Biomedical subjects

T A Grover

Publications and source records attributed to T A Grover.

At least 19 recordsLinked to original sources

PubMed1998

Identifying proteins using matrix-assisted laser desorption/ionization in-source fragmentation data combined with database searching.

Metastable ion decay in matrix-assisted laser desorption/ionization (MALDI) has become a routine method for obtaining primary structures of peptides. Significant fragmentation occurs in the MALDI ion source and can be observed via delayed ion extraction TOF-MS. In-source decay (ISD) can provide C- and N-terminal primary sequence data for even moderate-sized peptides (< 5000 Da). The unique cn series fragmentation that occurs in ISD has been exploited to obtain partial C-terminal sequences for proteins as large as human apotransferrin (75 kDa). Two approaches for combining this ISD MALDI-generated partial sequence information with protein database searching techniques are presented. In one approach, cyanogen bromide is used to cleave relatively large peptide fragments from a sample of human apotransferrin. One of the larger cleavage products (6034.84 Da) was isolated by HPLC and subjected to ISD MALDI analysis. An easily identified cn fragment ion series allowed two noncontiguous segments of the peptide's sequence to be determined (about 55% of the total sequence). This partial sequence information was used to search protein and oligonucleotide sequence databases. In addition to uniquely identifying human apotransferrin in a protein sequence database, an example of the use of this ISD MALDI-determined partial sequence information to search expressed sequence tag databases is presented. Such searches have the potential for rapidly identifying new genes that code for target proteins. An alternate approach for obtaining partial sequence information on proteins is also demonstrated that utilizes ISD MALDI fragmentation of the intact protein to generate partial sequence information. This approach is shown to generate about 5-7% of a protein's sequence, usually near the C-terminus of the protein. Examples of the ISD MALDI fragmentation data obtained from intact (reduced) human apotransferrin and intact (nonreduced) bovine serum albumin (66 kDa) proteins are presented.

Animals↗

PubMed1997

Roles of efficient substrates in enhancement of peroxidase-catalyzed oxidations.

Efficient peroxidase substrates may have a critical role in the oxidation of secondary compounds by peroxidases. Hydrazines are often oxidized slowly by peroxidases due, in part, to hydrazine-dependent inactivation of these enzymes. Peroxidase-catalyzed oxidation of hydrazines may be dramatically affected by an efficient peroxidase substrate. We investigated this hypothesis in a model system using the well-known peroxidase substrate chlorpromazine (CPZ) and the hydrazine derivative isoniazid. CPZ stimulated isoniazid oxidation as measured by nitroblue tetrazolium (NBT) reduction and O2 consumption. The kinetics of isoniazid and CPZ oxidation by horseradish peroxidase (HRP) in the presence of both compounds suggested CPZ was acting as an electron transfer mediator between HRP and isoniazid. Indeed, CPZ.+, the product of CPZ oxidation by HRP, was able to oxidize isoniazid. The rate constant for this pH-dependent reaction was (2.6 +/- 0.1) x 10(4) M-1 s-1 at pH 4.5. In the absence of CPZ, isoniazid-dependent irreversible inactivation of HRP was observed. The inactivation process involved the formation of compound III followed by accumulation of irreversibly inactivated HRP. CPZ completely inhibited inactivation. Thus, by acting as a redox mediator and preventing HRP inactivation, CPZ stimulated isoniazid oxidation by several orders of magnitude. Similarly, other efficient peroxidase substrates, such as phenol and tyrosine, were also able to dramatically stimulate isoniazid oxidation by HRP. We suggest that the presence of efficient peroxidase substrates may potentiate the activation of isoniazid and other hydrazines. As such, these substrates may have a vital role in the pharmacological and toxicological properties of hydrazines and other compounds.

Carbon Monoxide↗

PubMed1996

Redox mediation in the peroxidase-catalyzed oxidation of aminopyrine: possible implications for drug-drug interactions.

