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Andrew Claiborne - One of the best experts on this subject based on the ideXlab platform.
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crystal structures of oxidized and reduced forms of NADH Peroxidase
Methods in Enzymology, 2002Co-Authors: Andrew ClaiborneAbstract:Publisher Summary X-ray structural characterization of cysteine-sulfenic acid-containing proteins is one of the most defining approaches to characterizing this rapidly growing class of protein functional groups. Although outside the scope of this chapter, these structural analyses can lead to kinetic measurements in the crystal that allow intermediate states to be trapped, visualized, and studied. An understanding of the biochemistry of these reactive groups can be more fully gained by studying the localized protein environment in which these groups function. Increased perception of how elements of a protein can stabilize and contribute to modulation of function in these systems will allow novel means of enhancing or inhibiting function in important classes of protein molecules, including transcription factors and redox-regulated enzymes.
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analysis of the kinetic and redox properties of the NADH Peroxidase r303m mutant correlation with the crystal structure
Biochemistry, 2000Co-Authors: Edward J Crane, James Luba, Andrew ClaiborneAbstract:: The crystal structure of the flavoprotein NADH Peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of Cys42-SOH. The crystal structure of the R303M Peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein Peroxidase.
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Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation.
Biochemistry, 1999Co-Authors: Andrew Claiborne, Edward J Crane, James Luba, Thomas Colin Mallett, Véronique Charrier, Derek ParsonageAbstract:While it has been known for more than 20 years that unusually stable cysteine-sulfenic acid (Cys-SOH) derivatives can be introduced in selected proteins by mild oxidation, only recently have chemical and crystallographic evidence for functional Cys-SOH been presented with native proteins such as NADH Peroxidase and NADH oxidase, nitrile hydratase, and the hORF6 and AhpC peroxiredoxins. In addition, Cys-SOH forms of protein tyrosine phosphatases and glutathione reductase have been suggested to play key roles in the reversible inhibition of these enzymes during tyrosine phosphorylation-dependent signal transduction events and nitrosative stress, respectively. Substantial chemical data have also been presented which implicate Cys-SOH in redox regulation of transcription factors such as Fos and Jun (activator protein-1) and bovine papillomavirus-1 E2 protein. Functionally, the Cys-SOHs in NADH Peroxidase, NADH oxidase, and the peroxiredoxins serve as either catalytically essential redox centers or transient intermediates during peroxide reduction. In nitrile hydratase, the active-site Cys-SOH functions in both iron coordination and NO binding but does not play any catalytic redox role. In Fos and Jun and the E2 protein, on the other hand, a key Cys-SH serves as a sensor for intracellular redox status; reversible oxidation to Cys-SOH as proposed inhibits the corresponding DNA binding activity. These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-SOH stabilization and function.
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Thermal stability of a flavoprotein assessed from associative analysis of polarized time-resolved fluorescence spectroscopy.
European Biophysics Journal, 1999Co-Authors: Anatoli V. Digris, Andrew Claiborne, V. V. Skakoun, Eugene G. Novikov, A. N. Van Hoek, Antonie J. W. G. VisserAbstract:Upon gradually heating a particular mutant of the flavoprotein NADH Peroxidase, it was found from the peculiar time-resolved fluorescence anisotropy pattern of the flavin prosthetic group (FAD) that, at elevated temperature, FAD is released from the tetrameric enzyme. Since in this case a mixture of free and enzyme-bound FAD contributes to the time-dependent fluorescence anisotropy, its analysis can only be accomplished by an associative fitting model, in which specific fluorescence lifetimes of both species are linked to specific correlation times. In this letter the general approach to the associative polarized fluorescence decay analysis is described. The procedure can be used for other flavoproteins to determine the temperature at which the onset of thermal denaturation will start, leading to release of the flavin prosthetic group.
