Biomedical subjects

D E Edmondson

Publications and source records attributed to D E Edmondson.

At least 37 records · Page 2Linked to original sources

PubMed1998

Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2.

Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormö, M., Nordlund, P., & Sjöberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

Ascorbic Acid↗

PubMed1997

Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization.

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

Animals↗

PubMed1996

Purification and characterization of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans.

Xanthine dehydrogenase (XDH) is induced in Comamonas acidovorans cells incubated in a limited medium with hypoxanthine as the only carbon and nitrogen source. The enzyme has been purified to homogeneity using standard techniques and characterized. It contains two subunits with M(r) values of 90 and 60 kDa. Gel filtration studies show the enzyme to have an alpha 2 beta 2 native structure. No precursor form of the enzyme is observed on Western blot analysis of cell extracts obtained at various stages of enzyme induction. Metal analysis of the purified enzyme shows 1.1 Mo, 4.0 Fe, and 3.6 phosphorus atoms per alpha beta protomer. Cofactor analysis shows the enzyme to contain a single molybdopterin mononucleotide and one FAD per alpha beta protomer. Electron spin resonance and circular dichroism spectral studies of the oxidized and reduced forms of the enzyme suggest the Fe centers to be two nonidentical [2Fe-2S] clusters. Electron spin resonance signals due to Mo(V) and neutral FAD radical are also observed in the reduced form of the enzyme. Purified enzyme preparations ranged from 70% to 100% functionality. The enzyme is irreversibly inactivated by CN- and is inhibited on incubation with allopurinol. With xanthine and NAD+ as substrates the enzyme has a specific activity of 50 units/mg, a kcat value of 120 s-1, an activity/flavin ratio of 1930, and respective Km values of 66 and 160 mM. Using 8-D-xanthine as substrate, a DV value of 1.8 is found with no change in Km. Thus, the Km and KD values of the enzyme for xanthine are equal. These data show Comamonas XDH to exhibit structural properties similar to bovine milk xanthine oxidase/dehydrogenase and to chicken liver xanthine dehydrogenase. Although the bacterial enzyme exhibits a 6-7-fold greater turnover rate than bovine or avian enzymes, the catalytic efficiencies (as measured by V/K) are similar for all three enzymes.

Animals↗

PubMed1996

Sarcosine oxidase contains a novel covalently bound FMN.

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric protein containing three different enzymes: noncovalent FAD, noncovalent NAD+, and covalently bound flavin which is released as 8 alpha-(N3-histidyl)riboflavin upon complete hydrolysis of the protein. The following results show that the covalent flavin is not at the FAD level, as previously proposed, but it is rather as 8 alpha-(N3- histidyl)FMN coenzyme. First, no AMP is released when the protein moiety is treated with phosphodiesterase or subjected to mild acid hydrolysis. The enzyme contains a total of 5 mol of phosphate. Only one phosphate is covalently bound. The other four phosphates are noncovalent and attributed to noncovalently bound FAD and NAD+. The 31P NMR spectrum of native enzyme exhibits resonances due to a single phosphate monoester an two pyrophosphates. Only a resonance due to phosphate monoester is observed after removal of the noncovalent cofactors and proteolytic digestion of the protein moiety. The 8 alpha-(N3-histidyl)FMN found in corynebacterial sarcosine oxidase represents a novel type of covalent flavin. Studies with sarcosine oxidases from Arthrobacter sp. and Pseudomonas sp. show that these heterotetrameric enzymes also contain covalently bound FMN plus noncovalently bound FAD and NAD+, similar to corynebacterial sarcosine oxidase. In contrast, two monomeric sarcosine oxidases (from Bacillus sp. and an unidentified microorganism) were found to contain only covalently bound FAD.

Adenosine Monophosphate↗

PubMed1995

Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase.