Many drugs, industrial pollutants, and other xenobiotics are known to be oxidized by peroxidases to potentially harmful free-radical intermediates. We have examined the possibility that certain compounds, acting as efficient peroxidase substrates, may stimulate the formation of reactive free radicals by acting as mediators of electron transfer reactions (redox mediators). To explore this hypothesis, we have investigated the interaction of two well-known peroxidase substrates, chlorpromazine and aminopyrine. As shown by ESR and UV-visible spectroscopy, chlorpromazine radical was able to oxidize aminopyrine to aminopyrine cation radical. The rate constant for this rapid, pH-dependent, reaction was estimated to be 1 x 10(7) M-1 s-1 at pH 4.5. Transient-state and steady-state kinetic studies both showed that rate constants for chlorpromazine oxidation to its cation radical by horseradish peroxidase (HRP) were about 100-fold greater than for the corresponding HRP-catalyzed oxidation of aminopyrine to its cation radical. When both aminopyrine and chlorpromazine were present with HRP and H2O2, aminopyrine cation radical formation was stimulated 100-fold. Concomitantly, the accumulation of chlorpromazine cation radical was completely inhibited in the presence of aminopyrine. Similar results were obtained when lactoperoxidase, myeloperoxidase, or the myeloperoxidase mimic HOCl were substituted for HRP. These data suggest that chlorpromazine can act as a redox mediator for peroxidase-catalyzed oxidation of aminopyrine and other chemicals. We suggest that some peroxidase substrates, acting as redox mediators, may stimulate the production of toxic free-radical intermediates from various drugs and other xenobiotics. As such, this may have implications for a number of adverse effects caused by these xenobiotic chemicals.

Aminopyrine↗

PubMed1996

Free radicals produced during the oxidation of hydrazines by hypochlorous acid.

Hypochlorous acid (HOCl) derived from activated neutrophils and monocytes has been implicated in the activation of hydrazine-containing drugs to toxic intermediates. However, reactive intermediates formed during the reaction between HOCl and these drugs have not been identified. We investigated the oxidation of the hydrazine derivatives isoniazid, iproniazid, and hydralazine by HOCl. The reaction between HOCl and all three hydrazines resulted in O2 consumption, indicating that free radicals were produced, but the rate and extent of O2 consumption were different for each hydrazine. Moreover, reduction of nitroblue tetrazolium (NBT) was observed only during the reaction between HOCl and isoniazid, suggesting that different radical species may be produced from HOCl reaction with each hydrazine. The oxidation of iproniazid by HOCl in the presence of the radical trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) resulted in the formation of a carbon-centered radical adduct. In contrast, the reaction between HOCl and hydralazine resulted in the formation of a nitrogen-centered DMPO radical adduct. The oxidation of isoniazid by HOCl resulted in the formation of two oxygen-centered radical adducts, DMPO-OOH and DMPO-OH. Myeloperoxidase-catalyzed oxidation of these hydrazines in the presence of Cl- and H2O2 produced radical species that were identical to those observed with HOCl. Thus, some of the toxic side effects of these drugs may be the result of the production of free-radical intermediates from reaction with neutrophil-derived oxidants, such as HOCl. The types of radicals produced and the consequences of generating these reactive species are discussed.

Electron Spin Resonance Spectroscopy↗

PubMed1995

Determination of rate constants for rapid peroxidase reactions.

We describe an improved enzyme-monitored stopped-flow method for determining rate constants for peroxidase reactions that are too rapid to measure by conventional pseudo-first-order methods. Ascorbate will reduce many substrate radicals as rapidly as they are generated by a peroxidase. This ensures a constant substrate concentration during the peroxidase reaction. We investigated the reactions of horseradish peroxidase compound I (HRP-I) with three standard substrates (chlorpromazine (CPZ), 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), and p-methoxyphenol) in the presence and absence of ascorbate. The rate of reaction of CPZ with HRP-I is slow enough that it can be measured using pseudo-first-order conditions maintained by a minimum 10-fold excess of CPZ, or by the addition of ascorbate at very low CPZ concentrations. The same rate constant was obtained with either method. The rate of reaction of ABTS with HRP-I at lower pH (5.0-3.3) is rapid; consequently, we were unable to obtain rate constants using concentrations of ABTS which constitute pseudo-first-order conditions. However, using much lower ABTS concentrations with the addition of ascorbate, we obtained rate constants that ranged from 5 x 10(7) to 8 x 10(8) M-1 s-1. Hence, ascorbate provides a simplified way to maintain pseudo-first-order conditions for fast peroxidase reactions even at low substrate concentrations.

