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Reduction potential determinations of K75E, E139K and E301A ferredoxin-NADP+ reductases provide valuable information concerning the factors that contribute to tune the flavin reduction potential. Thus, while E139 is not involved in such modulation, the K75 side-chain tunes the flavin potential by creating a defined environment that modulates the FAD conformation. Finally, the E301 side-chain influences not only the flavin reduction potential, but also the electron transfer mechanism, as suggested from the values determined for the E301A mutant, where E ox/rd and E sq/rd shifted +41 and +102 mV, respectively, with regard to wild-type. Reduction potentials allowed estimation of binding energies differences of the FAD cofactor upon reduction. [ABSTRACT FROM AUTHOR]

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the π-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4)... [ABSTRACT FROM AUTHOR]

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to ≤ 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and ≤ 5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone. [ABSTRACT FROM AUTHOR]

Investigates the effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide (FMN). Occurrence of ionizations outside the pH range; Reduction of semiquinone to the hydroquinone; Measurement of the semiquinone concentrations in FMN solutions.

The first three-dimensional structure of moenomycin A in aqueous solution based on NMR-derived distance constraints and molecular dynamics simulations is presented. The antibiotic moenomycin A was obtained from the FlavomycinR complex by ultrafiltration, chromatography on a DEAE-cellulose ion exchanger and reverse-phase chromatography in 98 % purity. In contrast to the previously reported behaviour, the compound gave rise to well-resolved NMR spectra in standard solvents. Using several two-dimensional experiments, a complete assignment of proton and carbon chemical shifts was achieved in (CD3)2SO, CD3OD and H2O/D2O (9 : 1, pH 7.3). A total of 175 interproton distances were determined from 600-MHz rotating-frame NOE (ROE) spectra and were used as restraints in molecular dynamics calculations. These restraints included 84 ROEs between protons of the moenocinol part leading to a very well-defined structure of the lipid part of the molecule. The relative orientation of the subunits was determined by 66 ROEs among different sugar rings and between the sugar rings and moenocinol. As a result of the molecular dynamics calculation, rings D, E, and F as well as the moenocinol part are very well-defined (average rms deviation over all heavy atoms 0.48 Å) whereas rings A, B and C display a higher degree of conformational flexibility which might be an artefact due to the lower number of ROEs in this part of the molecule. A three-dimensional pharmacophore hypothesis comprising functional groups of rings E and F and the carboxyl group of glyceric acid is presented on the basis of the degradation and derivatization studies of Welzel and coworkers [Welzel, P., Kunisch, F., Kruggel, F., Stein, H., Scherkenbeck, J., Hiltmann, A., Duddeck, H., Müller, D., Maggio, J. E., Fehlhaber, H.-W., Seibert, G., van Heijenoort, Y. & van Heijenoort, J. (1987) Moenomycin A : Minimum structural requirements for... [ABSTRACT FROM AUTHOR]

Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Flox and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Flred)] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Flred 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Flred 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Flox, which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein. [ABSTRACT FROM AUTHOR]

Reports on the coryneform bacterium, Brevibacterium ammoniagenes, which contains two structurally related but functionally differentiated type I fatty acid synthases, FAS-A and FAS-B. Construction of specific fasA and fasB expression systems for the isolation of homogenous preparations of both enzymes; Finding that the purified FAS-A and FAS-B proteins were indistinguishable with respect to their flavin fluorescence; Differences in the ability of the B ammoniogenes type I fatty acid synthases to synthesize oleic acid and in their chain-length specificities.

Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity: The enzyme appeared to be encapsulated in particles obtained alter mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25 % (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu2+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu2+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported [Sullivan, J. D. & Ikawa, M. (1973) Biochim. Biophys. Acre 309, 11–22] characteristics of the enzyme probably originate from the characterization of a contaminating protein in the latter case. [ABSTRACT FROM AUTHOR]

The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH. A sequential ordered mechanism, with NADPH binding first, operates. Fire is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group. It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-ethylmaleimide (MalNEt). Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved. Altogether this suggested a crucial role of cysteine residues for catalysis. The three cysteine residues were mutated to serine residues. Single-mutant and double-mutant enzymes were as active as the wild-type and Km values for both substrates remained the same. Cysteine residues are thus not important for activity. Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation. In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation. This strongly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH. [ABSTRACT FROM AUTHOR]

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222→Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222→Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A., Mattevi, A., Oblomova, g., Kalk, K. H., Hol, W.G.J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemsitry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flaving. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222→Ala mutant shifts the equilibrium of flavin conformers towards the productive 'in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluor-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the 'out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme. [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222→Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring. [ABSTRACT FROM AUTHOR]

We have previously described the isolation and sequencing of cDNA clones encoding flavin-containing monooxygenases (FMOs) 1 and 4 of man [Dolphin, C., Shephard, E. A., Povey, S., Palmer, C. N. A., Ziegler, D. M., Ayesh, R., Smith, R. L. & Phillips, I. R. (1991) J. Biol. Chem. 266, 12379–12385; Dolphin, C., Shephard, E. A., Povey, S., Smith, R. L. & Phillips, I. R. (1992) Biochem. J. 287, 261–267]. We present heine the isolation of a cDNA for FMO3 of man. The sequence of this cDNA and the amino acid sequence deduced from it differ substantially from those previously reported for this member of the FMO family of man. In addition, we have investigated, by quantitative RNase protection assays, the expression in severed foetal and adult human tissues of genes encoding FMO1, FMO3 and FMO4. Our results demonstrate that, in the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus it is expressed in both organs. The lack of expression of FM01 in adult human liver is in marked contrast to the situation in other mammals, such as pig and rabbit, in which FMO1 constitutes a major form of the enzyme in the liver of the adult animal. The mRNA encoding FMO3 is abundant in adult liver and is also present, in low abundance, in some foetal tissues. Thus, FMO1 and FMO3 are both subject to developmental and tissue-specific regulation, with a developmental switch in the expression of the genes taking place in the liver. FMO4 mRNA is present in low abundance in several foetal and adult tissues and thus the corresponding gene appears to be expressed constitutively. [ABSTRACT FROM AUTHOR]

