Biomedical subjects

S D Aust

Publications and source records attributed to S D Aust.

At least 37 records · Page 2Linked to original sources

PubMed1998

Mechanisms for protection against inactivation of manganese peroxidase by hydrogen peroxide.

It has been reported that cation radicals of aromatic substrates maintain the active form of lignin peroxidase by oxidatively converting compound III, generated during peroxidase turnover, into ferric enzyme (D. P. Barr and S. D. Aust, 1994, Arch. Biochem. Biophys. 312, 511-515). In this work, we investigated protective mechanisms for manganese peroxidase. Oxidation of Mn(II) by manganese peroxidase displayed complex kinetics, which were explained by accumulation of compound III followed by its reactivation by the enzymatically produced Mn(III). Conversion of compound III to ferric enzyme by Mn(III) was not observed for lignin peroxidase or heme propionate-modified recombinant manganese peroxidase, suggesting that Mn(III) may interact with compound III of native manganese peroxidase at a heme propionate to oxidize iron-coordinated superoxide via long-range electron transfer. Additionally, Mn(II) also reactivated compound III. Although this reaction was slower, it could prevent compound III accumulation when excess Mn(II) was present. Another protective mechanism for manganese peroxidase is proposed for insufficient chelator conditions. In contrast to effective Mn(II) chelators, low-affinity ligands supported considerably slower enzyme turnover, and Mn(III) released was more reactive with hydrogen peroxide, resulting in a catalase-type reaction. Reactivation of compound III and catalatic activity may provide biologically relevant mechanisms for protection of manganese peroxidase against suicidal inactivation by hydrogen peroxide under a variety of manganese and oxalate conditions.

Basidiomycota↗

PubMed1998

Expression of the lignin peroxidase H2 gene from Phanerochaete chrysosporium in Escherichia coli.

The DNA sequence for the extracellular lignin peroxidase isozyme H2 from Phanerochaete chrysosporium, obtained from cDNA clone lambda ML-6, was synthesized by PCR and successfully expressed in Escherichia coli under control of the T7 promoter. The portion of the cDNA encoding the signal peptide, not found in the mature native enzyme, was not included. Recombinated lignin peroxidase H2 (rLiPH2) was produced in inclusion bodies in an inactive form. Active enzyme was obtained by refolding with glutathione-mediated oxidation in a medium containing urea, Ca2+, and hemin. The recombinant enzyme had spectral characteristics and kinetic properties identical to that of native enzyme isolated from P. chrysosporium. Surprisingly, rLiPH2, like the native enzyme, also exhibited some manganese peroxidase activity.

Escherichia coli↗

PubMed1998

Studies on the interaction between ferritin and ceruloplasmin.

We showed previously that ceruloplasmin associates with the H chain of rat liver ferritin during iron loading into ferritin such that the iron oxidized by ceruloplasmin was deposited into ferritin [S.-H. Juan et al. (1997) Arch. Biochem. Biophys. 341, 280-286]. Three synthetic decapeptides derived from domains 2, 4, and 6 of ceruloplasmin, referred to CP-2, CP-4, and CP-6, were utilized to identify a possible binding site on ceruloplasmin for ferritin. Two of the peptides, CP-4 and CP-6, were found to inhibit iron loading into the recombinant ferritin H chain homopolymer (rH-Ft) by ceruloplasmin. The extent of inhibition of iron loading into ferritin by ceruloplasmin by CP-6, but not CP-4, varied with pH, whereas the inhibitory effect remained constant in increasing concentrations of NaCl. The addition of rH-Ft quenched the fluorescence emission of CP-4 and CP-6, but not CP-2. The quenching of fluorescence was used to estimate dissociation constants for the peptides. Iron loading into ferritin in Hepes buffer was not affected in the presence of these peptides. In addition, synthetic peptides corresponding to the BC loop of ferritin H and L chains were utilized to localize an interaction site on ferritin for ceruloplasmin. The BC loop of H chain but not L chain of ferritin stimulated the ferroxidase activity of ceruloplasmin. Only the BC loop of ferritin H chain decreased the amount of iron loading into ferritin by ceruloplasmin.

Amino Acid Sequence↗

PubMed1998

Evidence for a protein-protein complex during iron loading into ferritin by ceruloplasmin.

