Volume 27, Issue 9

The effects of experimental geometry on the theoretical polarizations of the S 1 ← S 0 and S 2 ← S 0 bands of π→π* type in riboflavin have been examined. Polarizations of these two bands are characterized by an angle between them in the range of 20—28° and are relatively insensitive to the input geometry. Thus the predicted polarizations are generally in agreement with fluorescence polarization spectrum of riboflavin at 77°K. Alloxazine forms a strong complex with KI, and the fluorescence and phosphorescence from the charge transfer states have been characterized by means of luminescence and photoselection measurements. Riboflavin did not form a strong complex with KI, but it forms aggregates (dimer) more readily than alloxazine. The excited states of flavins can be populated by the weak dipole-dipole coupling mechanism of energy transfer from 1 L a states of indoles to the S 2 state of flavins. The measured critical distances estimated from the fluorescence depolarization experiments range from 31 Å for indole to 40 A for indole-2-carboxylic acid in glycerol-methanol mixture (9:1) at 263°K.

A. Equilibrium and second-order rate constants have been measured for the redox reaction between phthaloquinone and dihydroriboflavin (and its reverse) at a number of pH values. The equilibria and kinetic determinations are in agreement. No intermediates could be found in the reaction, and it is proposed that with this and other quinones electron transfer occurs within a complex of the flavin and hydroquinone (or dihydroflavin and quinone). B. A survey of the reaction between mercaptans and flavins is reported, as well as data showing that with mercaptoethanol the reaction is reversible, the disulfide and reduced flavin giving mercaptan and flavin. C. The reaction of alkylphenone enolates with isoalloxazine is described. This reaction leads t 50% isoalloxazine radical anion and an adduct of the enolate with the ketone. A possible route for this process is discussed.

3-methyllumiflavin, irradiated in the first flavin band, forms a N-5 adduct in pyridine solutions. The possible nature of this adduct is discussed, together with the effect of water on the course of the photoreduction reaction.

The interaction of flavinium salts with triphenylphosphine is described. The complexes which can be isolated in high yields in the crystalline state contain one mole triphenylphosphine per mole flavin. Triphenylphosphine is covalently bound to the N (5)-atom of the isoalloxazine ring system. Dissociation constants and rate constants have been determined spectrophotometrically. Evidence for the structure of the complex was obtained from light absorption, infrared and nuclear magnetic resonance studies. The similarities between the flavin-triphenylphosphine adduct and the earlier described flavin-sulfite adduct are described and the possible biological relevance of these complexes is discussed.

A slow and reversible hydration of N (1) -unsubstituted alloxazines in aqueous alkaline solutions has been studied. Reaction products stable (kinetically) in neutral and acidic solutions were isolated and investigated by IR, NMR, UV and mass spectroscopy. The possible mechanism of the reaction and the structure of the isolated products is discussed.

By photochemical attack on flavin cations, compounds substituted in the benzenoid subnucleus have been prepared. Water and alcohols can be added in this way and under certain conditions the alcoholic groups of the flavin side chain are involved. Structures and properties of photoproducts are described.

The photoreactions of the flavin triplet with unsaturated hydrocarbons, aldehydes, sulphur compounds, and carbonic acids are reported. These reactions proceed by attack at the allylic C-H-, the RCO-H-, or the α-C-H-bond, respectively; in the case of carbonic acids decarboxylation occurs. In all the reported reactions covalent adducts are formed, whose structure and mode of decay is characterized.

The pH-dependence of flavin-sensitized photodehydrogenation and oxidative photodecarboxylation shows a change of reaction rate with an apparent pK of about 5.5. This “photo-pK” cannot be assigned to any known ground state or excited flavin or substrate species, nor is it related to the kind of bond being broken (C-H or C-COO-). Therefore, it is attributed to the pK of a covalent flavin-substrate intermediate. Carboxylates R-COO~ react by decarboxylation, the nature of R determining only the rate of reaction. The acids R -COOH themselves are unreactive, unless functionally substituted at the α-carbon. Hence X-CH(R)-COOH behaves as does X-CH 2 -R, for X=OH, OR, NH 2 , NR 2 . R 3 N + - CH(R) -COO- is unreactive, while H 3 N + -CH(R)-COO-is slowly dehydrogenated through its neutral tautomer which is favored in the flavin-substrate exciplex.