The SF-Abs, RFQ-EPR, and RFQ-Möss data on the R2 reconstitution reaction are all consistent with the mechanism of Scheme I, in which the intermediate X is the immediate precursor to the product cofactor, and illustrate how the continuous SF approach and the discontinuous RFQ methods can be complementary. Given the inherent differences in the methods, it should not be taken for granted that data from the two will be consistent. A number of problems can be associated with the RFQ approach. For example, isopentane could conceivably interfere with or alter the chemistry to be studied. A second potential problem involves temperature-dependent equilibria among different intermediate species. This problem has been encountered by Dooley et al. with the 6-hydroxydopa-requiring protein, plasma amine oxidase and was previously observed with the adenosylcobalamin-dependent ribonucleotide reductase by Blakley and co-workers. This potential complication should be considered when discrepancies arise between SF and RFQ data and in low temperature structural studies of reactive intermediates in general. Each of the three methods employed can yield time-resolved quantitation of reaction components. In this regard, SF-Abs has the disadvantage of poor resolution, such that quantitation of individual components most often requires sophisticated mathematical analysis. Obvious advantages to the RFQ-Möss method are the presence of an internal standard (the known amount of 57Fe being proportional to the total absorption area) and the spectroscopic activity of all reaction components which contain iron. In our hands, quantitation by RFQ-EPR was most problematic and least reproducible. This irreproducibility most likely relates to heterogeneity among samples in terms of volume and density. As discussed in detail by Ballou and Palmer, the packing factor, which relates to the fraction of a sample made up by the reaction solution (the remainder being frozen isopentane), is dependent on the investigator. Given this caveat, it is not surprising that the RFQ-EPR data had the greatest uncertainty in our hands. Placing a chemically unreactive, EPR active standard in each reaction mixture could help alleviate this problem. Time-resolved Möss methods can be extremely powerful if excellent, nonoverlapping reference spectra of starting materials, products, and intermediates are available. All of the iron centers can be examined simultaneously. The problems associated with Möss arise from its extreme insensitivity. It takes millimolar solutions of proteins and several days for data collection of each time point.(ABSTRACT TRUNCATED AT 400 WORDS)

Coenzymes↗

PubMed1995

Structure activity studies of the substrate binding site in monoamine oxidase B.

The influence of para and meta substitution of benzylamine analogues on their interaction with bovine liver monoamine oxidase B has been investigated to provide insights into the nature of the substrate binding site. Binding data with para-substituted benzylamine analogues show the area of the binding site about the para position to be hydrophobic and exhibiting some steric constraints. Alkylation of the benzylamine nitrogen with methyl groups results in a dominance of steric constraints about the para-position as an influence on binding. meta-Substitution of the benzylamine ring results in a decreased binding affinity which exhibits a dependence on the van der Waals volume of the substituent indicating steric constraints also occur about this area of the bound substrate. The independence of the rate of enzyme reduction with the nature of the meta-substituent suggests these benzylamine analogues are bound in the substrate site in a manner which optimizes overlap of the pro-R benzyl C-H bond with the lone pair orbital on the nitrogen. In contrast, the observed rates of enzyme reduction by para-substituted benzylamine analogues exhibit a dominant steric dependence which suggests the mode of binding of this class of analogues does not provide this optimal overlap for efficient C-H bond cleavage. Support for this conclusion also comes from the observation that para-substituted N,N-dimethylbenzylamine analogues are competitive inhibitors and not substrates for monoamine oxidase B while the meta-substituted analogues are substrates, albeit poor ones. The demonstration of a tunneling contribution to the C-H bond cleavage step demonstrates the absence of any motion or changes in solvation coupled with that catalytic event and the close proximity of the enzyme group accepting the H to the pro-R position of the bound substrate. Little or no influence of meta or para benzylamine substituent on the rate of O2 reaction with the reduced flavin-protonated imine complex is observed which suggests alterations in the configuration of the bound substrate do not influence the reactivity of the reduced flavin.

Animals↗

PubMed1995

Aminium cation radical mechanism proposed for monoamine oxidase B catalysis: are there alternatives?

1. The interaction of bovine liver mitochondrial monoamine oxidase B (MAO B) with a series of benzylamine analogues was investigated to provide mechanistic information relative to the proposed cation radical mechanism and to provide information on the structural requirements of the substrate binding site. 2. Steady-state kinetic analysis of MAO B with 11 ring-substituted benzylamine analogues showed substitution does not alter the reaction pathway. All amine analogues tested exhibit sizeable deuterium kinetic isotope effects. 3. Anaerobic stopped-flow kinetic studies showed (1) C-H bond cleavage is rate-limiting in enzyme-bound flavin reduction and (2) that no specially detectable flavin radicals are observed. 4. The binding affinity of para-substituted benzylamine analogues to MAO B increased as the hydrophobicity of the substituent increased. In contrast, meta-substitution of the ring showed reduced affinity with an increase in the van der Waals volume of the substituent. 5. The rate of enzyme reduction by para-substitution exhibited a strong negative dependence with the Taft (Es) steric value of the substituent. In contrast, the rate of enzyme reduction by meta-substituted benzylamines is independent of the nature of the substituent. 6. para-Substituted N,N-dimethylbenzylamine analogues are not substrates for MAO B but are competitive inhibitors of benzylamine oxidation with a weaker affinity with increasing van der Waals volume of the substituent. In contrast, meta-substituted N,N-dimethyl benzylamine analogues are weak substrates for MAO B with oxidation occurring exclusively at the benzyl carbon. 7. The consequences of these results on the possible mechanisms (aminium cation radical, H abstraction, and nucleophilic mechanism) for C-H bond cleavage proposed for MAO B are discussed.