The formation of a protein-protein complex for the loading of iron into ferritin by ceruloplasmin was investigated. Ferritin stimulated the ferroxidase activity of ceruloplasmin unless the ferritin was fully loaded, in which case it inhibited the ferroxidase activity of ceruloplasmin. The apparent association constant for the interaction of ferritin and ceruloplasmin was 24 nM. Isothermal titration calorimetry indicated that the interaction of ceruloplasmin and ferritin was endothermic, driven by positive changes in entropy. The association constants for complex formation between ferritin and ceruloplasmin were 4.5 +/- 0.7 x 10(5) and 9.5 +/- 0.3 x 10(4) M-1 for the reduced and oxidized forms of ceruloplasmin, respectively. The oxidized form of ceruloplasmin was retained on an affinity column with ferritin immobilized as the ligand and remained bound to the column with mobile phases of increased hydrophobicity, but was eluted with increased ionic strength. The ability of ceruloplasmin to remain bound to the affinity resin was affected by the species from which ceruloplasmin was isolated. Gradient ultracentrifugation also provided evidence that the two proteins were associated, since ferritin promoted migration of ceruloplasmin through the gradient. Including ferrous iron in the gradient resulted in reduction of ceruloplasmin and increased the mobility of ceruloplasmin with ferritin. These data provide evidence that ferritin and ceruloplasmin form a protein-protein complex during iron loading into ferritin, which may limit redox cycling of iron in vivo.

Animals↗

PubMed1998

Mutational analysis of the four alpha-helix bundle iron-loading channel of rat liver ferritin.

We previously reported that the heavy chain of ferritin was required for loading it with iron using ceruloplasmin as a ferroxidase [J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335, 197-204]. Site-directed mutagenesis, K58E and G61H, on recombinant rat liver L chain ferritin (rL-Ft) was performed to construct a proposed iron-loading channel in the alpha-helix bundle similar to rat liver H chain ferritin (rH-Ft). Conversely, the channel in rH-Ft was closed by mutations E62K and H65G to form a K62 to E107 salt bridge, which is believed to exist in the L chain. Both variants were expressed in insect cells and were soluble and able to form multi-subunit homopolymers. The rH-Ft mutant homopolymer could not be loaded, whereas the rL-Ft mutant homopolymer could be loaded with iron by ceruloplasmin. However, we found that the initial rate of iron loading into the rL-Ft mutant homopolymer by ceruloplasmin was less than that into the rH-Ft homopolymer. When 500 atoms of iron per ferritin were used for loading, 98% was loaded into the rH-Ft homopolymer by ceruloplasmin in 15 min, but only 30% was loaded into the rL-Ft mutant homopolymer in the same time. Moreover, the ferroxidase activity of ceruloplasmin was enhanced in the presence of the rH-Ft and the rH-Ft mutant homopolymers, but not in the presence of the rL-Ft or the rL-Ft mutant homopolymers. These observations suggested that the four alpha-helix bundle channel of ferritin is required for iron loading, but an additional factor, i.e. , a site which stimulate the ferroxidase activity of ceruloplasmin, is also essential.

Animals↗

PubMed1998

The effect of putative nucleation sites on the loading and stability of iron in ferritin.

The L chain of the iron storage protein ferritin contains more putative nucleation sites in the core (Glu53, 56, 57, 60, and 63) than does the H chain (Glu61, 64, and 67). Recombinant DNA techniques were used to investigate the role of these putative nucleation sites on iron loading by ceruloplasmin and on the stability of the iron core. Recombinant rat liver ferritin H chain homopolymer and the two mutants (E61A and E61A-E64A), containing three, two and one nucleation sites, respectively, loaded up to 2010 +/- 50, 2010 +/- 40, and 1950 +/- 40 atoms of iron per ferritin, respectively. However, the mutations resulted in a 50% decrease in the rate of iron loading by ceruloplasmin. The ferritin variants incorporated the same amount of phosphate after iron loading (410 +/- 20, 400 +/- 30, and 420 +/- 20 atoms per ferritin, respectively). The stability of the iron cores prior to phosphate incorporation, assessed by the rate of iron release by 10 mM EDTA and the paraquat cation radical, corresponded to numbers of proposed nucleation sites. The subsequent incorporation of phosphate seemed to stabilize the iron core and minimized the effect of numbers of putative nucleation sites in ferritin on the rate of iron release by EDTA and the paraquat cation radical. After incorporation of phosphate the ferritins behaved similarly to the native rat liver ferritin with respect to the rate of iron release by the paraquat cation radical.

Animals↗

PubMed1998

Suppression of cell growth by heavy chain ferritin.