This study deals with the detailed electrochemistry and complete EPR-monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues. Cys69 has been replaced by both an alanine and a serine residue. Redox properties of she C69A and C69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox-linked pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred,1 = 5.39 ± 0.08. pKox = 7.29 ± 0.14, and pKred,2 = 7.84 ± 0.14 with Em,7 = -459 ± 4 mV, and a constant redox potential at high pH of -485 ± mV. The dependence of the semiquinone/hydroquionone redox potential on temperature is -0.5 ± 0.1 mV · K-1, yielding ΔH° = 28.6 ± 1.5 kJ · mol-1 and ΔS° = -50.0 ± 6.2 J · mol -1 · K-1. No significant differences in redox properties of wild-type. C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin 11. [ABSTRACT FROM AUTHOR]

Arg44, located at the si-face side of the flaving ring in 4-hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-um resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180° in 50% of the molecules. The Φ, Ψ angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis. [ABSTRACT FROM AUTHOR]

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 ≈ 20 h at 0 °C in air: the Arrhenius ΔHdecay= being about 170 kJ/mol) and can be prepared in pure form by Sephadex G-25 chromatography at 0-4 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. [ABSTRACT FROM AUTHOR]

Selectively 13C-enriched free and protein-bound flavins in the oxidized and two-electron reduced state were investigated by the 13C nuclear magnetic resonance technique. FMN in aqueous solution and N(3)-methyl-2',3',4',5'-tetraacetylriboflavin in an apolar solvent were used as reference compounds for the protein-bound FMN. A comparison of the chemical shifts of FMN in aqueous solution with those of N(3)-methyltetraacetyl-riboflavin in CHCl3 reveals that FMN is strongly polarized yielding a pseudo-ionic molecule stabilized by hydrogen bonding of H2O with C-2α of the isoalloxazine molecule. The interpretation is fully supported by the one-bond (¹J) 13C coupling constants. The chemical shifts combined with the coupling constants indicate the change of charge distribution within the molecule when going from apolar to polar solutions. Binding of FMN to Megasphaera elsdenii and to Axotobacter vinelandii apoflavodoxins affects the resonances due to C-2 and C-10a as compared to free FMN. This shifts together with the coupling constants indicate the formation of a hydrogen bond between C-2α of FMN and an amino acid residue of the apoprotein. In M. elsdenii flavodoxin such a hydrogen bond also exists with C-4α which is not observed in A. vinelandii flavodoxin. The chemical shifts to the two-electron reduced derivatives of FMN and N(3)-methyl-2',3',4',5'-tetraacetylriboflavin clearly indicate that the FMN derivative possesses a more planar conformation than its analog; it show pH-dependent chemical shifts of the atoms C-10a, C-2 and c-4a. the chemical shift of C-4 is pH-independent. From the pH-dependence of the chemical shifts an ionization constant of 6,7 is calculated. The chemical shifts of protein-bound FMN in the reduced state are very similar to those of the anionic, reduced FMN. The data show that in both flavodoxin studied the prosthetic group when reduced is bound in the anionic form an possesses approximately a coplanar conformation. The results are discussed with respect to the possible biological implications. It is suggested that specific interaction of the prosthetic group with the apoprotein, the ionization state and the degree of planarity of the reduced prosthetic group are factors determining the biological functions of flavodoxins and possibly the functions of flavoproteins in general. [ABSTRACT FROM AUTHOR]

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran-Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 μM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 μM NADH + 10 μM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitudes at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran-Sepharose: the NADH binding site and the flavin binding site. For the latter it is clear that the isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, an FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochern. 90, 563–569]. [ABSTRACT FROM AUTHOR]

Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethyl-benzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3–0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1′-14C]riboflavin as substrate the specific radioactivity of 5,6-dimetbylbenzimidazole is higher than the specific radioactivity of flavin-adenine dinucleotide and lower than the specific radioactivity of flavin mononucleotide. Therefore flavin mononucleotide is the immediate substrate for the formation of 5,6-dimethylbenzimidazole. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed. [ABSTRACT FROM AUTHOR]

Evidence is given for the fact that the excited flavin triplet (3Flox*) exhibits competitive 1e- 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’ 1e- donors, namely excess starting flavin in the ground state (Flox) and, as primary product, (alkyl)dihydroflavin (RFlredH). It will be shown that some of the flavin radicals, observed by earlier authors in the reaction of 3Flox* with CH or C-COO- substrates or in the autophotolytic side-chain cleavage of riboflavin, are due to the dye-dye reaction (I): [This symbol cannot be presented in ASCII format] 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 II) [This symbol cannot be presented in ASCII format] The products are much more photoreactive than the starting substrates, which leads to the secondary photocomproportionation (III): [This symbol cannot be presented in ASCII format] This latter reaction is the second source of radicals in the system. This (cf. II) ‘photohydrogenation’ of flavin is mechanistically related to the biological reduction of flavin by CH substrates. [ABSTRACT FROM AUTHOR]

