Biomedical subjects

J Vervoort

Publications and source records attributed to J Vervoort.

At least 73 records · Page 4Linked to original sources

PubMed1994

A spectrophotometric assay for the detection of 2-aminophenols in biological samples.

A sensitive and efficient spectrophotometric assay is described for the determination of 2-aminophenols in biological samples. Using ferric ions as a metal catalyst, 2-aminophenol dimerizes in an acidic aqueous environment to 2-hydroxyisophenoxazin-3-one, an intensively colored dye. The newly developed assay is suitable for the detection of 2-aminophenols in the micromolar range. The paper demonstrates that this chemical assay is also applicable for the determination of 2-aminophenols substituted with a halogen at the ortho, meta, or para position of the aromatic ring with respect to the amino moiety, lambda max, and the molar extinction coefficient varying with the substituent pattern. Results obtained for fluorinated 2-aminophenol detection in biological samples by either this new method or 19F NMR are similar. This observation corroborates that the newly developed assay is suitable for detection of 2-aminophenols in biological samples.

Aminophenols↗

PubMed1994

Flavodoxins.

Explore the source record for details and available documents.

Amino Acid Sequence↗

PubMed1993

A specific interaction between NADPH-cytochrome reductase and phosphatidylserine and phosphatidylinositol.

In the present study the interaction of NADPH-cytochrome reductase with phospholipids was investigated using 31P-NMR, thin-layer chromatography combined with chemical analysis, fluorescence spectroscopy and kinetic studies with purified rat liver cytochrome P450 IIB1. 31P-NMR analysis demonstrates that the composition of the phospholipids that remain associated to NADPH-cytochrome reductase upon its purification is significantly different from the phospholipid composition of the microsomal membrane. Thin-layer chromatography followed by chemical analysis of the phospholipid composition demonstrates that the isolated NADPH-cytochrome reductase was enriched in L-alpha-1,2-diacyl-sn-glycero-3-phosphoserine (acyl2GroPSer) and L-alpha-1,2-diacyl-sn-glycero-3-phosphoinositol (acyl2GroPIns) compared to the microsomal membrane. The observed preference of NADPH-cytochrome reductase for acyl2GroPSer and acyl2GroPIns appeared not to be a result of the procedure for solubilisation and/or purification of the protein. The specific interaction of NADPH-cytochrome reductase with acyl2GroPSer and acyl2GroPIns was further investigated by comparison of the effect of acyl2GroPSer and acyl2GroPIns with that of acyl2GroPCho and acyl2GroPEtn on the 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3- phosphocholine-(DphPamGroPCho)-dependent quenching of the tryptophan fluorescence of purified NADPH-cytochrome reductase. The results demonstrate that the addition of acyl2GroPSer or acyl2GroPIns affects the DphPamGroPCho-dependent quenching of the tryptophan fluorescence in a manner significantly different from the addition of acyl2GroPCho or acyl2GroPEtn. The relatively larger DphPamGroPCho-induced quenching of the tryptophan fluorescence of NADPH-cytochrome reductase in the presence of acyl2GroPSer and acyl2GroPIns must result from a change in the conformation of NADPH-cytochrome reductase induced by the latter two lipids. Finally, the possible consequences of this special interaction of acyl2GroPSer and acyl2GroPIns with NADPH-cytochrome reductase on the kinetic characteristics of the cytochrome P450 system were studied using cytochrome-P450-IIB1-dependent O-dealkylation of pentoxyresorufin as the model reaction. These studies demonstrate that a 1:1 mixture of acyl2GroPCho and acyl2GroPSer results in a significantly higher apparent maximum rate (V) of O-dealkylation than a 1:1 mixture of acyl2GroPCho and acyl2ProPEtn or acyl2GroPCho alone. This increase in the apparent V can be ascribed to an acyl2GroPSer-dependent improvement of the interaction of NADPH-cytochrome reductase with cytochrome P450. This improvement of the interaction of the proteins cannot, however, be exclusively ascribed to the negative charge of acyl2GroPSer, since the other negatively charged phospholipid investigated, namely acyl2GroPIns, resulted in a significant decrease in the apparent V.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

19F-NMR study on the pH-dependent regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon cutaneum. Implications for the reaction mechanism.