While producing recombinant rat liver H and L chain ferritin homopolymers using the baculovirus expression system, we noticed that rat liver H chain ferritin, but not L chain ferritin, had a suppressive effect on the growth of Spodoptera frugiperda (Sf-21) cells. Suppression was observed immediately after infection with recombinant H chain ferritin baculovirus prepared from lysed infected cells. Immediate suppression was observed when purified with either recombinant H chain apoferritin or various holoferritins (loaded with 1,970 +/- 50 or 2,520 +/- 90 atoms of iron/ferritin) indicating that suppression was not due to sequestration of iron required for cell growth. Suppression by H chain ferritin was also observed upon attempting to express the protein in Escherichia coli. Strategies for expression of recombinant rat liver H and L ferritin homopolymers in both prokaryotic and eukaryotic expression systems were developed.

Animals↗

PubMed1998

The effects of different buffers on the oxidation of DNA by thiols and ferric iron.

Because buffers can act as metal ligands, they can effect several reactions necessary for DNA oxidation by ferric iron and thiols, such as iron reduction. Therefore, these reactions were studied in Hepes and phosphate buffers and unbuffered NaCl. Reduction of Fe3+ by dithiothreitol (DTT) and cysteine was observed in either Hepes or NaCl solutions, but not in phosphate buffer. Thiyl radicals were observed in Hepes, but there was much less thiyl radical production in the saline or phosphate solutions. Redox cycling between either DTT or cysteine and Fe3+ also resulted in dioxygen consumption in Hepes buffer. Reduction of Fe3+ and O2 resulted in the formation of an oxidant capable of producing 8-hydroxy-2'-deoxyguanosine (8-OHdG) in calf-thymus DNA. The highest levels of 8-OHdG were detected when DTT or cysteine and Fe3+ were incubated in Hepes, while much less DNA oxidation was detected when the experiment was done in a saline solution, and almost no DNA oxidation occurred in the phosphate buffer. These results demonstrate that the use of different buffers can greatly affect the ability of thiols to promote iron-dependent oxidations.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1997

Detection and characterization of the lignin peroxidase compound II-veratryl alcohol cation radical complex.

Lignin peroxidases (LiP) from the white-rot fungus Phanerochaete chrysosporium oxidize veratryl alcohol (VA) by two electrons to veratryl aldehyde, although the VA cation radical (VA.+) is an intermediate [Khindaria, A., et al. (1995) Biochemistry 34, 6020-6025]. It was speculated, on the basis of kinetic evidence, that VA*+ can form a catalytic complex with LiP compound II. We have used low-temperature EPR to provide direct evidence for the formation of the complex. The EPR spectrum of VA*+ obtained at 4 K was explained by a model for coupling between the oxoferryl moiety of the heme (S = 1) and VA.+ (S = 1/2) similar to the model proposed for an oxyferryl and a porphyrin pi cation radical of horseradish peroxidase. The coupling constant suggested that VA.+ was equally ferro- and antiferromagnetically coupled to the oxoferryl moiety. The spectrum was simulated with g perpendicular only marginally greater than g parallel. This was surprising since the only other known organic radical coupled to the heme iron in a peroxidase is the tryptophan cation radical in cytochrome c peroxidase which exhibits a g tensor with g parallel greater than g perpendicular. Spin concentration analysis suggested that the 1 mol of VA*+ was coupled to the oxoferryl moiety per mole of enzyme. The VA.+ signal decayed with a first-order decay constant of 1.76 s-1, in close agreement with the earlier published decay constant of 1.85 s-1 from room-temperature EPR studies. The exchange coupling between VA.+ and the oxoferryl moiety strongly advocates calling this species (VA.+ and LiP compound II) a catalytic complex.

Basidiomycota↗

PubMed1997

Stimulation of the ferroxidase activity of ceruloplasmin during iron loading into ferritin.

Ceruloplasmin purified from horse serum was rapidly reduced upon addition of increasing equivalents of ferrous iron, generating an electronically and conformationally distinct form. This form of ceruloplasmin was characterized by significant (80%) loss of EPR detectable type I and type II copper(II), complete loss of visible absorbance at 610 nm, as well as decreased hydrophobic surface area. The reduced form of ceruloplasmin slowly reduced molecular oxygen to complete its catalytic cycle. The presence of varied concentrations of apoferritin, but not apotransferrin, significantly enhanced the rate of ceruloplasmin oxidation. The magnitude of this stimulatory effect increased as the molar ratio of ceruloplasmin to apoferritin approached 1.0, shown previously to be the optimum ratio for loading iron into ferritin. The rate of ferrous iron oxidation by ceruloplasmin was significantly stimulated by the presence of apoferritin; however, apotransferrin had no effect. The length of time required for ceruloplasmin to oxidize all the iron and return to the native form of the enzyme was also affected by the concentration of iron. In addition, the rate of iron loading into ferritin was dependent upon ferrous iron concentration. These results provide evidence for the formation of a specific complex between the reduced form of ceruloplasmin and apoferritin and that reduction of ceruloplasmin by ferrous iron may be the signal for complex formation.