The reduction of aldehydes by flavohydroquinone to yield the corresponding alcohols and flavoquinone is reported and discussed in view of its relevance to the mechanism of flavin-dependent dehydrogenation and carbonyl activation. With formaldehyde and flavohydroquinone formation of a covalent 5- (HO - CH 2 ) Fl red⁻ intermediate species is observed. In a reverse reaction 5-methylflavoquinonium cation undergoes an internal redox reaction to yield flavohydroquinone and formaldehyde.

The formation of a species with an absorption maximum around 410 nm, observed during the neutral photolysis of 10-(2'-hydroxyalkyl) flavins by earlier authors, is shown to depend on the presence of divalent anions. The structure of this new type of compounds is presented and a possible mechanism for the reaction is discussed.

The CD-spectra of riboflavin and riboflavin analogues in aqueous solutions differ very little depending on pH and ionic strength, but are extremely sensitive upon solvent changes. The two bands in the region of 300 —500 nm seen in aqueous solutions are split into seven bands in less polar solvents, which can be assigned to seven vibronic transitions. The spectra may be interpreted by “through space” and “through chain” interactions of the sidechain centers of chirality with the flavin chromophore, which influence the two first π —π* transitions in different manner.

A detailed magnetic resonance investigation of the metal ion interaction with flavoquinones has been carried out with the purpose of obtaining the stoichiometry, stability and (sub) molecular structure of the complexes. The isotropic shifts of the paramagnetic chelates are analysed in terms of contact and pseudocontact contribution. It is shown that the flavoquinone-metal binding is mainly of σ-type regardless of the nature of the ion.

4a,5-Cycloaddition at the flavoquinone, Mechanism of flavoprotein inhibitions Photochemically, covalent cycloaddition products of flavoquinone and acetylenic enzyme inhibitors have been prepared and elucidated, which appear analogous to flavoprotein-inhibitor complexes (monoamine and lactate oxidase). The additions occur with inhibitors of general type HC≡C-CH< at the C (4a) = N (5) azomethine grouping of oxidized flavin. The spectral properties of adducts and their chemical reactivity is described.

Nitroalkanes have been found to be general reductive substrates for D-amino acid oxidase, glucose oxidase and L-amino acid oxidase. These enzymes show different specificities for the structure of the nitroalkane substrate. The stoichiometry of the D-amino acid oxidase reaction is straightforward, consisting of the production of one mole each of aldehyde, nitrite and hydrogen peroxide for each mole of nitroalkane and oxygen consumed. The stoichiometry of the glucose oxidase reaction is more complex in that less than one mole of hydrogen peroxide and nitrite is produced and nitrate and traces of 1-dinitroalkane are formed. The kinetics of nitroalkane oxidation show that the nitroalkane anion is much more reactive in reducing the flavin than is the neutral substrate. The pH dependence of flavin reduction strongly suggests that proton abstraction is a necessary event in catalysis. A detailed kinetic mechanism is presented for the oxidation of nitroethane by glucose. It has been possible to trap a form of modified flavin in the reaction of D-amino acid oxidase with nitromethane from which oxidized FAD can be regenerated in aqueous solution in the presence of oxygen.

We describe anaerobic stopped-flow monitored interactions of D-B-chloroalanine with D-amino acid oxidase and show that the kinetics of absorbance changes at 550 nm due to enzyme-bound intermediates can not be correlated with steady state turnover behavior unless it is assumed that only a small fraction of the enzyme directly participates in the α-β elimination process.

The spectral changes of the enzyme-bound FAD were investigated in the anaerobic and aerobic reactions of lysine monooxygenase, a flavoprotein. Under anaerobic conditions, the enzyme FAD was fully reduced with lysine via a transient intermediate having a broad absorption band in the long wavelength region. This anaerobic species was presumably involved in the dehydrogenation of lysine leading to the formation of an α-keto acid. During the steady state of the aerobic reaction, a separate spectral species with a long wavelength absorption was observed. Both lysine and oxygen were required for its appearance. Kinetic evidence supports the intermediacy of this species in the oxygenation of lysine to an acid amide.