The regioselectivity and rate of the ortho-hydroxylation of 3-fluorophenol by phenol hydroxylase from Trichosporon cutaneum (EC 1.14.13.7) was studied using 19F-NMR. The regioselective hydroxylation as well as the rate of ortho-hydroxylation are pH dependent with a pKa of 6.5. At pH values below 6.5, 3-fluorophenol preferentially becomes hydroxylated at the C6 ortho position, resulting in a maximum C6/C2 hydroxylation ratio of 6.7. Upon increasing the pH, the total rate of conversion increases. Also, the C2 ortho-hydroxylation increases relatively to the C6 ortho-hydroxylation and yields a minimum C6/C2 hydroxylation ratio of 2.2 at pH values above 7.5. Based on data from 19F-NMR binding studies and molecular orbital calculations, a hypothesis is put forward which explains the pH-dependent effects observed. A mechanism is proposed involving an active-site amino acid residue acting as a base in the reduced form of the protein. Deprotonation of this residue results in hydrogen bond formation with the hydroxyl moiety of the phenolic substrate, leading to (partial) deprotonation of the substrate. Molecular orbital calculations demonstrate that such a (partial) deprotonation increases (a) the overall reactivity of 3-fluorophenol for an electrophilic attack and (b) the reactivity of C2 relative to the C6 position. The hypothesis may explain the decrease in the C6/C2 hydroxylation ratio. Furthermore the increased amount of ortho-hydroxylated products formed with increasing pH can also be explained by this hypothesis.

Fluorine↗

PubMed1993

Regioselectivity of cytochrome P-450 catalyzed hydroxylation of fluorobenzenes predicted by calculated frontier orbital substrate characteristics.

In the present study, a hypothesis is presented for the prediction of the regioselectivity of cytochrome P-450 catalyzed hydroxylation of fluorobenzenes. The regioselectivity of the in vivo hydroxylation of fluorobenzene, 1,2-difluorobenzene, 1,3-difluorobenzene, 1,2,3-triluorobenzene, and 1,2,4-triflurobenzene could be predicted within 6% accuracy on the basis of the substrate's frontier orbital characteristics for electrophilic attack. The in vivo regioselectivity of the hydroxylation of fluorobenzene was not significantly influenced by changes in the cytochrome P-450 enzyme pattern. This implies that the regioselectivity is not predominantly determined by the juxtaposition of the relatively small substrates in the active sites of the cytochrome P-450s catalyzing the reaction. Additional in vitro experiments using 1,2-difluorobenzene as the model substrate demonstrated that minor factors influencing the regioselectivity and possibly responsible for the 6% deviation from the calculated values in in vivo experiments might be (i) the influence of biotransformation routes occurring in vivo but not of importance in in vitro microsomal incubations and (ii) a small variation due to influences of the contribution of various cytochrome P-450 enzymes. On the basis of the results obtained, it is concluded that the aromatic hydroxylation of fluorobenzenes proceeds through an initial electrophilic attack of (FeO)3+ on the aromatic substrate, and not through initial electron abstraction followed by attack of the (FeO)2+ species on the substrate radical cation. The fact that the regioselectivity observed could be predicted and/or explained by the site of initial (FeO)3+ attack also argues against epoxides as important intermediates in the formation of phenol metabolites from fluorobenzenes.

Animals↗

PubMed1992

Frontier orbital study on the 4-hydroxybenzoate-3-hydroxylase-dependent activity with benzoate derivatives.

Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4-hydroxybenzoate-3-hydroxylase (EC 1.14.13.2). A clear correlation between in kcat for the conversion of a series of 4-hydroxylated substrates and their E(HOMO) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore, the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)-peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25 degrees C. Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild-type 4-hydroxybenzoate-3-hydroxylase, its mutants as well as of various other flavin-dependent aromatic hydroxylases, such as for example 3-hydroxybenzoate-4-hydroxylase (EC 1.14.13.23), 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7).

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1992

Study on the regioselectivity and mechanism of the aromatic hydroxylation of monofluoroanilines.