Animals↗

PubMed1997

Effects of Mn2+ and oxalate on the catalatic activity of manganese peroxidase.

Manganese peroxidase from Phanerochaete chrysosporium is an extracellular heme-containing enzyme known to catalyze the oxidation of Mn2+ to Mn3+ in a reaction requiring oxalate or another appropriate manganese chelator. We have found that the enzyme can also catalyze a manganese-dependent disproportionation of hydrogen peroxide when a manganese chelator is not included. The catalatic activity was observed in the pH range from 3.0 to 8.5, and the apparent second-order rate constant for catalatic reaction was about 2 x 10(5) M-1 s-1 at pH 4.5 to 7.0 at 25 degrees C. Oxalate inhibited oxygen production by increasing the apparent K(m) for Mn2+ for catalatic activity from micromolar to millimolar levels and facilitating peroxidase activity. Catalase-type function was recovered by excess of Mn2+ in the presence of oxalate. We propose that catalatic activity may protect the enzyme from inactivation by hydrogen peroxide in an environment where free oxalate may be limited.

Animals↗

PubMed1997

Thermodynamics of binding of the distal calcium to manganese peroxidase.

We previously demonstrated that manganese peroxidase from Phanerochaete chrysosporiumwas susceptible to thermal inactivation due to release of the distal calcium, which maintained the distal heme environment of the enzyme [Sutherland, G. R. J., Zapanta, L. S., Tien, M., & Aust, S. D. (1997) Biochemistry 36, 3654-3662]. In this investigation the binding of calcium to the distal calcium binding site of manganese peroxidase was studied by optical absorption spectroscopy and isothermal titration calorimetry. The dissociation constant for the distal calcium binding site was 11 +/- 1 microM and the Hill coefficient was 1.1 +/- 0.1. The binding of calcium was accompanied by decreases in enthalpy and entropy that were large compared to those of other calcium binding proteins. The decreases were consistent with the large conformational changes proposed to occur in manganese peroxidase as a result of the binding and release of the distal calcium. Studies involving binding of the hydrophobic fluorescent probe, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium salt (bis-ANS), to manganese peroxidase indicated that the active, calcium-containing form of the enzyme had less exposed hydrophobic surface area, which would contribute to an increase in enthalpy and entropy upon calcium binding. Therefore, the negative changes in enthalpy and entropy associated with calcium binding were attributed to a large increase in the structural rigidity and compactness of the enzyme. The dissociation constant for calcium decreased and the rate of thermal inactivation decreased with decreasing pH. However, both the ability of calcium to prevent thermal inactivation of manganese peroxidase and the rate of calcium binding decreased as the pH decreased. Therefore it was proposed that, at lower pH, calcium binding to manganese peroxidase was more thermodynamically favorable, but the rate of calcium binding decreased because the flexibility of the calcium binding site, and in turn exposure of the ligands to the incoming ion, decreased.

Anilino Naphthalenesulfonates↗

PubMed1997

Kinetics of calcium release from manganese peroxidase during thermal inactivation.

It was previously reported that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was susceptible to thermal inactivation because it contains relatively labile Ca2+ ions required for stability and activity [Sutherland and Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. In this work we determined that four Ca2+ ions are present in the enzyme as isolated but this was reduced to 2 mol/mol upon treatment with Ca2+-chelating agents or extensive dialysis of dilute enzyme. One of two relatively tightly bound Ca2+ remaining in the enzyme was released during thermal inactivation at pH 7.2. Inactive enzyme contained one Ca2+ which could be removed in acidic conditions. Inactivation kinetics were biphasic and the rates for the two inactivation steps and the release of Ca2+ during inactivation suggested that the first, faster phase of inactivation was coupled to the removal of Ca2+. The weakly associated Ca2+ normally present in the enzyme did not affect enzyme activity and did not seem to protect the enzyme from thermal inactivation at submicromolar enzyme concentrations. Excess Ca2+ or Mn2+ decreased the rate of the thermal inactivation and Mn2+ stabilized the enzyme more efficiently than Ca2+ at higher temperature. Enzyme stabilization by Mn2+ was proposed to be due to binding of Mn2+ to the Mn2+ substrate binding site. In competition studies, Ca2+ was shown to bind to this site with apparent dissociation constants of 10(-2) and 10(-4) M at pH 4.5 and 7.2, respectively. Moreover, Ca2+ was a poor inhibitor of manganese peroxidase activity at pH 4.5. It is therefore suggested that Ca2+ is absent from the substrate site in physiological conditions but can bind to this site at higher pH and therefore may stabilize the enzyme by binding to both the Mn2+ site and, as previously proposed, to the distal Ca2+ site.

