Neutral Flavin Radical Research Articles

G-Tensor of the Neutral Flavin Radical Cofactor of DNA Photolyase Revealed by 360-GHz Electron Paramagnetic Resonance Spectroscopy

The flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form, FADH•, was investigated by high-frequency/high-field continuous-wave electron paramagnetic resonance at 360 GHz. The data presented are the first flavin radical spectra where the full rhombic symmetry of the g-tensor is resolved. A fit of the spectrum yields accurate principal values of g, which show only a small anisotropy: gX = 2.004 31(5), gY = 2.003 60(5) and gZ = 2.002 17(7). The hyperfine splitting observed in the gY region could be assigned to a hyperfine tensor component of the H(5) proton in the 7,8-dimethyl isoalloxazine moiety of FADH•. From a comparison of this effective hyperfine coupling with the principal value obtained from pulsed (Davies) electron−nuclear double resonance, the orientation of the g-tensor principal axes with respect to the H(5) hyperfine principal axes could be derived. Remaining ambiguities in the sign of the angle between the principal axes of g and the molecular axes are discussed by ta...

Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance.

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6-4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6-4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.

Resonance Raman spectroscopic study of the neutral flavin radical complex of DNA photolyase from Escherichia coli

AbstractThe neutral flavin radical complex of DNA photolyase from Escherichia coli was studied by resonance Raman spectroscopy for the first time. The experiments were carried out with enzyme variants that lack the light‐harvesting methenyltetrahydrofolate cofactor so that the resonance Raman spectra exclusively display the vibrational bands of the flavin radical in the catalytic site. Spectral changes induced upon H–D exchange and upon substituting the natural flavin by its 15N‐labelled isotopomer allow a tentative assignment of the prominent bands in the region between 1200 and 1650 cm−1. It is shown that most of the bands observed with 568 nm excitation originate from modes localized in the isoalloxazine ring. In conjunction with previous data on flavin radicals, the present results allow the identification of the marker bands that appear to be particularly sensitive towards changes in the electron density distribution of this ring and, hence, reflect the specific intermolecular interactions in the catalytic site. Copyright © 2001 John Wiley & Sons, Ltd.

On the iron–sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase.

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.

Model Systems for Flavoenzyme Activity: One- and Two-Electron Reduction of Flavins in Aprotic Hydrophobic Environments

The processes occurring during the electrochemically irreversible reduction of flavins in aprotic organic medium have been the cause of considerable controversy. To provide insight into these events we have investigated the reduction of flavins alkylated at N(3). We have demonstrated that the reversible reduction of N(3)-methylated flavin can be converted to the irreversible reduction observed with unalkylated flavins through addition of the weak external proton donor phthalimide. Proton transfer to the flavin radical anion generated at the electrode leads to partial formation of the neutral flavin radical, which is instantaneously further reduced to the fully reduced flavin anion. This mirrors the reduction of unalkylated flavins, where the proton source is the imide proton at N(3) of fully oxidized flavin in bulk solution. Simultaneous electrochemistry and EPR experiments confirm the disappearance of electrogenerated flavin radical anion both methylated and non-methylated at N(3), upon addition of phtha...

PHOTOCHEMISTRY, PHOTOPHYSICS, AND MECHANISM OF PYRIMIDINE DIMER REPAIR BY DNA PHOTOLYASE

DNA photolyases photorepair pyrimidine dimers (Pyr < > Pyr) in DNA as well as RNA and thus reverse the harmful effects of UV-A (320-400 nm) and UV-B (280-320 nm) radiations. Photolyases from various organisms have been found to contain two noncovalently bound cofactors; one is a fully reduced flavin adenine dinucleotide (FADH-) and the other, commonly known as second chromophore, is either methenyltetrahydrofolate (MTHF) or 8-hydroxydeazaflavin (8-HDF). The second chromophore in photolyase is a light-harvesting molecule that absorbs mostly in the near-UV and visible wavelengths (300-500 nm) with its high extinction coefficient. The second chromophore then transfers its excitation energy to the FADH-. Subsequently, the photoexcited FADH- transfers an electron to the Pyr < > Pyr generating a dimer radical anion (Pyr < > Pyr.-) and a neutral flavin radical (FADH.). The Pyr < > Pyr.- is very unstable and undergoes spontaneous splitting followed by a back electron transfer to the FADH.. In addition to the main catalytic cofactor FADH-, a Trp (Trp277 in Escherichia coli) in apophotolyase, independent of other chromophores, also functions as a sensitizer to repair Pyr < > Pyr by direct electron transfer.

Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bleaching and FADH2 oxidation are inhibited by substrate.

Oxidation-reduction potentials of butyryl-CoA dehydrogenase.