The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30°C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J. Biochem. 46, 393–399]. We report here studies carried out at 5°C with L-[2-2H]lactate, where fiavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the α-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme. The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillère-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J. Biochern. 54, 549–566]. An alternative model based on computer-simulation studies will be presented in a paper in this journal. [ABSTRACT FROM AUTHOR]

Fluorescence lifetimes and polarized emission properties of reduced flavin were measured using several model compounds and flavoproteins. Depending on the conditions of solvent and temperature or reduction method the lifetimes vary between 1 and 15 ns. The longer lifetime values are found in several forms of reduced lactate oxidase, in which a good correlation exists between fluorescence intensity and lifetime. In practically all flavoproteins the fluorescence is heterogeneous. Several mechanisms are proposed to explain the observed heterogeneity in lifetimes. The reduced models in glycerol at subzero temperature exhibit high degrees of polarization of the fluorescence, whereas distinct depolarization is encountered in several reduced flavoproteins suggesting a certain mobility of the flavin chromophor. [ABSTRACT FROM AUTHOR]

1. The aromatic proton resonances in the 360-MHz ¹H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessible to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180° about their Cβ−Cγbond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues. [ABSTRACT FROM AUTHOR]

The catalytic action of 5-deazaflavin in the photochemical reduction of flavin and iron proteins [Massey, V. and Hemmerich, P. (1978) Biochemistry, 17, 9-17] is shown to be due to the highly reactive 5-deazaflavosemiquinone. This radical is generated in a complex sequence of reactions, which involves (a) covalent photoaddition of the substrate residue to the deazaflavin, (b) fast secondary photoreaction of this adduct with starting deazaflavin to yield a covalent radical dimer, accompanied by the liberation of the oxidized substrate, and (c) deazaflavin-sensitized cleavage of the radical dimer to the monomers. The structure and properties of this radical (redimerisation or dismutation) and the precursor intermediates as well as the mechanism of the photoreaction are described. Deazaflavins and their natural parent compounds are compared with respect to their different redox behavior and radical stability. The syntheses of 5-deuterated deazaflavins are described and their redox reactions are compared with those of normal deazaflavins. [ABSTRACT FROM AUTHOR]

By sulfur substitution of the N-5 atom in flavins and flavocoenzymes a flavin analog is obtained, 5-thiaflavin, which is found to be isoelectronic and isosteric with natural flavin in the fully reduced and half-reduced states, but not in the oxidized state. Among the three 'redox shuttles' characterizing the flavin system, viz. upper 1e-, lower 1e- and 2e- shuttle, only the second one is retained in thiaflavin, which limits the redox activity of this system to 1e- transfer. The structure and properties of the molecular species participating in the thiaflavin redox system are discussed in comparison with the flavin system. The corresponding chemistry of a '2e- flavin', 5-deazaflavin, has been treated in the preceding paper. 5-Thiaflavin is found to exhibit a stable neutral radical, which is analogous to the 'blue' flavosemiquinone. Unlike normal flavin, where the radical is in a dismutation equilibrium, thiaflavin radical shows reversible formation of a covalent dimer, which is stable in aprotic solution and disproportionates only in water, with irreversible formation of a sulfoxide. The ultraviolet and infrared spectra of the dimer are in agreement with the structure of two 5-thiaflavin molecules linked covalently at their 4a carbons. This corroborates the earlier hypothesis that the essential intermediate in the dismutation of normal flavin is likewise a covalent dimer. Thiaflavin is tightly bound by apoflavodoxin. The protein catalyses the autoxidation to the radical state. Thiaflavodoxin radical is even more stable (towards further oxidation) than is the free thiaflavin radical. The redox potential of the couple reduced thiaflavin/thiaflavin radical (sFlred/sFl) is surprisingly high. From the reversible equilibrium established with ferricyanide, sFlred + Fe(CN)63- ⇄ sFl• + Fe(CN)64-, the standard potential of the sFlred/sFl• couple, Em at pH 7, has been estimated as + 0.38 V. [ABSTRACT FROM AUTHOR]

An extracellular enzyme which utilizes molecular oxygen to oxidize cellodextrins to the corresponding aldonic acids has been isolated from culture filtrates of the white-rot fungus Sporotrichum puh,erulentum. This enzyme, tentatively named cellobiose oxidase, has been highly purified by classical techniques and has been demonstrated to be a glycoprotein with a molecular weight of approximately 93000. Ultraviolet spectra of the enzyme in the presence and absence of substrate are characteristic of a hemoprotein. Acidic hydrolyses of the enzyme followed by a spectrofluorimetric investigation of the hydrolysate has demonstrated the presence of approximately one flavin component per enzyme molecule. The possible role of this complex enzyme in cellulose degradation is discussed. [ABSTRACT FROM AUTHOR]

Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillére Blandin et al. Eur. J. Biochem. 54, 549-566 (1975)] and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 µM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavoproteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244, 3999-4006 (1969)]. [ABSTRACT FROM AUTHOR]

1. The addition of Ca2+ to purified nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa results in a 2–8-fold increase in flavin fluorescence, closely resembling that induced by 2′-AMP (adenosine 2′-monophosphate). The response to Ca2+ is sigmoidal, with a Hill-coefficient of 2.6. Reduction of thionicotinamide adenine dinucleotide (oxidised form) by NADH responds in a similar fashion to Ca2+ and 2′-AMP. 2. Mg2+ inhibits the Ca2+-dependent activation in a competitive fashion, whereas no effect of Mg2+ is observed when the enzyme is activated with 2′-AMP. 3. The pH dependencies of the activations by 2′-AMP and Ca2+ are different. The activation by Ca2+ shows little or no dependence on pH, whereas higher concentrations of 2′-AMP are required to obtain the same activation when the pH is increased. 4. The addition of both Ca2+ and 2′-AMP causes a dramatic decrease in the concentration of Ca2+ required for half-maximal stimulation. 5. A negative cooperative effect of NADP+ has been demonstrated indirectly, i.e. addition of NADP+ increases the concentration of Ca2+ required for half-maximal stimulation. 6. These results are interpreted as indicating the existence of separate allosteric binding sites for Ca2+ and 2′-AMP. Furthermore, Ca2+ is proposed to act as a heterotropic ligand. The results are discussed in terms of current allosteric models. [ABSTRACT FROM AUTHOR]