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time resolved fluorescence of flavin adenine dinucleotide in wild type and mutant NADH Peroxidase elucidation of quenching sites and discovery of a new fluorescence depolarization mechanism
Journal of Physical Chemistry B, 1998Co-Authors: Antonie J. W. G. Visser, P.a.w. Van Den Berg, Nina V. Visser, H.a. Van Den Burg, A. N. Van Hoek, Derek Parsonage, Andrew ClaiborneAbstract:Time-resolved polarized fluorescence experiments have been carried out on the FAD of tetrameric NADH Peroxidase from Enterococcus faecalis and three mutant enzymes, C42A, C42S, and Y159A, respectively. In particular Tyr159 and, in part, Cys42 turned out to be the amino acids which are responsible for the strong dynamic quenching of flavin fluorescence, because two picosecond fluorescence lifetime components <150 ps are clearly present in the wild-type enzyme and in the Cys42 mutants, while only one picosecond lifetime <150 ps is present in the Tyr159 mutant. This observation is corroborated by the distance information obtainable from the known three-dimensional structure of the wild-type enzyme. Steady-state fluorescence spectroscopy indicated that the Tyr159 mutant has the same fluorescence yield as both Cys42 mutants suggesting that static fluorescence quenching prevails in the tyrosine mutant. Cys42 is the amino acid which is probably responsible for the static quenching in the wild-type enzyme and Y15...
Al Claiborne - One of the best experts on this subject based on the ideXlab platform.
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coenzyme a disulfide reductase from staphylococcus aureus evidence for asymmetric behavior on interaction with pyridine nucleotides
Biochemistry, 1999Co-Authors: James Luba, Véronique Charrier, Al ClaiborneAbstract:An unusual flavoprotein disulfide reductase, which catalyzes the NADPH-dependent reduction of CoASSCoA, has recently been purified from the human pathogen Staphylococcus aureus [delCardayre, S. B., Stock, K. P., Newton, G. L., Fahey, R. C., and Davies, J. E. (1998) J. Biol. Chem. 273, 5744-5751]. Coenzyme A-disulfide reductase (CoADR) lacks the redox-active protein disulfide characteristic of the disulfide reductases; instead, NADPH reduction yields 1 protein-SH and 1 CoASH. Furthermore, the CoADR sequence reveals the presence of a single putative active-site Cys (Cys43) within an SFXXC motif also seen in the Enterococcus faecalis NADH oxidase and NADH Peroxidase, which use a single redox-active cysteine-sulfenic acid in catalysis. In this report, we provide a detailed examination of the equilibrium properties of both wild-type and C43S CoADRs, focusing on the role of Cys43 in the catalytic redox cycle, the behavior of both enzyme forms on reduction with dithionite and NADPH, and the interaction of NADP+ with the corresponding reduced enzyme species. The results of these analyses, combined with electrospray mass spectrometric data for the two oxidized enzyme forms, fully support the catalytic redox role proposed for Cys43 and confirm that this is the attachment site for bound CoASH. In addition, we provide evidence indicating dramatic thermodynamic inequivalence between the two active sites per dimer, similar to that documented for the related enzymes mercuric reductase and NADH oxidase; only 1 FAD is reduced with NADPH in wild-type CoADR. The EH2.NADPH/EH4.NADP+ complex which results is reoxidized quantitatively in titrations with CoASSCoA, supporting a possible role for the asymmetric reduced dimer in catalysis.
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13c nmr analysis of the cysteine sulfenic acid redox center of enterococcal NADH Peroxidase
Biochemistry, 1997Co-Authors: Edward J Crane, Jacques Vervoort, Al ClaiborneAbstract:In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH Peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled Peroxidase HHAA mutant (His10His23Ala87Ala258);...