In order to obtain butyryl-CoA dehydrogenase from Megasphaera elsdenii in pure enough form to perform redox studies, the existing purification procedures first had to be modified and clarified [Engel, P. (1981) Methods Enzymol. 71, 359-366]. These modifications are described, and the previously unpublished spectral properties of the electrophoretically pure CoA-free butyryl-CoA dehydrogenase are presented. In our spectral reductive titration of pure enzyme, we show that although blue neutral flavin radical is stabilized in nonquantitative amounts in dithionite titrations (19%) or in electrochemical reductions mediated by methylviologen (5%), it is not thermodynamically stabilized; therefore, only a midpoint potential for butyryl-CoA dehydrogenase is obtained. The electron-transfer behavior from pH 5.5 to pH 7.0 indicates reversible two-electron transfer accompanied by one proton: EFlox + 2e- + H+ = EFlredH- Em7 = -0.079 V vs. SHE where EFlox is oxidized butyryl-CoA dehydrogenase, EFlredH- is two electron reduced enzyme, and Em7 is the midpoint potential at pH 7.0 at 25 degrees C. Redox data and activity data both indicate that the enzyme loses activity rapidly at pH values above 7.0. The Em7 of the butyryl-CoA dehydrogenase is 40 mV positive of the Em7 of the butyryl-CoA/crotonyl-CoA couple [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. Binding of substrate analogue acetoacetyl-CoA caused the potential of butyryl-CoA dehydrogenase to shift 100 mV negative of the free enzyme. The negative shift in potential makes electron transfer from enzyme to substrate more probable, which is consistent with the direction of electron transfer in the bacterial system.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase.

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.

Identification of a neutral flavin radical and characterization of a second chromophore in Escherichia coli DNA photolyase.

DNA photolyase from Escherichia coli is a blue protein exhibiting absorption maxima at 580, 475, and 384 nm. One of the two chromophores present in this enzyme has been identified as the blue neutral flavin adenine dinucleotide (FAD) radical on the basis, in part, of visible absorption and electron spin resonance (ESR) data. The enzyme-bound radical (epsilon 580 = 3.6 X 10(3) M-1 cm-1) is stable toward O2 or K3Fe(CN)6, is reversibly reduced by dithionite, and is converted to oxidized FAD upon aerobic denaturation. Disproportionation of the radical is observed upon anaerobic denaturation, consistent with an N-5 unsubstituted radical. The absorbance of the enzyme at lambda greater than 500 nm is due only to the FAD radical whereas the band at 384 nm reflects contributions from both the radical and a second chromophore. The latter is labile when protein free a neutral pH (lambda max = 360 nm, k = 5.5 X 10(-2) min -1 +/- O2), a reaction that is readily monitored by the loss of an intense absorption band at 360 nm following enzyme denaturation under conditions where radical oxidation is immediate. This decomposition is pH dependent and the chromophore is stable at acid pH. Native photolyase is fluorescent (emission lambda max = 470 nm, excitation lambda max = 398 nm). An unlikely fluorescent flavin radical can be excluded by the position of the emission maximum. The enzyme fluorescence is attributed to the second chromophore.

Structure and catalytic inactivity of the bacterial luciferase neutral flavin radical.

A luciferase-bound neutral flavin semiquinone radical can be formed upon the oxidation of the luciferase-FMNH2 complex by molecular oxygen. This species can also be formed anaerobically by comproportionation of FMN and FMNH2 in the presence of luciferase. The radical is kinetically stable (t1/2 approximately 20 h at 0 degree C in air; the Arrhenius delta H not equal to decay being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 degrees C. The pure enzyme-bound radical is inactive for light emission either with or without aldehyde, and is not in (relevantly rapid) equilibrium with the luciferase 4a-peroxyflavin, the active intermediate in the bioluminescent reaction.

Flash photolysis of flavins. V. Oxidation and disproportionation of flavin radicals.

Flash photolysis techniques have been utilized to investigate the reactivity patterns of flavin radical species. Rate constants for disproportionation were found to be la the following order: lumiflavin>FMN>FAD and neutral radicals>anionic radicals. Neutral flavin radicals react with oxygen at a rate which is at least 104 times slower than the anionic species. No evidence for an intermediate complex or adduct is obtained in this reaction. The pK values for the ionization of the neutral flavin radicals are in the order FAD>FMN>riboflavin=limiflavin. The rates of reaction of ferricyanide with flavin radicals are essentially independent of pH, whereas benzoquinone reacts slightly more slowly (≅5 times) with the neutral flavin radical than with the anionic form. Cytochromec reacts at least ten times more slowly with flavin radicals than does ferricyanide.

The chemical and electronic structure of the neutral flavin radical as revealed by electron spin resonance spectroscopy of chemically and isotopically substituted derivatives.

The neutral flavosemiquinone has been studied in detail by electron spectroscopy. Isotopic (15N, 2H) and chemical substitutions have been carried out. A hyperfine coupling scheme of the neutral flavosemiquinone is described where N(5), N(10), CH3(10), CH3(8) and H(6) are involved. The highest spin density is located at N(5) as has also been found for the anionic and chelated form of the flavosemiquinone. The exchangeable proton in the neutral flavosemiquinone is attached to N(5) having a coupling constant of 7.6 G which is about the same magnitude as the coupling constant to N(5). The hyperfine couplings are interpreted in terms of spin densities and comparison is made with available quantum chemical calculations. Evidence is presented that two tautomeric forms of the neutral flavosemiquinone have to be considered which can be obtained in a pure form by suitable substitution of flavin derivatives. The two types of neutral flavosemiquinones are easily distinguishable by their electron spin resonance and light absorptions.