Maleimide spin label was covalently bound to sulfhydryl residues of ᴅ-amino acid oxidase and lipoamide dehydrogenase. Labeling of native ᴅ-amino acid oxidase resulted in a non-homogeneous EPR-spectrum, which consisted of 4 moles of spin label bound immobile to the enzyme (mol.-weight 90.000). A detailed analysis of the spectrum, the kinetics of the reaction of the spin label with the protein and the temperature dependence of the spectrum showed that the spectrum originates from two different pairs of sulfhydryl groups. The spin labeled lipoamide dehydrogenase yielded also a mixed EPR-spectrum of two different bound spin labels, i.e. an immobile and a semimobile species. The temperature dependence gave for both types of spectra a transition point at 10°C. Titration with urea gave only for the immobile species a transition at 1.5 M. Part of the semimobile species seems to be bound near the active site. ᴅ-amino acid oxidase was also specifically labeled, near the active site, with a substrate analogue. From its EPR-spectrum it appeared that the analogue was bound very mobile (τ c =0.3 nsec) with respect to the protein. Removal of FAD had a drastic reversible effect on the spectrum.

Lipoamide dehydrogenase (EC 1.6.4.3) has been succesfully linked to a CNBr-activated polysaccharide matrix, Sepharose-4B, under different reaction conditions. The enzyme is probably bound more homogeneously at lower pH values (pH 7.5) than at pH 8.5. Such immobilized preparations yield V values 8-30% of the value of the V of the free enzyme (18,600 mole/min/mole of flavin). A low level of CNBr-activation in combination with substrate protection and a pH of 7.5 during the coupling reaction leads to the most active preparations. The K m values for both substrates increase considerably. The overall kinetic pattern of the matrix-bound enzyme preparations is not different from that of the free enzyme. Both activation by high concentration of lip (SH) 2 NH 2 at low NAD + levels and a stimulation of the V by increased phosphate concentration in the assay buffer is observed with free and some immobilized enzymes.

4-hydroxybenzoate hydroxylase from Ps. putida has been studied with regard to the oxygen interaction with the reduced flavin. By rapid scanning spectrophotometry and EPR-measurements of rapidly frozen mixtures no intermediate could be established either in the presence of 4-hydroxybenzoate or the dead-end inhibitor 6-hydroxynicotinic acid.

Following elucidation of the structures of the flavin components of succinate dehydrogenase (SD) as N (3) -histidyl-8α-FAD and of monoamine oxidase (MAO) as cysteinyl-8α-FAD and determination of the peptide sequences around the flavin sites of these enzymes, attention has been focused on the covalently bound FAD of Chromatium cytochrome c-552. As documented in preliminary communications, the FAD moiety of this enzyme is also substituted at the 8α-position, as judged from ESR hyderfine structure of the free radical cation and the characteristic hypsochromic shift of the second absorption band of the neutral flavoquinone in purified preparations of the flavin. Definite proof has come from the liberation of 8-carbxyriboflavin on performic acid treatment of the enzyme. In regard to ESR and optical spectra and the tendency of the purified flavin (liberated by proteolysis) to undergo autooxidation with a further hypsochromic shift of the second absorption band and increased fluorescence, the flavin resembles the MAO flavin. The fact that fluorescence is >90% quenched at all pH values even at the FMN level and doees not vary with pH between 3.2 and 8 also suggests a thioether linkage as in cysteinyl riboflavin. In many respects, however, the Chromatium flavin differs from cysteinyl riboflavin. Highly purified preparations from tryptic-chymotryptic digests give a positive chloroplatinic test. Electrophoresis clearly shows the presence of carboxyl and amino groups but the peptide gives no characteristic ninhydrin reaction and amino acid analysis of performic acid oxidized samples yields cysteic acid and threonine in amounts less than equimolar to the flavin. The amino acid environment around the flavin may account for these results although a linkage other than a thioether remains a possibility.

D- and ʟ-hydroxynicotine oxidase were obtained in homogeneous form and compared with regard to molecular weight, subunit structure, reaction mechanism, and specificity. The most striking differences between these enantiozymes are the type of coenzyme binding and the reactivity towards artificial two-electron acceptors.

D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95°C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH-dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin layer chromatography it was identical with 8α-(N-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125°C liberated 1 mole of histidine per mole of flavin as shown by amino acid analysis. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8a- (N-3-histidyl) -flavin-adenine-dinucleotide in the active center.

The enzyme was anaerobically isolated and was characterized as a flavoprotein containing at least 1 FMN, 5-8 Fe and 7-8 moles labile sulfide per mole (M. W. appr. 300.000). It catalyzes the oxidation of formate by NAD, O 2 (forming H 2 O 2 ) and dyes and the oxidation of NADH by O 2 (forming Η 2 Ο 2 ) and dyes. It is irreversibly inhibited by formate.