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13c nmr analysis of the cysteine sulfenic acid redox center of enterococcal NADH Peroxidase
Biochemistry, 1997Co-Authors: Edward J Crane, Jacques Vervoort, Al ClaiborneAbstract:In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH Peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled Peroxidase HHAA mutant (His10His23Ala87Ala258); the spectral change over the pH range 5.8-7. 8 is attributed to deprotonation of the surface-exposed His23. Furthermore, replacement of Arg303, which is hydrogen bonded to His10, has no effect on the 13C spectrum. These results provide direct evidence in support of the Peroxidase Cys42-sulfenic acid/thiol redox cycle and add significantly to our structure-based understanding of protein-sulfenic acid stabilization and function.
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evidence for regulation of the NADH Peroxidase gene npr from enterococcus faecalis by oxyr
Fems Microbiology Letters, 1997Co-Authors: Paul R Ross, Al ClaiborneAbstract:We report that the purified Escherichia coli OxyR protein can bind specifically upstream of the gene encoding NADH Peroxidase (npr) from Enterococcus faecalis 10C1, to a site located some 144 bp from the promoter. A 34 kDa protein has been identified in crude extracts of E. faecalis that cross-reacts with polyclonal antisera to purified OxyR from E. coli and a protein(s) present in these extracts retards npr DNA fragments in gel shift assays. Taken together with the results of sequence analyses, these observations suggest that enterococcal npr is regulated by OxyR.
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the active site histidine 10 of enterococcal NADH Peroxidase is not essential for catalytic activity
Biochemistry, 1996Co-Authors: Edward J Crane, Derek Parsonage, Al ClaiborneAbstract:In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G. E. (1993) Eur. J. Biochem. 211, 221−226] that the active-site His10 of NADH Peroxidase functions as an essential acid−base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The kcat values for both H10Q and H10A Peroxidases, and the pH profile for kcat with H10Q, are very similar to those observed with wild-type Peroxidase. Both mutants, however, exhibit Km(H2O2) values much higher (50−70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H2O2 reactivity of two-electron reduced H10Q demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E·NADH intermediate in the conversion of E→EH2 is confirmed by diode-array spectra...
Derek Parsonage - One of the best experts on this subject based on the ideXlab platform.
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Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation.
Biochemistry, 1999Co-Authors: Andrew Claiborne, Edward J Crane, James Luba, Thomas Colin Mallett, Véronique Charrier, Derek ParsonageAbstract:While it has been known for more than 20 years that unusually stable cysteine-sulfenic acid (Cys-SOH) derivatives can be introduced in selected proteins by mild oxidation, only recently have chemical and crystallographic evidence for functional Cys-SOH been presented with native proteins such as NADH Peroxidase and NADH oxidase, nitrile hydratase, and the hORF6 and AhpC peroxiredoxins. In addition, Cys-SOH forms of protein tyrosine phosphatases and glutathione reductase have been suggested to play key roles in the reversible inhibition of these enzymes during tyrosine phosphorylation-dependent signal transduction events and nitrosative stress, respectively. Substantial chemical data have also been presented which implicate Cys-SOH in redox regulation of transcription factors such as Fos and Jun (activator protein-1) and bovine papillomavirus-1 E2 protein. Functionally, the Cys-SOHs in NADH Peroxidase, NADH oxidase, and the peroxiredoxins serve as either catalytically essential redox centers or transient intermediates during peroxide reduction. In nitrile hydratase, the active-site Cys-SOH functions in both iron coordination and NO binding but does not play any catalytic redox role. In Fos and Jun and the E2 protein, on the other hand, a key Cys-SH serves as a sensor for intracellular redox status; reversible oxidation to Cys-SOH as proposed inhibits the corresponding DNA binding activity. These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-SOH stabilization and function.