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421-505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C (4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the substituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavo-proteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides. [ABSTRACT FROM AUTHOR]

The change in fluorescence emission at 520 nm after excitation at 365 nm was used to investigate the effect of pH and ionic strength on the dissociation of flavin cofactors from microsomal NADPH/cytochrome c (P-450) reductase. In the unmodified enzyme both the FAD and FMN moieties appeared to dissociate at a similar rate and followed first-order kinetics. The rate constant for the dissociation was increased by low pH and high ionic strength, particularly in the range pH 4.4-3.8 (0.02 M acetate buffer) where the rate constants increased 80-fold. Modification of the enzyme by treatment with p-chloromercuribenzoate enhanced the rate of flavin dissociation and, in the region of pH 4, resulted in a biphasic increase in fluorescence consistent with two simultaneous parallel first-order dissociations. It was concluded that p-chloromercuribenzoate treatment modified the protein so that the two flavin cofactor dissociated at different rates. Using the measured rate constants for the dissociations, and the known variation in fluorescence of flavin nucleotides with pH, and analogue computer simulation of the dissociation as well as manual curve-fitting procedure showed that the biphasic response could be explained as a simultaneous rapid dissociation of FAD and a slower loss of FMN from the protein. [ABSTRACT FROM AUTHOR]

A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenii. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine:[This symbol cannot be presented in ASCII format]Proof of this structure has been obtained by chemical synthesis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methyl-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (ε = 41 000 M&Sup-1; cm⊃-1;) and a pK at 4.8 due to the ionisation of the C(8)-OH group. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form. [ABSTRACT FROM AUTHOR]

Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. The enzyme has a molecular weight of about 200000 and is a hornopolymeric assemblage of either three or four subunits of molecular weight 54000–58000. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixed-function oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocyclohexane (5-valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2-lactouizing) (EC 1.14.13.—). Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p-hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent. The enzyme has a pH optimum of 7.7. The Km, for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits. Anaerobic reduction of enzyme-bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments. Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated. [ABSTRACT FROM AUTHOR]

The photoreaction of flavoquinones (lumiflavin, riboflavin, FMN etc. and their 3-alkylated derivatives) with propargylamine-type acetylenic substrates, R4-Cα≡ Cβ-Cγ~HR3-NR2R1, yields a mixture of two adducts, which result from covalent Cα fixation of the Cγ-deprotonated substrate to either position C(4a) or N(5) in the flavin nucleus. The N(5) adduct is a dihydroflavin-5-trimethine-cyanine with very intense (ε > 20000 M-1 cm-1) absorption maxima in the region 380–450 nm depending on the R1, R2. This absorption allows recognition of minute amounts of this species of flavocyanine even in complex mixtures. Flavocyanines can be reconverted to starting flavin by base. Its spectral properties are identical with those obtained for the pargyline-flavin inhibitor complex from bovine kidney or pig liver monoamine oxidase. The C(4a) adduct is a 4a:5-propenylidene-dihydroflavin, the cyclisation probably arising through secondary attack of the Cγ carbon at N(5). It cannot be reconverted to starting flavin in relevant yields. Its chromophor (λmax ≈ 360 nm) is identical with that of lactate-oxidase inhibitor complex. If N(3) is alkylated, these cyclic adducts undergo cleavage of the 4—4a bond upon treatment with base, followed by aromatization of the substrate substituent to yield novel pyrroloquinoxal-4-ones. Upon treatment with LiAIH4, however, the adducts are hydrogenated at the N(1)=C(10a) double bond, which by concomitant base hydrolysis yields 1,2-dihydroquinoxaline derivatives. Their autoxidation yields a pyrrolo-quinoxalinium cation which exhibits spectral identity with the product isolated from the α-hydroxybutynoate-flavin inhibitor complex of Mycobacterium smegmatis lactate oxidase, after borohydride treatment. The scope and limitations of covalent adduct formation in flavin-dependent bioeatalysis will be discussed. [ABSTRACT FROM AUTHOR]

The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr-activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin-free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively. Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (± 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N-10 side-chain of 5 carbon atoms and a C-5' phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12-15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months. [ABSTRACT FROM AUTHOR]

The presence of higher (>0.2 M) concentrations of divalent anions A2- (hydrogenphosphate, sulfate) is found to accelerate as well as to change entirely the course of riboflavin photolysis: instead of 10-dealkylation to yield lumichrome, intramolecular addition of the 2'-hydroxyl group is found to occur at the peri-position C(9). The reaction is analogous to the "photohydration" of the flavin nucleus in the cationic state as described by Schöllnhammer and Hemmerich [Eur. J. Biochem. (1974) 44, 561-577]. The final product of the new addition reaction arises from autoxidation of dihydroflavin intermediate and exhibits the structure ... It is thus representative for the new class of flavins ("cyclo-dehydrofalvins"). Earlier reports on "anomalous" flavin photodegradation products absorbing around 410 nm [Holmström (1964) Ark. Kem. 22, 281; Massey and Atherton (1962) J. Biol. Chem. 237, 2965] are readily explained. The reaction is found to depend strictly on the presence of a nucleophilic function in the N(10)-side chain, e.g. N(10)-CH2-C(OH)RR' or even N(10)-(CH2)2-SO3-. Quenching experiments suggest that the new reaction occurs via the singlet state ¹Flox*, while the normal photolysis is mediated by the triplit ³ Flox*. The new photoaddition is thought to occur via a Flavin-A2- complex which creates sterically favorable conditions for C(9)/O(2'α)-interaction. [ABSTRACT FROM AUTHOR]