Ascorbic Acid↗

PubMed1995

Reductions catalyzed by a quinone and peroxidases from Phanerochaete chrysosporium.

A quinone produced from veratryl alcohol by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was tested for its ability to mediate reduction. The quinone (2-hydroxymethyl-5-methoxy-1,4-benzoquinone), reduced chemically or by cellobiose:quinone reductase isolated from cultures of the fungus, mediated the reduction of cytochrome c in reactions containing either Mn(III), a manganese-dependent peroxidase, Mn(II) and H2O2, or lignin peroxidase and H2O2. Formation of the semiquinone, the species responsible for reducing cytochrome c, was observed by electron spin resonance spectroscopy in these reactions. The production of the quinone was observed in the extracellular fraction of cultures grown under nutrient nitrogen-deficient conditions (2.4 mM ammonium tartrate) for over 10 days, starting on Day 2, but not under nutrient nitrogen-sufficient conditions. These results suggest that a quinone produced by lignin peroxidase can serve as a physiological mediator of reductive reactions catalyzed by the fungal peroxidases.

Benzoquinones↗

PubMed1995

Evidence for formation of the veratryl alcohol cation radical by lignin peroxidase.

Lignin peroxidases (LiP) catalyze the H2O2-dependent two-electron oxidation of veratryl alcohol (VA) to veratryl aldehyde. We present here, electron spin resonance (ESR) evidence for the formation of the one-electron oxidized intermediate, the veratryl alcohol cation radical (VA.+). The ESR spectrum of VA.+ was first obtained in a fast-flow system with Ce(IV) as an oxidant and 10% HNO3 to stabilize the radical. This ESR signal was deconvoluted, and the hyperfine splitting constants were determined. The identity of the radical was confirmed by computer simulation of the ESR spectrum and calculation of spin and charge densities on the radical. An identical radical signal was observed with LiP, also in a fast-flow incubation containing 10 microM LiP, 2 mM VA, and 500 microM H2O2 at pH 3.5. The Fourier transforms of the ESR signals further confirmed that the spectra obtained with both Ce(IV) and LiP were due to the same radical species. The VA.+ had a distinct visible spectrum in 98% H2SO4 with an absorbance maximum at 529 nm. The extinction coefficient of the VA.+ spectral band at 529 nm was calculated to be 11,000 M-1 cm-1. The VA.+ was found to be a strong acid, as are other cation radicals, with the pKa at -1.0 pH. This value was determined by quantitating both the concentration of VA.+ by visual and ESR spectrometry and the g-value of the ESR signal at various pH values.

Benzyl Alcohols↗

PubMed1995

Evidence for veratryl alcohol as a redox mediator in lignin peroxidase-catalyzed oxidation.

We have examined the hypothesis that veratryl alcohol (VA) may act as a redox mediator in lignin peroxidase (LiP)-catalyzed oxidations. The oxidation of chlorpromazine (CPZ) by this system was used to evaluate this hypothesis. Chlorpromazine can be oxidized by one electron to form a stable cation radical (CPZ.+). This cation radical can be oxidized by another electron to the sulfoxide (CPZSO). These oxidation steps are easily monitored, making CPZ a useful chemical to investigate redox mediation by VA. Lignin peroxidase oxidized CPZ to CPZ.+ whether or not VA was present. The inclusion of VA, however, stimulated CPZ oxidation to CPZ.+ and subsequent oxidation of CPZ.+ to CPZSO. In the absence of VA, the initial rates of CPZ oxidation by LiP were CPZ concentration-dependent. However, when saturating concentrations of VA were added, the oxidation of CPZ and CPZ.+ became independent of CPZ concentration. When the oxidation of VA to veratryl aldehyde was examined, increasing concentrations of CPZ produced a lag in veratryl aldehyde appearance proportional to the concentration of CPZ. Conversely, increasing concentrations of VA never inhibited CPZ oxidation. Transient-state kinetic studies indicated that both VA and CPZ reduced the compound I and compound II forms of LiP. However, when saturating concentrations of VA were utilized, LiP turnover was independent of CPZ concentration. We suggest these data demonstrate that VA may act as a redox mediator for the indirect oxidation of compounds by LiP.