Amines↗

PubMed1994

Hydrogen tunneling in the flavoenzyme monoamine oxidase B.

Competitive kH/kT and kD/kT kinetic isotope effects on p-methoxybenzylamine oxidation by the 8 alpha-S-cysteinyl flavin adenine dinucleotide (FAD)-dependent enzyme monoamine oxidase B (MAO-B) have been measured as a function of temperature. At pH 7.5, exponents relating observed kH/kT and kD/kT isotope effects indicate the presence of a temperature-dependent change in rate-limiting step. At lower temperature (e.g., 2 degrees C), the presence of multiple rate-limiting steps (commitments) is clearly indicated from the size of the exponent and individual isotope effects. Noncompetitive kH/kD isotope effect measurements indicate a trend in observed isotope effects between pH 9.0 and 6.0, with isotope effects increasing at lower pH. Primary and secondary kH/kT and kD/kT isotope effects were therefore measured as a function of temperature at pH 6.1. Exponents relating primary and secondary kH/kT and kD/kT in the 10-43 degrees C range are 3.13 +/- 0.04 and 2.36 +/- 0.13, respectively, and do not systematically change with temperature. These data indicate that commitments, if present, remain constant across this temperature range. The temperature dependence of the observed primary isotope effects gives values for the ratios of Arrhenius prefactors of 0.13 +/- 0.03 (AH/AT) and 0.52 +/- 0.05 (AD/AT). Both values are well below the lower limits predicted in the absence of tunneling contributions to the reaction coordinate, indicating that both deuterium and protium tunneling take place in this reaction. The presence of a temperature independent commitment contribution cannot be rigorously ruled out; however, the effect of such a commitment on the observed AH/AT and AD/AT values is shown to be quite small.

Animals↗

PubMed1994

Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.

Vitamin B2-aldehyde-forming enzyme from Schizophyllum commune catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have monitored enzyme activity by spectrophotometrically measuring the reduction of 2,6-dichlorophenol-indolphenol as electron acceptor to assess 35 riboflavin analogs as potential substrates or competitive inhibitors with the purpose of delimiting structural requirements of the substrate binding site. Analogs with side chains of two- to six-carbon length modified by deletion of secondary hydroxyls or by changes in their epimeric configuration are not oxidized. The omega-hydroxyalkyl-flavins (n = 2-6) are competitive inhibitors (Ki = 7-16 microM) of riboflavin oxidation, as are some analogs with L-secondary hydroxyls in the side chain. Analogs with bulky substituents on the isoalloxazine ring are also not substrates. The enzyme does not significantly bind flavins with an 8 alpha-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and beta-hydroxyethylamino at position 2. Also the replacement of N with CH in 1-deazariboflavin disallows substrate reaction. Analogs with fluoro, chloro, methyl, amino, or methylamino at position 8; chloro at 7; methyl or carboxylmethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relative Vmax values ranging from 27 to 110% that of riboflavin. The Km values for the analogs oxidized are all found to be in the micromolar range (22-176 microM). Overall specificity of the enzyme for riboflavin is found to be rather narrow and sterically limited, which suggests that the vitamin is the natural substrate.

Alcohol Oxidoreductases↗

PubMed1994

Kinetic studies on the electron-transfer reaction between cytochrome c3 and flavodoxin from Desulfovibrio vulgaris strain Hildenborough.

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c3 and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c3 with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c3 are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s-1 and respective Kd values of 65.9 +/- 9.4 microM and 54.5 +/- 13 microM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 x 10(5) and 8.5 x 10(4) M-1 s-1 are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e(-)-reduced cytochrome c3 reacts with an approximately 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 +/- 1.4 s-1 and a Kd value of approximately 8 microM. Increasing the ionic strength of the pH 6.0 solution to 100 microM KCl results in a biphasic reaction with relative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M-1 s-1. Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome c3 in a slow, monophasic manner with limiting rate of electron transfer of 1.2 +/- 0.06 s-1 and a Kd value of 80.9 +/- 10.7 microM. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].

Azotobacter vinelandii↗

PubMed1994

Structure-activity relationships in the oxidation of benzylamine analogues by bovine liver mitochondrial monoamine oxidase B.