Simulation studies by digital computer were undertaken in order to test and clarify the interpretations deduced from experimental data concerning the electron transfer mechanism from L-lactate to flavocytochrome b2, which were presented in a preceding paper in this journal. The reaction scheme proposed as the ‘best’ one is composed of 7 steps. It allows the best fitting of the time courses established for the oxidized ravin (Flox), the ravin semiquinone (Flsq), the fully reduced ravin (Flred), and the reduced haem (Hred); it can be extended to 1 s. This scheme also allows a good simulation of the general shape of preequilibrium titration curves obtained at a 200-ms reaction time for Hred and Flsq, and a valuable simulation of the reduction electron paramagnetic resonance time course established for Hred and Flsq at low lactate concentration. The agreement between experimental and simulated curves led to an estimation of some rate constants experimentally unknown, relative (in particular) to the electron exchange between ravin and haem and between couples of flavins. Another interest of these stimulation studies was to point out the obligatory involvement of a slow final step to perform the flavocytochrome b2 full reduction; this step could be controlled by some conformational change of the protein. [ABSTRACT FROM AUTHOR]

Extensively or completely activated preparations of beef heart succinate dehydrogenase have been investigated by electron paramagnetic resonance (EPR) techniques at 6 to 97 K. Reductive titrations with dithionite and rapid kinetic studies were performed with various types of soluble and membrane-bound preparations of the enzyme. The following components were detected and their behavior analyzed: a free radical, presumably arising from the covalently bound flavin on reduction, two iron-sulfur centers of the ferredoxin type, the signals of which appear on reduction, and a high-potential iron-sulfur component, detectable in the oxidized state. The high-potential component was only detected in complex II and inner-membrane preparations. This component and one of the ferredoxin­type centers were present in amounts close to stoichiometric with the flavin and were reduced by substrate. The other ferredoxin­type center was present in amounts between 0.1 and 0.5 times that of the flavin and was reduced only by dithionite. Of the components reduced by succinate, however, only a fraction (up to 50% of the high-potential iron-sulfur center and 40-60% of the ferredoxin­type iron-sulfur center) was reduced within the turnover time of the enzymes, in complex II not more than about 10% of the flavin appeared in the semiquinone form at any time. Soluble, purified preparations behaved similarly except that the high-potential component was nearly or completely absent and extensive accumulation of the free radical occurred (up to 70 to 80% of the flavin) in titration and kinetic experiments. No significant difference was observed between the rates of semiquinone formation and the reduction of the ferredoxin­type or high-potential centers by the substrate. Also no qualitative differences in the properties studied in this work became apparent between preparations containing 4 or 8 iron atoms, respectively. [ABSTRACT FROM AUTHOR]

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single-photon decay spectroscopy. When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. The phenomenon was observed at 4°C (τ1 = 0.8 ns, τ2 = 4.7 ns) and at 20°C (τ1 = 0.8 ns, `τ2 = 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers. By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods, show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm. [ABSTRACT FROM AUTHOR]

1. Flavin-nicotinamide biscoenzymes covalently linked by two, three or four methylene groups through positions N(1) of the flavin (Fl) and N(1) of the nicotinamide (Nic) form long-wave- length-absorbing, intramolecular complexes when the flavin part of the molecule is reduced specifically. The energy of the long-wavelength transition is minimal and its intensity maximal for Nicox+-(CH2)3-FlredH- (&lambdamax= 600nm; ϵ= 1200 M-1 × cm-1). 2. The increasing proximity of the positively charged nicotinamide lowers the pK-value of dihydroflavin deprotonation up to 1.7 units and the flavin oxidation-reduction potential becomes more positive up to 116 mV. 3. Specific reduction of the nicotinamide part of the biscoenzymes yields transient, long-wave- length-absorbing complexes. The energy of the long-wavelength transition is minimal and its intensity maximal for the complex NicredH-(CH2)3-Flox (λmax = 575 nm; ϵ = 650 M-1 × cm-1). 4. The rate of intramolecular flavin-dependent dihydronicotinamide dehydrogenation is highest for NicredH-(CH2)3-Flox (345 s-1),about 3 times slower for NicredH-(CH2)4-Flox and 100 times slower for NicredH-(CH2)2-Flox 5. The results obtained in this study are consistent with a reaction mechanism that involves formation of a charge transfer complex between reduced nicotinamide and oxidized flavin and rate-limiting heterolytic breakdown into products. [ABSTRACT FROM AUTHOR]