Benzyl Alcohols↗

PubMed1994

Oxalate-dependent reductive activity of manganese peroxidase from Phanerochaete chrysosporium.

The mechanism of oxalate-dependent reductive activity of a manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing Mn-peroxidase, Mn2+, oxalate, H2O2, ferric chloride, and 1,10-phenanthroline. Only catalytic amounts of H2O2 were required. Oxygen consumption was also observed in reaction mixtures containing Mn-peroxidase, Mn2+, oxalate, and H2O2 and was inhibited by the addition of ferric iron. Electron spin resonance studies, using the spin traps 5,5-dimethyl-1-pyrroline-N-oxide and alpha-4-pyridyl-1-oxide-N-t-butylnitrone were used to obtain evidence for the production of the formate radical (CO2.-) and superoxide (O2.-) in a reaction mixture containing Mn2+, oxalate and H2O2. It was concluded that both CO2.- (anaerobic conditions) and O2.- (aerobic conditions) could reduce ferric iron. The dismutation of some O2.- would produce H2O2 to provide a constant supply of H2O2.

Aerobiosis↗

PubMed1994

The role of oxalate in lignin peroxidase-catalyzed reduction: protection from compound III accumulation.

Reduction may be an important step in the degradation of some highly oxidized environmental pollutants by Phanerochaete chrysosporium. Lignin peroxidases (LiP) from P. chrysosporium are able to catalyze reductive reactions using veratryl alcohol (VA) as a mediator and either oxalate or EDTA as electron donors. Reduction of oxygen to superoxide, monitored by oxygen consumption, was used as a measure of the reductive activity of LiP. In the presence of EDTA, the rate of O2 reduction catalyzed by LiP decreased with time and increasing concentrations of H2O2. When oxalate replaced EDTA, LiP-catalyzed O2 reduction did not decrease with time, and increasing concentrations of H2O2 increased the duration and extent of O2 reduction. LiP was converted to the compound III state in the presence of EDTA, H2O2, and veratryl alcohol. When oxalate replaced EDTA, compound II was observed. The importance of the veratryl alcohol cation radical (VA.+) in the conversion of LiP compound III to active enzyme has been previously examined (D.P. Barr and S.D. Aust, 1994, Arch. Biochem. Biophys. 311, 378-382). We propose that rapid reduction of VA.+ by EDTA results in accumulation of LiP compound III and the loss of activity resulting in a decrease in LiP-catalyzed reduction reactions. Oxalate is less effective in reducing the VA.+, therefore, some VA.+ remains to convert compound III to active enzyme and maintain LiP-catalyzed reduction reactions. Thus oxalate, a normal secondary metabolite of P. chrysosporium, is a suitable candidate for mediating reduction reactions by LiP in vivo.

Edetic Acid↗

PubMed1993

Reduction of CCl4 to the trichloromethyl radical by lignin peroxidase H2 from Phanerochaete chrysosporium.

We were able to spin trap the trichloromethyl radical with PBN in a reaction mixture containing lignin peroxidase H2, H2O2, CCl4, veratryl alcohol and EDTA. Upon removal of CCl4, EDTA radicals were detected. Based on a previously reported study (Shah et al., 1992, J. Biol. Chem. 267, 21564-21569), we propose that the oxidation of EDTA to EDTA derived radicals is mediated by the veratryl alcohol cation radical, and the reduction of CCl4 to the trichloromethyl radical is caused by the EDTA derived radicals. The novel finding here is that CCl4, which is neither a substrate for the enzyme nor a good reductant, is degraded via free radicals generated by lignin peroxidases under reducing conditions.