The in vitro and in vivo metabolism of monofluoroanilines was investigated. Special attention was focused on the regioselectivity of the aromatic hydroxylation by cytochromes P-450 and the mechanism by which this reaction might proceed. The results clearly demonstrate that the in vitro and in vivo regioselectivity of the aromatic hydroxylation by cytochromes P-450 is dependent on the fluoro-substituent pattern of the aromatic aniline-ring. Results from experiments with liver microsomes from differently pretreated rats demonstrate that the observed regioselectivity for the aromatic hydroxylation is not predominantly determined by the active site of the cytochromes P-450. To investigate the underlying reason for the observed regioselectivity, semi-empirical molecular orbital calculations were performed. Outcomes of these calculations show that neither the frontier orbital densities of the LUMO/LUMO + 1 (lowest unoccupied molecular orbital) of the monofluoroanilines nor the spin-densities in their NH. radicals can explain the observed regioselectivities. The frontier orbital densities of the HOMO/HOMO - 1 (highest occupied molecular orbital) of the monofluoroanilines however, qualitatively correlate with the regioselectivity of the aromatic hydroxylation. Based on these results it is concluded that the cytochrome P-450 dependent aromatic hydroxylation of monofluoroanilines does not proceed by hydrogen or electron abstraction from the aniline substrate to give an aniline-NH. radical. The results rather suggest that cytochrome P-450 catalyzed aromatic hydroxylation of monofluoroanilines proceeds by an electrophilic attack of the (FeO)3+ species of cytochrome P-450 on a specific carbon atom of the aromatic aniline-ring.

Aniline Compounds↗

PubMed1992

Rat liver microsomal metabolism of 2-halogenated 4-methylanilines.

1. Rat liver microsomal metabolism of 2-fluoro-, 2-chloro- and 2-bromo-4-methylaniline was investigated using h.p.l.c. Metabolites identified include products from side-chain C-hydroxylation (benzyl alcohols and benzaldehydes) and N-hydroxylation (hydroxylamines and nitroso derivatives). Aromatic ring hydroxylation was not a major reaction pathway. 2. A new type of microsomal metabolite was detected which was identified as a secondary amine, i.e. a halogenated N-(4'-aminobenzyl)-4-methylaniline. 3. In addition to these products azoxy, azo and hydrazo derivatives were formed. 4. Benzyl alcohols and halogenated N-(4'-aminobenzyl)-4-methylanilines were the major microsomal metabolites for all three 2-halogenated 4-methylanilines. 5. Quantification of the metabolite patterns demonstrated an influence of the type of halogen substituent on the rate of microsomal metabolism. The rate of side-chain C-hydroxylation increases in the order 2-fluoro-4-methylaniline < 2-chloro-4-methylaniline < 2-bromo-4-methylaniline. 6. The rate of N-hydroxylation increases from 2-bromo-4-methylaniline < 2-fluoro-4-methylaniline < 2-chloro-4-methylaniline. That 2-chloro-4-methylaniline is N-hydroxylated to a larger extent is in accordance with its greater mutagenicity, twice that of 2-bromo-4-methylaniline.

Animals↗

PubMed1991

NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida.

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed1991

Biotransformation of 2-fluoroaniline in rats studied by in vivo 19F NMR.

The present study describes results from an in vivo 19F NMR study on rats exposed to the xenobiotic compound 2-fluoroaniline. Qualitative pharmacokinetics and the biotransformation of 2-fluoroaniline were studied after exposure to 50 mg/kg body wt 2-fluoroaniline. Accumulation and elimination of the parent compound in and from the liver of exposed animals were readily observed. Metabolites formed in the liver were shown to be efficiently excreted from the liver, as the amount of metabolites in this organ was always less than 10% of the maximum amount of the parent compound observed. In the bladder, rapid accumulation of 2-fluoroaniline-derived metabolites was detected. The metabolite pattern was shown to change in time, with the parent compound being dominantly present during the first hours of exposure. N-acetylated products (4-acetamido-3-fluorophenyl sulphate and 4-acetamido-3-fluorophenyl glucuronide) were observed to accumulate more slowly in the bladder than the non-acetylated products (4-amino-3-fluorophenyl sulphate and 4-amino-3-fluorophenyl glucuronide). Urine metabolite patterns obtained from the bladder in vivo were compared to those obtained during metabolic cage experiments.

Administration, Oral↗

PubMed1991

Bioactivation of 4-fluorinated anilines to benzoquinoneimines as primary reaction products.

Metabolism and bioactivation of fluoroanilines was studied both in vitro in microsomal systems and in vivo. 4-Fluoroaniline and pentafluoroaniline and their non-para fluorinated analogues were used as the model compounds. Special attention was focussed on bioactivation to reactive benzoquinoneimines. Cytochrome P-450 dependent monooxygenation at a non-fluorinated para position in (fluoro)aniline derivatives proceeds by formation of the para hydroxylated derivative as the primary metabolite. Monooxygenation at a fluorinated para position in an aniline derivative, however, proceeds by formation of fluoride anion and the reactive benzoquinoneimine as primary reaction products. Thus, for fluoroanilines with a fluorine substituent at the para position bioactivation to the reactive benzoquinoneimine can be a direct result of the cytochrome P-450 dependent conversion. In systems containing NAD(P)H and/or other reducing equivalents part of the benzoquinoneimine can be chemically reduced to give the corresponding 4-hydroxyaniline. In vivo this reduced form of the metabolite can be sulphated or glucuronidated and excreted into urine. The results obtained point to increased chances of bioactivation for aniline derivatives with a fluorinated para position as compared to their non-para fluorinated analogues, both in vitro but also in vivo.