The previous paper in this series described the isolation of tryptic-chymotryptic and peptic flavin peptides from Chromatium cytochrome c552 in homogeneous form and presented evidence that the FAD component is covalently linked to a cysteinyl residue via a hemiacetal linkage to the 8α position of the isoalloxazine ring. The amino acid sequence of the peptic and tryptic-chymotryptic flavin peptides were found to be Tyr-Thr-Cys(FAD)-Tyr and Thr-Cys(FAD)-Tyr. respectively. However, the presence of the NH2-tyrosyl residue in the former, imparts markedly greater stability to the cysteinyl flavin linkage, and even results in essentially quenched fluorescence of the flavin after performic acid oxidation of the sulfur moiety. Osidatlon at 40°C, which destroys tyrosine, results in the same level of fluorescence as found when the tryptic-chymotryptic peptide is oxidized at 0 C. Removal of this tyrosyl residue from the peptic peptide, oxidized at 0°C, by aminopeptidase M also leads to an increase in fluorescence. Evidence for a tyrosyl-flavin interaction has been further obtained from CD spectra. The peptic peptide has a broad, positive Cotton effect with a maximum at 484 to 490 nm, which is enhanced after treatment with pyrophosphatase and phosphatase and negative bands at 305 and 375 nm. The tryptic-chymotryptic peptide, on the other hand. has a positive band at 340 nm, as m FAD. but only a small negative band at 380 nm. Thus the tyrosyl-flavin interaction appears to be responsible for the stabilization of the thiohemiacetal bond in the peptide and perhaps also in the protein. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [ABSTRACT FROM AUTHOR]

The behaviour of bakers' yeast L-lactate dehydrogenase toward [2-²H]lactate, 3-chlorolactate and 2-hydroxy-3-butynoate is reported. By comparison with known facts concerning D-amino acid and lactic oxidases, the following conclusions are drawn. 1. Hydrogen removal at C-2 is rate-limiting in the overall reaction. This distinguishes L-lactate dehydrogenase from the oxidases where it is product dissociation which limits the rate. 2. The lack of dehydrohalogenation of 3-chlorolactate by L-lactate dehydrogenase, which differentiates the enzyme from the oxidases, can be explained if the reduction rate of ravin is much faster than for the oxidases. 3. Although only indirect evidence is presented, it seems most probable that the lactate C-2 hydrogen is removed as a proton, as in the case of D-amino acid oxidase and lactic oxidase. 4. The first steps of lactate oxidation are qualitatively similar for the two known enzymatically active forms of L-lactate dehydrogenase. [ABSTRACT FROM AUTHOR]

1. New flavins have been isolated from purified prepartions of an electron-transferring flavo-protein (ETF) from Peptostreptococcus elsdenii and glycolate oxidase from pig liver. The structures of these new species have been established as FAD and FMN derivatives of 6-hydroxy-7,8-dimethyl-isoalloxazine, the chemical synthesis of which is described in tlhe accompanying paper by Schöllnhammer and Hemmerich. The chromophores are yellow at pH 5 and green at pH 9 due to an ionization at pK 7.1. 2. 6-OH-FAD is bound by apo-ETF and its pK is decreased. The complex is reduced by NADH and it couples the oxidation of NADH to the reduction of dichlorophenolindophenol. Unlike the complex of FAD and apo-ETF, 6-OH-FAD · ETF does not couple the oxidation of NADH to the reduction of butyryl-CoA dehydrogenase. 3. 6-OH-FMN is bound by the FMN-specific protein apoflavodoxin from P. elsdenii and the pK is increased to ≈9. This complex is reduced by sodium dithionite and an intermediate, presumed to be the semi-quinone, is formed at half rduction. [ABSTRACT FROM AUTHOR]

1. The method of "photohydroxylation" of azaheteroaromatics, known in the phenazine and a alloxazine series, has been applied to flavin (= isoalloxazine) derivatives FI[SUBox]H. The products habe been identified as 6- and 9-hydroxyflavins (Fl[SUBox]-6/9-OH). On this basis it was possible to assign a 6-hydroxyflavin structure to the new flavocoenzyme chromophores described by Mayhew et al. in the accompanying paper. 2. In agreement with the data obtained by Dekker et al. in a recent laser study on alloxazine, we find that, the reaction proceeds via, addition of water, alcohols or carboxylic acids by the lowest excited singlet state of flavin with synchronous intramolecular hydrogen shift from the site of RO-addition [= C(6) or C(9)] towards N(5) and subsequent oxidation of the 6/9-hydroxy1.5-dihydroflavin formed. 3. The photoaddition reaction occurs in competition with photodehydrogenation of available CH-bonds by the lowest excited flavin triplet, in which case the CH-substrate may be provided by the solvent ROH or, when R = H, by methyl groups of a second flavin molecule (= flavin autophotolysis). This photodehydrogenation may be suppressed by O[SUB2]-dependent triplet quenching and/or by applying a positive charge to the flavin (RFl[SUBox]H[SUP+], R = H or alkyl) in order to inhibit the reaction with itself. 4. Differentiation of 6- and 9-hydroxyflavin isomers was possible by double resonance evaluation of the proton coupling between the residual Ar-H and the adjacent methyl group, by evaluation of the electron paramagnetic resonance hyperfine coupling for the residual Ar-H in the radical cations 1,5-RHFl-6/9-OH, and by Formation of cupric chelates from 6-hydroxy-flavin as compared to the lack of metal affinity in the 9-isomer. [SUP1]H-Nuclear magnetic resonance, electron paramagnetic resonance and visible spectra and their dependence on the state of ionisation of hydroxyflavins are discussed. Hydroxyflavin anions as well as their N(1)-alkylated... [ABSTRACT FROM AUTHOR]

1. Initial velocity studies on β-cyclopiazonate oxidocyclase in the presence of the competitive inhibitor, 3-acetyl-5-scatyl-tetramic acid, indicates that this enzyme catalyzes the oxidocyclization of β-cyclopiazonic acid by a Ping-Pong-Bi-Bi mechanism. 2. It was possible to reduce the flavin coenzyme to the 3,4-dihydroflavin by consecutive cycles of reduction with BH4- and reoxidation by atmospheric oxygen. 8. The BH4-reduced enzyme shows a similar affinity for indole derivatives as does the native flavoquinone enzyme, but is inactive. β-Cyclopiazonic acid binds to the enzyme in a simple equilibrium, and the reduction of the 3,4-dihydroflavin to the 1,3,4,5.tetrahydroflavin or a conversion to 1,5-dihydroflavin does not proceed. 4. Partial reactivation of the enzyme by the photocatalyzed oxidation of the 3,4-dihydroflavin to the flavoquinone form was observed [ABSTRACT FROM AUTHOR]