Carbon Tetrachloride↗

PubMed1993

Xanthine oxidase- and iron-dependent lipid peroxidation.

Xanthine oxidase and iron-dependent lipid peroxidation has been studied extensively in many model systems, yet several details of this process remain unclear. Because redox reactions of iron are important parameters of iron-catalyzed lipid peroxidation, we have examined the roles of superoxide and hydrogen peroxide, produced by xanthine oxidase, to oxidize and reduce iron and thereby affect iron-catalyzed lipid peroxidation. Thus, we compared lipid peroxidation catalyzed by xanthine oxidase and ADP:Fe(III) to that catalyzed by xanthine oxidase and ADP:Fe(II). An examination of the action of superoxide on iron oxidation and reduction revealed that superoxide is a better oxidant of ADP:Fe(II) than a reductant of ADP:Fe(III). A superoxide generating system (composed of xanthine oxidase and catalase) and ADP:Fe(II) also resulted in a greater amount of lipid peroxidation than superoxide and ADP:Fe(III). Hydrogen peroxide, as expected, only served as an Fe(II) oxidant. A comparison of the oxidant activities of either superoxide or hydrogen peroxide on ADP:Fe(II) and the corresponding effects on lipid peroxidation revealed that both oxidants were roughly equivalent. We conclude that superoxide and hydrogen peroxide, produced from xanthine oxidase, support iron-catalyzed lipid peroxidation through their participation in redox reactions of iron, that is, they facilitate Fe(II) oxidation or Fe(III) reduction necessary for lipid peroxidation. The relevance of the reactions of O2-. and H2O2 on physiological chelates of iron are discussed.

Adenosine Diphosphate↗

PubMed1993

Reductive activity of a manganese-dependent peroxidase from Phanerochaete chrysosporium.

A manganese-dependent peroxidase (MnP) from Phanerochaete chrysosporium catalyzed the reduction of cytochrome c in a reaction mixture containing H2O2, Mn(II)-tartrate, and p-hydroquinone. Electron spin resonance studies have shown that the hydroquinone-dependent reductive activity of MnP is due to the benzosemiquinone formed upon the one-electron oxidation of p-hydroquinone by Mn(III)-tartrate, which is formed upon the oxidation of Mn(II) by MnP. The reductive activity increased linearly with an increase in the concentration of p-hydroquinone. The reductive activity was also observed using other hydroquinones such as methylhydroquinone, 2,5-dimethylhydroquinone, and trimethylhydroquinone. The apparent Km values for Mn(II) and H2O2 for the hydroquinone-dependent reductive activity were similar to those for oxidative reactions of MnP. A stoichiometry study showed that about 1.5 mol of cytochrome c was reduced per mole of H2O2 consumed. The stoichiometry decreased with an increase in the concentration of H2O2. The optimal pH for the reductive activity was 5.0, approximately the physiological pH of the fungus. The reduction of cytochrome c was also observed using a quinone and cellobiose:quinone oxidoreductase isolated from the extracellular medium of the fungus.

Basidiomycota↗

PubMed1992

Production of hydroxyl radical by lignin peroxidase from Phanerochaete chrysosporium.

The mechanism for the production of hydroxyl radical by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was investigated. Ferric iron reduction was demonstrated in reaction mixtures containing lignin peroxidase isozyme H2 (LiPH2), H2O2, veratryl alcohol, oxalate, ferric chloride, and 1,10-phenanthroline. The rate of iron reduction was dependent on the concentration of oxalate and was inhibited by the addition of superoxide dismutase. The addition of ferric iron inhibited oxygen consumption in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, and oxalate. Thus, the reduction of ferric iron was thought to be dependent on the LiPH2-catalyzed production of superoxide in which veratryl alcohol and oxalate serve as electron mediators. Oxalate production and degradation in nutrient nitrogen-limited cultures of P. chrysosporium was also studied. The concentration of oxalate in these cultures decreased during the period in which maximum lignin peroxidase activity (veratryl alcohol oxidation) was detected. Electron spin resonance studies using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide were used to obtain evidence for the production of the hydroxyl radical in reaction mixtures containing LiPH2, H2O2, veratryl alcohol, EDTA, and ferric chloride. It was concluded that the white rot fungus might produce hydroxyl radical via a mechanism that includes the secondary metabolites veratryl alcohol and oxalate. Such a mechanism may contribute to the ability of this fungus to degrade environmental pollutants.

