous Buifer: Fi As Donorsupporting

confidence: 52%

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Distinction of 2e- and le- Reduction Modes of the Flavin Chromophore as Studied by Flash Photolysis

Hemmerich

Knappe

Kramer³

et al. 1980

Eur J Biochem

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Evidence is given for the fact that the excited flavin triplet (3F10*x) exhibits competitive le-and 2e-transfer chemistry, depending on the nature of the photosubstrate. As an 'external' photoreductant, the 2e-donor borohydride has been investigated. Borohydride is found to compete effectively with the 'internal' 1 e-donors, namely excess starting flavin in the ground state (Flax) and, as primary product, (alky1)dihydroflavin (RFl,,dH). It will be shown that some of the flavin radicals, observed by earlier authors in the reaction of 3Fl& with CH or C -COO-substrates or in the autophotolytic side-chain cleavage of riboflavin, are due to the dye-dye reaction (I) :(1) H i In contrast flavin photoreduction by borohydride or hydrocarbon substrates need not involve radicals, but may in fact be a hydride or 'carbanion-plus-proton' addition towards the highly polar and considerably basic (pK = 4.4) acceptor triplet (cf. reaction 11) RCOO-The products are much more photoreactive than the starting substrates, which leads to the secondary photocomproportionation (111) : 3Fl,*, + R-Fl,,dH + HFl + RFl, (R = alkyl or H). (111) This latter reaction is the second source of radicals in the system. This (cf. 11) 'photohydrogenation' of flavin is mechanistically related to the biological reduction of flavin by CH substrates.The photochemistry of flavins is in fact older than the corresponding ground-state chemistry and even biochemistry, since the side-chain photolysis of vitamin Bz was the first flavin reaction to be discovered [l]. The very complex course of this reaction, yielding lumiflavin at alkaline pH and lumichrome at neutrality, is still not completely resolved [2] (and references therein). This is largely due to a common mistake of insisting that such reactions must involve radical Dedicated to Professor Hellmut Bredereck, Universitat Stuttgart, on his 75th birthday.Abbreviations. Fl,,, flavoquinone, flavin; 3Fl,*,, excited flavin triplet; HFI Fl-, acidic and basic flavosemiquinone; Fl', oxidized flavin radical; HZFlred, flavohydroquinone, 1,5-dihydroflavin.

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Section: Flash Photolysis Of Flavin In Aqueous Buifer: Fi As Donorsupporting

confidence: 52%

“…Holmstrom [4] was the first to observe such radicals in riboflavin photolysis, while Vaish and Tollin [5,6] demonstrated fast photoelectron transfer and spontaneous back donation with suitable substrates, such as phenol :…”

Section: (111)mentioning

confidence: 99%

Distinction of 2e- and le- Reduction Modes of the Flavin Chromophore as Studied by Flash Photolysis

Hemmerich

Knappe

Kramer³

et al. 1980

Eur J Biochem

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“…4 compares the LOV2 absorption difference spectra at 30 ns and 500 ms with the absorption difference spectra of the FMN triplet and semiquinone forms. Clearly, the early transient only fits well to the spectral features of well established triplet-state difference spectra of flavins in aqueous solution (15,16).…”

Section: Resultsmentioning

confidence: 91%

“…Various flavin photoproducts are known to show absorption in this wavelength range, including charge transfer states, triplet states, and neutral flavosemiquinones (13,14). To explore the nature of the early photoproduct, we generated the triplet and flavo-semiquinone states of FMN in aqueous solution by laser flash excitation (15,16) and compared their spectral features with those of the intermediate states of LOV2. Although charge transfer bands of flavoenzymes also absorb maximally at wavelengths longer than that of the ground state (17,18), they were excluded from consideration because the LOV2C39A mutant, which cannot form a charge transfer complex, formed an almost identical transient species (see below).…”

Section: Resultsmentioning

confidence: 99%

The Photocycle of a Flavin-binding Domain of the Blue Light Photoreceptor Phototropin