Ubiquinone-free succinate dehydrogenase may be activated by reduction by NADH (in the presence of an enzyme catalysing the transfer of reducing equivalents to succinate dehydrogenase) or Na 2 S 2 O 4 .

Catalysis by lipoamide dehydrogenase involves the concerted action of the flavin and a cystine residue. Peptides containing this cystine residue have been previously isolated from E. coli and now from pig heart. The sequences of amino acid residues reveal a high degree of homology indicating a strict conservation of the region around the active site cystine during the long evolutionary period between these two species. The peptide sequences suggest a likely conformation of the polypeptide chain in the region of the flavin as well as the forces involved in substrate and flavin binding.

Further studies of butyryl CoA dehydrogenase show that the enzyme forms tight complexes with long-wavelength absorption bands upon addition of α, β-unsaturated alicyclic carboxylic acyl CoA compounds. As in the straight chain series, the size of the acyl moiety is critical. The thiol portion by contrast determines neither whether a long-wavelength band is formed nor the wavelength of maximum absorption if such a band is present. It does however markedly affect the dissociation constant. Unusual features of the acid stability of the acyl CoA found on the native green form of the enzyme are also reported.

NADH dehydrogenases have been isolated in a low-molecular weight form (56,500) from both low-iron and normal-iron grown cells of Azotobacter vinelandii. Unlike low-molecular weight forms of NADH dehydrogenases reported from mammalian mitchondria, the enzymes in this study have not undergone any unusual alterations in various reactivities with substrates and electron acceptors. The NADH dehydrogenases from A. vinelandii do differ in cofactor cotent, light-absorption spectra and electron paramagnetic resonance spectra but show similar catalytic-center activities with substrates and electron acceptors. The relationship of these NADH dehydrogenases to site I oxidative phosphorylation in both the bacterial and mammalian systems is discussed.

The roles of enzyme-bound FAD and non-heme iron in the mechanism of adenylyl sulfate reductase have been investigated by inhibitor studies, stopped-flow techniques and EPR spectroscopy. The results indicate that the non-heme iron found in the purified reductase is catalytically active and that the turnover number of the enzyme-bound FAD is identical with the maximum turnover number for the enzyme.

E. coli NADPH-sulfite reductase, depleted of FMN but retaining its FAD, has been prepared by photoirradiation of native enzyme in 30% — saturated ammonium sulfate. FMN-depleted enzyme loses its ability to reduce (using NADPH) ferricyanide, cytochrome c, sulfite, or the enzyme’s own heme-like chromophore. However, the FAD remains rapidly reducible by NADPH, and the FMN-depleted enzyme retains NADPH-acetylpyridine NADP* transhydrogenase activity. Thus, FAD can serve as entry port for NADPH electrons, and FMN is required for further transmission along the enzyme’s electron transport chain. These data, plus other studies, have enabled us to suggest a mechanism for catalysis which involves FAD cycling between the fully-oxidized and fully-reduced forms while FMN cycles between fully-reduced and semiquinone. This mechanism, which includes a disproportionation step, permits a “step-down” from the twoelectron donor, NADPH, to a succession of equipotential one-electron transfer steps.

Pyruvate is shown to be a substrate for the reduction of flavodoxin from Azotobacter vinelandii, using the phosphoroclastic enzyme system from Clostridium pasteurianum. In this reaction, the reduction of this flavoprotein proceeds beyond the semiquinone state partly to the hydroquinone form. Pyruvate is possibly the substrate for the reduction of flavodoxin in the intact organisms.

Crystals of oxidized Desulfovibrio vulgaris flavodoxin have been studied by X-ray diffraction techniques. The structure has been determined at 2.5 A resolution. The molecule consists of a pleated sheet core with two sections of helix on each side of the sheet. The FMN group is distinguishable: it is mostly burried but 7- and 8-Methyl groups appear to be at the surface of the protein.

The amino acid sequences of 10 tryptic peptides from C. pasteurianum flavodoxin are determined and ordered. The sequence of two thirds of the protein is presented. New electrophoretic techniques for sequence determination are also presented.

Circadian rhythm, oxygen consumption, rat liver, suspension culture, bimodal pattern