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time resolved fluorescence of flavin adenine dinucleotide in wild type and mutant NADH Peroxidase elucidation of quenching sites and discovery of a new fluorescence depolarization mechanism
Journal of Physical Chemistry B, 1998Co-Authors: Antonie J. W. G. Visser, P.a.w. Van Den Berg, Nina V. Visser, H.a. Van Den Burg, A. N. Van Hoek, Derek Parsonage, Andrew ClaiborneAbstract:Time-resolved polarized fluorescence experiments have been carried out on the FAD of tetrameric NADH Peroxidase from Enterococcus faecalis and three mutant enzymes, C42A, C42S, and Y159A, respectively. In particular Tyr159 and, in part, Cys42 turned out to be the amino acids which are responsible for the strong dynamic quenching of flavin fluorescence, because two picosecond fluorescence lifetime components <150 ps are clearly present in the wild-type enzyme and in the Cys42 mutants, while only one picosecond lifetime <150 ps is present in the Tyr159 mutant. This observation is corroborated by the distance information obtainable from the known three-dimensional structure of the wild-type enzyme. Steady-state fluorescence spectroscopy indicated that the Tyr159 mutant has the same fluorescence yield as both Cys42 mutants suggesting that static fluorescence quenching prevails in the tyrosine mutant. Cys42 is the amino acid which is probably responsible for the static quenching in the wild-type enzyme and Y15...
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the active site histidine 10 of enterococcal NADH Peroxidase is not essential for catalytic activity
Biochemistry, 1996Co-Authors: Edward J Crane, Derek Parsonage, Andrew ClaiborneAbstract:In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G. E. (1993) Eur. J. Biochem. 211, 221−226] that the active-site His10 of NADH Peroxidase functions as an essential acid−base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The kcat values for both H10Q and H10A Peroxidases, and the pH profile for kcat with H10Q, are very similar to those observed with wild-type Peroxidase. Both mutants, however, exhibit Km(H2O2) values much higher (50−70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H2O2 reactivity of two-electron reduced H10Q demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E·NADH intermediate in the conversion of E→EH2 is confirmed by diode-array spectra...
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the active site histidine 10 of enterococcal NADH Peroxidase is not essential for catalytic activity
Biochemistry, 1996Co-Authors: Edward J Crane, Derek Parsonage, Al ClaiborneAbstract:In order to test the proposal [Stehle, T., Claiborne, A., & Schulz, G. E. (1993) Eur. J. Biochem. 211, 221−226] that the active-site His10 of NADH Peroxidase functions as an essential acid−base catalyst, we have analyzed mutants in which this residue has been replaced by Gln or Ala. The kcat values for both H10Q and H10A Peroxidases, and the pH profile for kcat with H10Q, are very similar to those observed with wild-type Peroxidase. Both mutants, however, exhibit Km(H2O2) values much higher (50−70-fold) than that for wild-type enzyme, and stopped-flow analysis of the H2O2 reactivity of two-electron reduced H10Q demonstrates that this difference is due to a 150-fold decrease in the second-order rate constant for this reaction with the mutant. Stopped-flow analyses also confirm that reduction of the enzyme by NADH is essentially unaffected by His10 replacement and remains largely rate-limiting in turnover; the formation of an E·NADH intermediate in the conversion of E→EH2 is confirmed by diode-array spectra...
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analysis of the kinetic mechanism of enterococcal NADH Peroxidase reveals catalytic roles for NADH complexes with both oxidized and two electron reduced enzyme forms
Biochemistry, 1995Co-Authors: Edward J Crane, Derek Parsonage, Leslie B. Poole, Andrew ClaiborneAbstract:: Anaerobic titrations of the two-electron-reduced NADH Peroxidase (EH2) with NADH and 3-acetylpyridine adenine dinucleotide (AcPyADH) yield the respective complexes without significant formation of the four-electron-reduced enzyme (EH4). Further analysis of the EH2/EH4 redox couple, however, yields a midpoint potential of -312 mV for the free enzyme at pH 7. The catalytic mechanism of the Peroxidase has been evaluated with a combination of kinetic and spectroscopic approaches, including initial velocity and enzyme-monitored turnover measurements, anaerobic stopped-flow studies of the reactions of both oxidized enzyme (E) and EH2 with NADH and AcPyADH, and diode-array spectral analyses of both the reduction of E-->EH2 by NADH and the formation of EH2.NADH. Overall, these results are consistent with rapid formation of an E.NADH complex with distinct spectral properties and a rate-limiting hydride transfer step that yields EH2, with no direct evidence for intermediate FADH2 formation. The EH2.NADH complex described previously [Poole, L. B., & Claiborne, A. (1986) J. Biol. Chem. 261, 14525-14533] is not catalytically competent and reacts relatively slowly with H2O2. Stopped-flow analyses do, however, support the very rapid formation of an EH2.NADH* intermediate, with spectral properties that distinguish it from the static EH2.NADH form, and yield a first-order rate constant for the conversion between the two species that is smaller than kcat. The combined rapid-reaction and steady-state data are best accommodated by a limiting type of ternary complex mechanism very similar to that proposed previously [Parsonage, D., Miller, H., Ross, R.P., & Claiborne, A. (1993) J. Biol. Chem. 268, 3161-3167].