Swartz¹,

Corchnoy²,

Christie³

et al. 2001

Journal of Biological Chemistry

518

783

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The plant blue light receptor, phot1, a member of the phototropin family (1), is a plasma membrane-associated flavoprotein that contains two (ϳ110 amino acids) flavinbinding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Near-UV blue light regulates a variety of different responses in higher plants. These include phototropism, the inhibition of hypocotyl elongation, the expression of various genes, and stomatal opening. Phot1 (nph1), the recently discovered blue light receptor, is a member of the phototropin receptor family (1). Phot1 is a plasma membrane-associated flavoprotein that functions as the primary photoreceptor mediating phototropic plant movement (2-4). Phot1 has two 12.1-kDa flavin-binding domains, LOV1 and LOV2, within its N-terminal region and a typical serinethreonine protein kinase domain at the C-terminal region. Heterologous expression studies have shown that phot1 binds FMN 1 as a chromophore and undergoes autophosphorylation in response to light treatment. It has therefore been proposed that this receptor functions as a light-activated serine/threonine kinase (4). The isolated LOV domains from oat phot1 expressed in Escherichia coli have been shown to undergo a cyclic photoreaction upon the absorption of light; LOV1 recovers with a half-time of 11.5 s, whereas LOV2 recovers with a half-time of 27 s (5). In addition, the quantum efficiencies for photoproduct (adduct) formation for LOV1 and LOV2 are ϳ0.045 and 0.44, respectively (5). The ground forms of the LOV domains have major absorption peaks at 360 and 450 nm with the 450 peak having vibronic structure at 425 and 475 nm. Upon absorption of light, the chromophore bleaches 2 in the 450 nm region generating a species that absorbs maximally at 390 nm. This intermediate has been assigned as a flavin-cysteinyl adduct between the protein and the C(4a) carbon of the FMN chromophore. This adduct breaks down spontaneously, returning the protein to its ground form. A LOV2 mutant (LOV2C39A) in which the cysteine that forms the adduct has been mutated to alanine does not undergo this photoreaction (5).Recently the crystal structure of the LOV2 domain from the fern Adiantum capillus-veneris phy3 (6) was solved to 2.7-Å resolution (7). Phy3 is a chimeric photoreceptor with homology to phytochrome at its N-terminal end and an almost complete phototropin at its C-terminal end. Its LOV2 domain shares a 70% sequence homology to the oat phot1 LOV2 (6). The structure indicates that the FMN molecule is held noncovalently within...

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“…2 shows kinetic transients obtained at 640 nm, associated with the lumiflavin triplet state (cf. [22,23]), and at 552 nm, which monitors the state of oxidation/reduction of the cytochrome. In curve A, the initial rise in absorbance at 640 nm is due to the formation of the triplet state, and the subsequent absorbance decay corresponds to triplet disappearance.…”

Section: Resultsmentioning

confidence: 99%

Flavin‐photosensitized oxidation of reduced c‐type cytochromes

Roncel

Hervás

Navarro

et al. 1990

European Journal of Biochemistry

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In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20 -100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome ~5 5 2 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin -cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.The mechanism of electron transfer between redox proteins has been the subject of extensive study in recent years (cf. [l -41 for reviews). The primary focus of these investigations has been on the reactions of soluble proteins containing a single redox center (e.g. c-type cytochromes, copper proteins, iron-sulfur proteins), although work has also been done with multicenter and membrane-associated systems. This emphasis is mainly a consequence of the substantial body of structural information which is available for the smaller and simpler redox proteins. With electron carriers of this type, electron transfer generally proceeds from a small substrate or from another protein into the prosthetic group of the redox protein, and then from the reduced protein to an acceptor, which again may be a small molecule such as dioxygen or another redox protein such as a cytochrome. Thus, a question arises as to the identity of the sites of reduction and oxidation: are they the same or different? This question has been studied most extensively with c-type cytochromes, and the general consensus appears to be that for these proteins the sites are probably the same (cf. [5, 61 f...

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Flash photolysis of flavins. IV. Some properties of the lumiflavin triplet state

Cited by 75 publications

References 14 publications

Distinction of 2e- and le- Reduction Modes of the Flavin Chromophore as Studied by Flash Photolysis

Distinction of 2e- and le- Reduction Modes of the Flavin Chromophore as Studied by Flash Photolysis

The Photocycle of a Flavin-binding Domain of the Blue Light Photoreceptor Phototropin

Flavin‐photosensitized oxidation of reduced c‐type cytochromes

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