This study describes the regioselective hydroxylation and the rates of conversion of a series of fluorinated phenol derivatives by phenol hydroxylase from the yeast Trichosporon cutaneum. The natural logarithm of the kcat value for the conversion of the phenolic substrates correlates with the calculated energy of the reactive electrons in the highest occupied molecular orbital of the substrate (r = 0.85). This observation supports the hypothesis that at physiological pH (7.6) and 25°C, in the absence of monovalent anions, the nucleophilic attack of the electrons in the highest occupied molecular orbital of the substrate on the C(4a)-hydroperoxyflavin enzyme intermediate is of major importance in determining the overall rate of catalysis. Results from 19F-NMR analysis of the incubation mixtures demonstrate for phenols with two identical ortho substituents, that the ortho position which becomes preferentially hydroxylated is the one with the highest density of the reactive electrons in the highest occupied molecular orbital. A halogen substituent at a meta position decreases the chances for hydroxylation at the adjacent ortho position further khan expected on the basis of the calculated reactivity. This result indicates a contribution of a protein/substrate dipolar interaction, influencing the time-averaged orientation of the substrate with respect to the reactive C(4a)-hydroperoxyflavin intermediate. [ABSTRACT FROM AUTHOR]

A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl-CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851–4858)]. The initial reaction is catalyzed by benzoate-CoA ligase (AMP-forming), converting benzoate into benzoyl-CoA. The next step is 3-hydroxylation of benzoyl-CoA to 3-hydroxybenzoyl-CoA catalyzed by a flavin-nucleotide-dependent monooxygenase, benzoyl-CoA 3-monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl-CoA, but not benzoate, is hydroxylated. The protein is a monomer of Mr 65000 and is induced when cells are grown aerobically with benzoate. 3-Hydroxybenzoyl-CoA is further hydroxylated para to the hydroxyl group affording 2,5-dihydroxybenzoate (gentisate). This reaction requires another monooxygenase, 3-hydroxybenzoyl-CoA 6-monooxygenase, which is unspecific with respect to the pyridine nucleotide. Cells contain a second 6-monooxygenase activity which acts on free 3-hydroxybenzoate. Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced. The proposed intermediates are benzoyl-CoA, 3-hydroxybenzoyl-CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate. [ABSTRACT FROM AUTHOR]

Reduction of the ravin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state. This intermediate had an optical spectrum characteristic of a ravin anionic semiquinone. The rate and extent of reduction were pH-dependent. No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant. No intermediate radical was detected during the reoxidation process. Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state. The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M-1 cm-1. The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004. The linewidth of the signal was 1.48 mT when the protein was studied in H2O or D2O. These data are in agreement with those reported for anionic semiquinones. The linewidths were the same when measured either at X-band or at S-band frequencies, indicating that line broadening is due to hyperfine interactions. The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added. Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the ravin radical with protons. A group of signals, with couplings of 912 MHz, is attributed to protons on 8-CH3 (Aiso = 10.9 MHz) and on C6 (Aiso = 9 MHz) of the ravin ting. No change in these hyperfine coupling constants was detected when the protein was studied in D2O. However, the hyperfine coupling constant attributed to protons on 8-CH3 decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (Aiso = 9.92 MHz). A second group of signals was observed with hyperfine couplings less than 2.5 MHz, Some of these weak couplings disappeared when the protein was transferred to D2O, or when the substrate, dehydroisoandrosterone, was present. These signals are attributed to displaced water protons, or to exchangeable protons from amino-acid residues on the protein near the ravin binding site, involved in substrate stabilisation. [ABSTRACT FROM AUTHOR]

The time-resolved fluorescence characteristics of flavin in oxidized flavodoxin isolated from the anaerobic bacterium Clostridium beijerinckii have been examined. The fluorescence intensity decays were analyzed using the maximum-entropy method. It is demonstrated that there exist large differences in fluorescence behaviour between free and protein-bound FMN. Three fluorescence lifetime components are found in oxidized flavodoxin, two of which are not present in the fluorescence-intensity decay of free FMN. The main component is distributed at 30 ps. with relative contribution of 90%. Another minor component (4% contribution) is distributed at 0.5 ns. The third component is distributed at 4.8 ns (6%), coinciding with the main distribution present in the fluorescence decay of free FMN. The results allowed us to determine the dissociation constant. Kd = 2.61 × 10 10M (at 20°C). Collisional fluorescence-quenching experiments revealed that the flavin moiety responsible for the longest fluorescence lifetime is, at least partially, exposed to the solvent. The shortest lifetime is not affected significantly, indicating that it possibly originates from an active-site conformation in which the flavin is more or less buried in the protein and not accessible to iodide. The fluorescence anisotropy behaviour of free and protein-bound FMN was examined and analyzed with the maximum-entropy method. It was found that an excess of apoflavodoxin is required to detect differences between free and protein-bound FMN. In free FMN one single distribution of rotational correlation times is detected, whereas in flavodoxin the anisotropy decay is composed of more than one distribution. Associative analysis of fluorescence anisotropy decays shows that part of the 4.8 ns fluorescence lifetime present in the flavodoxin fluorescence decay, is coupled to a rotational correlation time similar to that of free FMN in solution, while another part of this lifetime is coupled to a longer correlation time of about, 1 ns. This finding is in accordance with earlier studies [Barman, B. G. & Tollin, G. (1972) Biochemistry II. 4746–4754] in which it was proposed that the first binding step of the flavin to the protein involves the phosphate group rather than another part of the FMN. The two shortest fluorescence lifetimes, which do not carry information on the long-term rotational behaviour of the protein, seem nonetheless to be associated with a longer rotational correlation time which is comparable to overall protein tumbling. These lifetime components probably originate from a complex in which the flayin-ring system is more or less immobilized within the protein matrix. [ABSTRACT FROM AUTHOR]