Aniline Compounds↗

PubMed1990

Reaction pathways for biodehalogenation of fluorinated anilines.

Pathways for biodehalogenation of fluorinated aniline derivatives were investigated. Microsomal NADPH-dependent dehalogenation of fluoroanilines was shown to proceed by three different reaction pathways. The first route appeared to result in monooxygenation at a fluorinated position and release of the fluorine atom as a fluoride anion. The primary additional reaction product formed is the reactive quinoneimine, not the 4-hydroxyaniline. In NADPH-containing microsomal systems with 4-fluoro-substituted anilines, formation of the 4-hydroxyaniline derivative is observed because NADPH chemically reduces this quinoneimine metabolite. A second pathway for dehalogenation proceeds by protein binding of a fluoro-containing (semi)quinoneimine metabolite, the formation of which may result from the mono-oxygenase reaction (pathway 1) and/or from (re)oxidation of a hydroxyaniline metabolite by superoxide anion radicals produced by the microsomal system. This latter reaction pathway becomes more important with increasing number of fluoro-substituents in the fluoroaniline derivative. The higher ratio of fluoride anion formed to 4-hydroxyaniline derivative detected in incubations with liver microsomes from dexamethasone-treated rats, as compared to incubations with liver microsomes from control rats, can in part be explained by the higher production of superoxide anion radicals observed in the dexamethasone systems. The third mechanism was shown to proceed by formation of a hydroxylated metabolite that loses fluoride anion upon exposure to oxygen. The reactive intermediate formed upon oxygen exposure might be the semiquinoneimine which loses its fluorine atom as a fluoride anion upon dimerization or polymerization and/or protein binding. The fluorohydroxyanilines, in which the hydroxyl group is ortho or para with respect to the fluoro substituent, appear especially to be highly unstable and lose fluoride anion in the presence of oxygen. Finally, it is concluded that all three pathways for dehalogenation of fluorinated aniline derivatives are bioactivation pathways. The reactivity of the (semi)quinoneimines formed in these reactions is dependent on their substitution pattern and increases with increasing number of fluoro-substituents. Therefore, bioactivation for a series of fluorinated aniline derivatives, can be expected to vary with the substitution pattern and to increase with increasing number of halogen substituents.

Aniline Compounds↗

PubMed1990

Tertiary structure of two-electron reduced Megasphaera elsdenii flavodoxin and some implications, as determined by two-dimensional 1H-NMR and restrained molecular dynamics.

The tertiary structure of the non-crystallizable two-electron-reduced Megasphaera elsdenii flavodoxin (15 kDa, 137 amino acid residues) has been determined using nuclear Overhauser enhancement restraints extracted from two-dimensional 1H-NMR spectra. A tertiary structure satisfying the experimental restraints very well (maximum NOE violation of 66 pm) was obtained with use of restrained molecular dynamics, using 509 distance restraints (including one non-NOE) on a starting structure modeled from the crystal structure of one-electron-reduced Clostridium MP flavodoxin. The protein consists of a central parallel beta-sheet surrounded on both sides by two alpha-helices. The flavin is positioned at the periphery of the molecule. The tertiary structure of the protein is highly defined with the exception of the flavin. The latter is expected to result from performing the restrained molecular dynamics simulation without water molecules and without proper charges on the flavin. The flavin, including the phosphate, the ribityl side chain and the isoalloxazine ring, is solvent accessible under the experimental conditions used and evidenced by a two-dimensional amide exchange experiment. This accessibility is expected to be important in the redox potential regulation of the semiquinone/hydroquinone couple of the protein. The amide exchange against deuterons and several typical line shapes in the two-dimensional NMR spectra are consistent with the structure generated. The structure is discussed in detail.

Bacterial Proteins↗

PubMed1990

A two-dimensional 1H-NMR study on Megasphaera elsdenii flavodoxin in the oxidized state and some comparisons with the two-electron-reduced state.