The influence of para and meta substitution of benzylamine on its interaction with bovine liver mitochondrial monoamine oxidase B (MAO B) has been investigated by steady-state and reductive half-reaction anaerobic stopped-flow kinetic approaches. Steady-state kinetic properties of each benzylamine analogue suggest that para or meta substitution does not alter the mechanistic pathway of catalysis [Husain, M., et al. (1982) Biochemistry 21, 595-600]. All analogues tested exhibited Dkcat values ranging from 5.5 to 8.9 and D[kcat/Km(amine)] values ranging from 3.3 to 8.1 D[kcat/Km(O2)] values of approximately 1 are observed for all substrate analogues. Values for Kd were calculated from steady-state isotope effect data [Klinman, J.P., & Matthews, R.G. (1985) J. Am. Chem. Soc. 107, 1058-1060] and are in good agreement with Ks values determined from analysis of the rate of MAO B reduction as a function of benzylamine analogue concentration in reductive half-reaction experiments. A linear correlation of benzylamine analogue Kd values with the hydrophobicity parameter (phi) is observed for the para-substituted analogues where the binding affinity increases with increasing hydrophobicity of the substituent. Statistical treatment of the correlation shows a small negative contribution to binding by the van der Waals volume (VW) of the para substituent. meta-Substituted benzylamine analogues show a decreased binding affinity with the VW of the substituent and no correlation with the hydrophobicity value of the substituents tested. No spectral evidence was found for any flavin radical intermediates during the time course of MAO B flavin reduction in anaerobic reductive half-reduction stopped-flow experiments with any of the alpha,alpha-diprotio- or alpha,alpha-dideuteriobenzylamine analogues tested. The limiting rates of enzyme reduction exhibit large Dk values (6.5-14.1) for all of the analogues tested. para-Substituted benzylamine analogues reduce MAO B with limiting rates that correlate with the steric influence (Es value) of the substituent. Statistical analysis shows the rate of MAO B reduction by para-substituted analogues to be retarded by increased values of Es and, with a smaller contribution, by the hydrophobicity value of the substituent. The rate of MAO B reduction by meta-substituted benzylamine analogues is essentially independent of the nature of the substituent. No evidence was found for any electronic contribution to the rate of MAO B flavin reduction by any of the analogues tested. These data demonstrate the steric orientation of the substrate to be important in the rate of amine oxidation by MAO B and that ring meta substituents favor this orientation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Studies on the induction and phosphorylation of xanthine dehydrogenase in cultured chick embryo hepatocytes.

Chick embryo hepatocytes, cultured in a chemically defined medium, were used to investigate hormonal requirements for xanthine-dehydrogenase induction and to determine whether the enzyme is phosphorylated. Triiodothyronine is found to be required to induce the synthesis of active enzyme. Inclusion of sodium tungstate in the medium resulted in the complete loss of enzyme activity but no decrease of immunochemically detectable levels of enzyme. Immunoprecipitated xanthine dehydrogenase from cell extracts migrates with enzyme purified from adult chicken liver on SDS/PAGE. Both the native 150-kDa subunit and the 130-kDa form of the enzyme is observed. N-terminal sequence analysis of the 150-kDa subunit shows the following; Ala-Pro-Pro-Glu-Thr-Gly-Asp-Glu-Leu-Val-Phe-Phe-Val-Asn-Gly-Lys-Lys-Val- Val which is similar to the published N-terminal sequences of rat, mouse and insect xanthine dehydrogenases. Autoradiography of denaturing gels of xanthine dehydrogenase isolated from 32P(i)-labeled hepatocytes demonstrates that the 150-kDa and the 130-kDa forms of the enzyme are phosphorylated. Chemical phosphate analysis of acid-precipitated, electrophoretically pure chicken liver xanthine dehydrogenase also shows the presence of covalently bound phosphate. Phosphoamino acid analysis of both 32P-labeled forms of the enzyme demonstrates the presence of phosphoserine. Thus, chicken liver xanthine dehydrogenase contains a phosphoserine residue as found previously in bovine milk xanthine oxidase [Davis, M. D., Edmondson, D. E. & Müller, F. (1984) Eur. J. Biochem. 145, 237-250].

Amino Acid Sequence↗

PubMed1993

Spectral and kinetic studies of imine product formation in the oxidation of p-(N,N-dimethylamino)benzylamine analogues by monoamine oxidase B.