Agaricales↗

PubMed1992

On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA.

The mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, veratryl alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, veratryl alcohol, and EDTA. The Km values of veratryl alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of veratryl alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the veratryl alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the veratryl alcohol cation radical intermediate back to veratryl alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the veratryl alcohol cation radical.

Alcohol Oxidoreductases↗

PubMed1992

Rat ceruloplasmin: resistance to proteolysis and kinetic comparison with human ceruloplasmin.

Rat ceruloplasmin was purified from serum using fast protein liquid chromatography and compared to human ceruloplasmin isolated in the same manner. Rat ceruloplasmin was found to be more resistant to plasmin-mediated proteolysis than was human ceruloplasmin. Although both proteins were cleaved initially to products with apparent molecular weights of 116,000 and 20,000 Da, rat ceruloplasmin was resistant to further proteolysis, whereas the human enzyme was cleaved to smaller fragments. Primary structure differences could account for the different relative stabilities between the two enzymes. Kinetic analysis of rat ceruloplasmin produced a biphasic v vs v/s plot with apparent Km's of 40 and 1.5 microM for iron. When compared with the human enzyme, rat ceruloplasmin showed about one-fourth the ferroxidase activity and had a much broader pH profile than that of human ceruloplasmin. Rates of p-phenylenediamine oxidation by rat ceruloplasmin were about one-half those obtained with human ceruloplasmin, with maximal p-phenylenediamine oxidase activity at pH 5.0 for both enzymes.

Amino Acid Sequence↗

PubMed1992

Inhibition of veratryl alcohol oxidase activity of lignin peroxidase H2 by 3-amino-1,2,4-triazole.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was inhibited by 3-amino-1,2,4-triazole (AT). Inhibition was found to be competitive with respect to veratryl alcohol (K1 = 18 microM) and noncompetitive with respect to H2O2. Unlike bovine lactoperoxidase, catalase, and thyroid peroxidase, AT was not a suicide (mechanism based) inhibitor for lignin peroxidase H2. Binding studies revealed that lignin peroxidase H2 catalyzed insignificant binding of [14C]AT to the enzyme. Apparently AT is a poor substrate for lignin peroxidase H2 and is only slowly oxidized to form a yellow product in the presence of H2O2. The formation of the yellow product was shown to increase with increasing concentrations of veratryl alcohol, suggesting that an intermediate in the oxidation of veratryl alcohol is able to mediate the oxidation of AT. Extensive metabolism of AT to CO2 by the white rot fungus Phanerochaete chrysosporium (approximately 60% in 30 days) was also demonstrated.

Alcohol Oxidoreductases↗

PubMed1991

Inhibition of lignin peroxidase H2 by sodium azide.

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 from Phanerochaete chrysosporium and H2O2 was strongly inhibited by sodium azide. Inhibition was competitive with respect to veratryl alcohol (Ki = 1-2 microM) and uncompetitive with respect to H2O2. In contrast, sodium azide bound to the native enzyme at pH 6.0 with an apparent dissociation constant (KD) of 126 mM. Formation of azidyl radicals was detected by ESR spin trapping techniques. The enzymes is nearly completely inactivated in four turnovers. The H2O2-activated enzyme intermediate (compound I) reacted with sodium azide to form a new species rather than be reduced to the enzyme intermediate compound II. The new species has absorption maxima at 418, 540, and 570 nm, suggesting the formation of a ferrous-lignin peroxidase-NO complex. Confirmation of this assignment was obtained by low-temperature ESR spectroscopy. An identical complex could be simulated by the addition of nitrite to the reduced enzyme. The enzyme intermediate compound II is readily reduced by sodium azide to native enzyme with essentially no loss of activity.

Agaricales↗