Edward J Crane - One of the best experts on this subject based on the ideXlab platform.
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analysis of the kinetic and redox properties of the NADH Peroxidase r303m mutant correlation with the crystal structure
Biochemistry, 2000Co-Authors: Edward J Crane, James Luba, Andrew ClaiborneAbstract:: The crystal structure of the flavoprotein NADH Peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of Cys42-SOH. The crystal structure of the R303M Peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein Peroxidase.
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Protein-sulfenic acids: diverse roles for an unlikely player in enzyme catalysis and redox regulation.
Biochemistry, 1999Co-Authors: Andrew Claiborne, Edward J Crane, James Luba, Thomas Colin Mallett, Véronique Charrier, Derek ParsonageAbstract:While it has been known for more than 20 years that unusually stable cysteine-sulfenic acid (Cys-SOH) derivatives can be introduced in selected proteins by mild oxidation, only recently have chemical and crystallographic evidence for functional Cys-SOH been presented with native proteins such as NADH Peroxidase and NADH oxidase, nitrile hydratase, and the hORF6 and AhpC peroxiredoxins. In addition, Cys-SOH forms of protein tyrosine phosphatases and glutathione reductase have been suggested to play key roles in the reversible inhibition of these enzymes during tyrosine phosphorylation-dependent signal transduction events and nitrosative stress, respectively. Substantial chemical data have also been presented which implicate Cys-SOH in redox regulation of transcription factors such as Fos and Jun (activator protein-1) and bovine papillomavirus-1 E2 protein. Functionally, the Cys-SOHs in NADH Peroxidase, NADH oxidase, and the peroxiredoxins serve as either catalytically essential redox centers or transient intermediates during peroxide reduction. In nitrile hydratase, the active-site Cys-SOH functions in both iron coordination and NO binding but does not play any catalytic redox role. In Fos and Jun and the E2 protein, on the other hand, a key Cys-SH serves as a sensor for intracellular redox status; reversible oxidation to Cys-SOH as proposed inhibits the corresponding DNA binding activity. These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-SOH stabilization and function.
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13c nmr analysis of the cysteine sulfenic acid redox center of enterococcal NADH Peroxidase
Biochemistry, 1997Co-Authors: Edward J Crane, Jacques Vervoort, Andrew ClaiborneAbstract:In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH Peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled Peroxidase HHAA mutant (His10His23Ala87Ala258);...
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13c nmr analysis of the cysteine sulfenic acid redox center of enterococcal NADH Peroxidase
Biochemistry, 1997Co-Authors: Edward J Crane, Jacques Vervoort, Al ClaiborneAbstract:In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH Peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled Peroxidase HHAA mutant (His10His23Ala87Ala258);...
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13c nmr analysis of the cysteine sulfenic acid redox center of enterococcal NADH Peroxidase
Biochemistry, 1997Co-Authors: Edward J Crane, Jacques Vervoort, Al ClaiborneAbstract:In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH Peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled Peroxidase HHAA mutant (His10His23Ala87Ala258); the spectral change over the pH range 5.8-7. 8 is attributed to deprotonation of the surface-exposed His23. Furthermore, replacement of Arg303, which is hydrogen bonded to His10, has no effect on the 13C spectrum. These results provide direct evidence in support of the Peroxidase Cys42-sulfenic acid/thiol redox cycle and add significantly to our structure-based understanding of protein-sulfenic acid stabilization and function.