The oxidative deamination of p-(N,N-dimethylamino)benzylamine and N-methyl-p-(N,N-dimethylamino)benzylamine by bovine liver monoamine oxidase B has been investigated by absorption spectral, steady-state, and stopped-flow kinetic studies. An absorbing intermediate with a maximum at 390 nm is observed with either analogue in turnover experiments at neutral pH and is identified as due to the formation of protonated imine as the initial product. p-(N,N-Dimethylamino)benzaldehyde is the final product formed from either substrate analogue. Anaerobic stopped-flow measurements show N-methyl-p-(N,N-dimethylamino)benzylamine to reduce enzyme-bound flavin with a limiting rate of 1.8 s-1 concurrent with the appearance of a 390-nm absorption due to protonated imine product with a limiting rate of 1.7 s-1. Both observed rates are somewhat faster than catalytic turnover (1.5 s-1). Under anaerobic conditions, the decay of protonated N-methyl-p-(N,N-dimethylamino)benzenimine is much slower than turnover (k = 4.8 x 10(4) s-1). p-(N,N-Dimethylamino)benzylamine reduces the enzyme with a limiting rate of 2.1 s-1, which is faster than catalytic turnover (1.2 s-1). Protonated imine formation is also observed with this substrate with an apparent limiting rate of 1.3 s-1. The decay of the protonated p-(N,N-dimethylamino)benzenimine absorbance is slower than catalytic turnover but faster than the rate of aldehyde formation under anaerobic conditions. Deuterium kinetic isotope effect values of approximately 10 are observed both for flavin reduction and for protonated imine formation. No isotope effect is observed for the rate of imine decay.(ABSTRACT TRUNCATED AT 250 WORDS)

Aniline Compounds↗

PubMed1993

Resonance Raman spectroscopic evidence for an anionic flavin semiquinone in bovine liver monoamine oxidase.

The flavoprotein monoamine oxidase B (MAO B) from bovine liver, as isolated, has an unusual additional absorption band at 412 nm, which is similar to the absorption of its anionic flavin semiquinone form, (Fl.-), and other typical (Fl.-) flavoproteins. Denaturation of the enzyme results in the elimination of this anomalous absorption. The resonance Raman (RR) spectrum of MAO B as isolated is virtually identical to that of its dithionite-reduced (Fl.-) form. Both spectra show features similar to those of the RR spectrum of the (Fl.-) form of Aspergillus niger glucose oxidase (GO) in the region between 300 and 1700 cm-1 with 406.7 nm excitation. These features are readily distinguishable from those of oxidized flavin, neutral flavin semiquinone, and hemoprotein, strongly suggesting the presence of an (Fl.-) form in MAO B as isolated, even with preparations isolated in the absence of light. There are significant differences between the RR spectra of the (Fl.-) form of MAO B and those of GO or the published RR spectra of the (Fl.-) form of D-amino acid oxidase with excess substrate analog. At least some of these differences can be attributed to the different binding of flavin in the three enzymes. No EPR signals due to (Fl.-) are observed in MAO B as isolated. The dithionite-reduced (Fl.-) form exhibits approximately 50% less EPR signal than that expected from the absorption spectrum, which suggests a possible coupling of the (Fl.-) flavin with a paramagnetic center of unknown identity in the protein. The implications of these observations on MAO B with the current view of its catalytic mechanism are discussed.

Animals↗

PubMed1992

Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.

Diglycerides↗

PubMed1992

Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies.

Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.

Azospirillum brasilense↗

PubMed1992

Riboflavin 5'-pyrophosphate: a contaminant of commercial FAD, a coenzyme for FAD-dependent oxidases, and an inhibitor of FAD synthetase.

Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5'-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apoprotein of porcine D-amino-acid oxidase, values determined for RPP were 8.4 microM for Km and 0.10 for Vmax compared to 0.47 microM and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 nM for Km and 0.02 for Vmax and 45 nM and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a Ki value for RPP against FMN was determined to be 9 microM where Km for FMN was 5.5 microM. These studies illustrate the value of riboflavin 5'-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.

Animals↗

PubMed1992

Effect of KCl on the interactions between NADPH:cytochrome P-450 reductase and either cytochrome c, cytochrome b5 or cytochrome P-450 in octyl glucoside micelles.

Significant dissociation of FMN from NADPH:cytochrome P-450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P-450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN-depleted enzyme with added FMN. The reductions of cytochrome c and detergent-solubilized cytochrome b5 by NADPH:cytochrome P-450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P-450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high-salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non-physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret-band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P-450 reductase with either cytochrome b5 or cytochrome P-450. It is suggested that the high-salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic-hydrophobic interactions rather than electrostatic-charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P-450 reductase with the cytochrome.

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