Assignments for the 137 amino acid residues of oxidized Megasphaera elsdenii flavodoxin have been made using the sequential resonance assignment procedure. Great benefit was experienced from assignments of the fully reduced protein. The secondary and tertiary structures of the typical alpha/beta protein remain virtually identical on going from the oxidized to the two-electron-reduced state as judged from two-dimensional NOE spectroscopy. However, functionally important conformation changes in the flavin-binding region do occur on reduction. Considerable reduction-state-dependent chemical shift variations of protons in the immediate vicinity of the isoalloxazine moiety take place. From analysis of these shifts, it can be concluded that ring current effects of the pyrazine part of the flavin diminish on two-electron reduction.

Amino Acid Sequence↗

PubMed1990

A 31P-nuclear-magnetic-resonance study of NADPH-cytochrome-P-450 reductase and of the Azotobacter flavodoxin/ferredoxin-NADP+ reductase complex.

31P-nuclear-magnetic-resonance spectroscopy has been employed to probe the structure of the detergent-solubilized form of liver microsomal NADPH--cytochrome-P-450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2'-phosphate (2'-AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2'-AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220-7228] for the protease-solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line-broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2'-AMP was broadened by approximately 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2'-AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P-NMR spectrum as compared with the native enzyme. The 2'-AMP is removed from the enzyme during the FMN-depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN-depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air-stable paramagnetic semiquinone form results in broadening of the FMN and 2'-AMP resonances in the detergent-solubilized enzyme. In agreement with previous results. FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme. 31P-NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin-NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent-solubilized and protease-solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P-NMR experiments in multi-flavin containing enzymes.

Animals↗

PubMed1990

Secondary and tertiary structure characteristics of Megasphaera elsdenii flavodoxin in the reduced state as determined by two-dimensional 1H NMR.

The secondary structure of two-electron-reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving C alpha H, C beta H and NH protons observed in NOESY (two-dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however. M. elsdenii flavodoxin consists of a central parallel beta-sheet including five strands surrounded on both sides by a pair of alpha-helices.

Amides↗

PubMed1990

13C and 15N NMR studies on the interaction between 6,7-dimethyl-8-ribityllumazine and lumazine protein.

The interaction between the prosthetic group 6,7-dimethyl-8-(1'-D-ribityl)lumazine and the lumazine apoproteins from two marine bioluminescent bacteria, one from a relatively thermophilic species, Photobacterium leiognathi, and the other from a psychrophilic species, Photobacterium phosphoreum, was studied by 13C and 15N NMR using various selectively enriched derivatives. It is shown that the electron distribution in the protein-bound 6,7-dimethyl-8-ribityllumazine differs from that of free 6,7-dimethyl-8-ribityllumazine in buffer. The 13C and 15N chemical shifts indicate that the protein-bound 6,7-dimethyl-8-ribityllumazine is embedded in a polar environment and that the ring system is strongly polarized. It is concluded that the two carbonyl groups play an important role in the polarization of the molecule. The N(3)-H group is not accessible to bulk solvent. The N(8) atom is sp2 hybridized and has delta+ character. Nuclear Overhauser effect studies indicate that the 6,7-dimethyl-8-ribityllumazine ring is rigidly bound with no internal mobility. The NMR results indicate that the interaction between the ring system and the two apoproteins is almost the same.

Bacterial Proteins↗

PubMed1990

A two-dimensional 1H NMR study on Megasphaera elsdenii flavodoxin in the reduced state. Sequential assignments.

Assignments for the 137 amino acid residues of Megasphaera elsdenii flavodoxin in the reduced state have been made using the sequential resonance assignment procedure. Several hydroxyl and sulfhydryl protons were observed at 41 degrees C at pH 8.3. Spin systems were sequentially assigned using phase-sensitive two-dimensional-correlated spectroscopy and phase-sensitive nuclear Overhauser enhancement spectroscopy. Spectra of the protein in H2O and of protein preparations either completely or partly exchanged against 2H2O were obtained. Use of the fast electron shuttle between the paramagnetic semiquinone and the diamagnetic hydroquinone state greatly simplified the NMR spectra, making it possible to assign easily the 1H resonances of amino acid residues located in the immediate neighbourhood of the isoalloxazine ring. The majority of the nuclear Overhauser effect contracts between the flavin and the apoprotein correspond to the crystal structure of the flavin domain of Clostridium MP flavodoxin, but differences are also observed. The assignments provide the basis for the structure determination of M. elsdenii flavodoxin in the reduced state as well as for assigning the resonances of the oxidized flavodoxin.

Amino Acids↗