Brahma B. Panda - One of the best experts on this subject based on the ideXlab platform.
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aluminum induces oxidative burst cell wall NADH Peroxidase activity and dna damage in root cells of allium cepa l
Environmental and Molecular Mutagenesis, 2012Co-Authors: Mohan V M Achary, Narasimham L Parinandi, Brahma B. PandaAbstract:Plants under stress incur an oxidative burst that involves a rapid and transient overproduction of reactive oxygen species (ROS: O2•−, H2O2, •OH). We hypothesized that aluminum (Al), an established soil pollutant that causes plant stress, would induce an oxidative burst through the activation of cell wall-NADH Peroxidase (NADH-PX) and/or plasma membrane-associated NADPH oxidase (NADPH-OX), leading to DNA damage in the root cells of Allium cepa L. Growing roots of A. cepa were treated with Al3+ (800 μM of AlCl3) for 3 or 6 hr without or with the pretreatment of inhibitors specific to NADH-PX and NADPH-OX for 2 hr. At the end of the treatment, the extent of ROS generation, cell death, and DNA damage were determined. The cell wall-bound protein (CWP) fractions extracted from the untreated control and the Al-treated roots under the aforementioned experimental conditions were also subjected to in vitro studies, which measured the extent of activation of Peroxidase/oxidase, generation of •OH, and DNA damage. Overall, the present study demonstrates that the cell wall-bound NADH-PX contributes to the Al-induced oxidative burst through the generation of ROS that lead to cell death and DNA damage in the root cells of A. cepa. Furthermore, the in vitro studies revealed that the CWP fraction by itself caused DNA damage in the presence of NADH, supporting a role for NADH-PX in the stress response. Altogether, this study underscores the crucial function of the cell wall-bound NADH-PX in the oxidative burst-mediated cell death and DNA damage in plants under Al stress. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.
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aluminum induces oxidative burst cell wall NADH Peroxidase activity and dna damage in root cells of allium cepa l
Environmental and Molecular Mutagenesis, 2012Co-Authors: Mohan V M Achary, Narasimham L Parinandi, Brahma B. PandaAbstract:Plants under stress incur an oxidative burst that involves a rapid and transient overproduction of reactive oxygen species (ROS: O(2) (•-) , H(2) O(2) , (•) OH). We hypothesized that aluminum (Al), an established soil pollutant that causes plant stress, would induce an oxidative burst through the activation of cell wall-NADH Peroxidase (NADH-PX) and/or plasma membrane-associated NADPH oxidase (NADPH-OX), leading to DNA damage in the root cells of Allium cepa L. Growing roots of A. cepa were treated with Al(3+) (800 μM of AlCl(3) ) for 3 or 6 hr without or with the pretreatment of inhibitors specific to NADH-PX and NADPH-OX for 2 hr. At the end of the treatment, the extent of ROS generation, cell death, and DNA damage were determined. The cell wall-bound protein (CWP) fractions extracted from the untreated control and the Al-treated roots under the aforementioned experimental conditions were also subjected to in vitro studies, which measured the extent of activation of Peroxidase/oxidase, generation of (•) OH, and DNA damage. Overall, the present study demonstrates that the cell wall-bound NADH-PX contributes to the Al-induced oxidative burst through the generation of ROS that lead to cell death and DNA damage in the root cells of A. cepa. Furthermore, the in vitro studies revealed that the CWP fraction by itself caused DNA damage in the presence of NADH, supporting a role for NADH-PX in the stress response. Altogether, this study underscores the crucial function of the cell wall-bound NADH-PX in the oxidative burst-mediated cell death and DNA damage in plants under Al stress.