The enzymatic properties and the three-dimensional structure of spinach glycolate oxidase which has the active-site Tyr129 replaced by Phe (Y129F glycolate oxidase) has been studied. The structure of the μtant is unperturbed which facilitates interpretation of the biochemical data. Y129F glycolate oxidase has an absorbance spectrum with maxima at 364 and 450 nm (ε[submax] = 11400 M[sup-1] cm[sup-1]). The spectrum indicates that the flavin is in its normal protonated form, i.e. the Y129F μtant does not lower the pKa of the N(3) of oxidized flavin as does the wild-type enzyme (Macheroux, P., Massey, V., Thiele, D. J., and Volokita, M. (1991) Biochemistry 30, 4612-4619). This was confirmed by a pH titration of Y129F glycolate oxidase which showed that the pKa is above pH 9. In contrast to wild-type glycolate oxidase, oxalate does not perturb the absorbance spectrum of Y129F glycolate oxidase. Moreover oxalate does not inhibit the enzymatic activity of the μtant enzyme. Typical features of wild-type glycolate oxidase that are related to a positively charged lysine side chain near the flavin N(1)-C(2 = O), such as stabilization of the anionic flavin semiquinone and formation of tight N(5)-sulfite adducts, are all conserved in the Y129F μtant protein. Y129F glycolate oxidase exhibited about 3.5% of the wild-type activity. The lower turnover number for the μtant of 0.74 s[sup-1] versus 20 s[sup-1] for the wild-type enzyme amounts to an increase of the energy of the transition state of about 7.8 kJ/mol. Steady-state analysis gave K[subm] values of 1.5 mM and 7 microM for glycolate and oxygen, respectively. The K[subm] for glycolate is slightly higher than that found for wild-type glycolate oxidase (1 mM) whereas the K[subm] for oxygen is μch lower. As was the case for wild-type glycolate oxidase, reduction was found to be the rate-limiting step in catalysis, with a rate of 0.63 s[sup-1]. [ABSTRACT FROM AUTHOR]

Iron is an essential element in all living cells. Solubilization, uptake and transport of iron by microorganisms is controlled by highly efficient and specific Fe[sup 3+]-chelating agents named siderophores. However, mechanisms of mobilization of iron from ferrisiderophores are still enigmatic. Here, we demonstrate that Escherichia coli contains a powerful enzymatic system for the reduction of ferrisiderophores. Siderophores have a much lower affinity for ferrous iron, which then can be liberated. This system has been previously purified and characterized as a NAD(P)H:flavin oxidoreductase [Fontecave, M., Eliasson, R. and Reichard, P. (1987) J. Biol. Chem. 262, 12325–12331)], It catalyzes the reduction of free flavins, FMN, FAD or riboflavin by NADH or NADPH. Reduced flavins, in turn, transfer their electrons to physiological ferric complexes: ferrisiderophores, ferric citrate and ferritins. The reaction is inhibited by molecular oxygen and greatly stimulated by Fe[sup 2+]-acceptors such as ferrozine or the iron-free form of ribonucleotide reductase subunit R2. We suggest that the reduction and the mobilization of iron from ferrisiderophores in the cell might be regulated by the presence of physiological ferrous traps such as apoproteins. [ABSTRACT FROM AUTHOR]

Electron-transferring flavoprotein (ETF) was purified from the bacterium Paracoccus denitrificans and the structural and redox relationships to the porcine and human ETFs were investigated. The three proteins have essentially identical subunit masses and the a-helix content of the bacterial and porcine ETFs are very similar, indicating global structural similarity. An anti-(porcine ETF) polyclonal antibody that crossreacts with the human large and small subunits also crossreacts strongly with the large subunit of Paracoccus ETF. However, crossreactivity with the small subunit is very weak. Nonetheless, an amino-terminal peptide and four internal peptides of the small bacterial subunit show extensive sequence identity with the human small subunit. Local similarities in environment are also indicated by the intrinsic tryptophan fluorescence emission spectra of porcine and Paracoccus ETFs. Although the visible spectra of porcine and Paracoccus ETFs are virtually identical, flavin fluorescence in the bacterial protein is only 15% that of the mammalian protein. Further, the circular dichroic spectrum of the flavin in the bacterial protein is significantly more intense, suggesting that the microenvironment of the isoalloxazine ring is different in the two proteins. Enzymatic or photochemical reduction of Paracoccus ETF rapidly yields an anionic semiquinone; formation of the fully reduced flavin in the bacterial ETF is very slow. The spacing of the oxidation-reduction potentials of the flavin couples in the bacterial ETF is essentially identical to that in porcine ETF as judged from the disproportionation equilibrium of the bacterial ETF flavin semiquinone. Together, the enzymatic reduction and disproportionation equilibria suggest that the flavin potentials of the two ETFs must be very close. The data indicate that the structural properties of the bacterial and mammalian proteins and the thermodynamic properties of the flavin prosthetic group of the proteins are very similar. [ABSTRACT FROM AUTHOR]