Binding Sites↗

PubMed1997

Loading of iron into recombinant rat liver ferritin heteropolymers by ceruloplasmin.

We have reported previously that the heavy chain of ferritin is required for iron incorporation by ceruloplasmin (J.-H. Guo, M. Abedi, and S. D. Aust (1996) Arch. Biochem. Biophys. 335(1)). The purpose of this study was to determine how many heavy chains were required for ceruloplasmin to interact with ferritin such that iron loading occurred. The cDNA sequences encoding the heavy and light chains of rat liver ferritin were cloned into the baculovirus transfer vector pA-cUW51 under the control of polyhedrin and p10 promoters, respectively, which was then incorporated by homologous recombination into the infections Autographa californica nuclear polyhedrosis virus genome. Both ferritin chains were expressed and assembled into two heteropolymers following the infection of insect cells by recombinant virus, which were separated by DEAE-Sepharose chromatography. The percentage of heavy (H) and light (L) chains making up the two heteropolymers, determined by gel scanning following the resolution of chains on SDS-PAGE, were equivalent to 1 H and 23 L chains and 2 H and 22 L chains. The maximal extent of iron loading was observed using 1 mol of rat ceruloplasmin per mole of H chain in the two heteropolymers. The extent of iron incorporation decreased with additional ceruloplasmin. Iron incorporation into rat liver ferritin, found to contain 10 H chains, increased as the molar ratio of ceruloplasmin to ferritin increased to 4:1 and remained the same up to 8:1. Iron loading into horse spleen ferritin, found to have one H chain, appeared similar to that for recombinant ferritin, having only one H chain. Therefore, we propose that the optimal molar ratio of ceruloplasmin to ferritin depends upon the numbers of H chain making up the ferritin molecule for the maximal incorporation of iron into ferritin. These results also suggest that the iron loading channel is contained within a single H chain subunit.

Animals↗

PubMed1997

Spectral changes of lignin peroxidase during reversible inactivation.

The heme environment of lignin peroxidase (LiP) has been investigated by electronic absorption and electron paramagnetic resonance (EPR) spectroscopy. Native LiP was a pentacoordinate, high-spin ferric iron with a high-spin absorption band at 634 nm and g values at 5.86 and 2.07 in the EPR spectrum. Upon thermal inactivation, calcium ions were released from the enzyme and the Soret absorption decreased and red-shifted about 2 nm, the high-spin absorption band at 634 nm disappeared, and a low-spin absorption band appeared at 532 nm. The EPR spectrum and the temperature dependence of electronic absorption spectra revealed that the heme iron of the thermally inactivated enzyme was a mixture of high- and low-spin states, which was further supported by the changes in the electronic absorption and EPR spectra when cyanide was added to the thermally inactivated enzyme. Addition of various imidazoles or CN- to thermally inactivated enzyme demonstrated that the low-spin heme iron of inactivated enzyme was hexacoordinate with a distal histidine as its sixth ligand, in contrast to the active enzyme, which was pentacoordinate and high-spin. Upon addition of calcium to recover the thermally inactivated LiP, the reactivated enzyme had absorptions at 408, 502, and 634 nm and g values at 5.86 and 2.07 in the EPR spectrum, which demonstrated that the heme iron of the reactivated enzyme was again high-spin and pentacoordinated.

Calcium↗

PubMed1997

Role of calcium in maintaining the heme environment of manganese peroxidase.

We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland & Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pKa of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near-ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

Basidiomycota↗

PubMed1997

Effect of calcium on the reversible thermal inactivation of lignin peroxidase.

This study investigated the effects of calcium on the thermal inactivation of lignin peroxidase from Phanerochaete chrysosporium. The monophasic loss of veratryl alcohol oxidase activity corresponded to the loss of calcium when the enzyme was thermally inactivated. Addition of calcium slowed and oxalate and EGTA increased the apparent inactivation rate. The thermally inactivated lignin peroxidase could be readily reactivated by addition of Ca2+. The amount of activity recovered was dependent on temperature, Ca2+ concentration, and incubation conditions. Enzyme activity could be recovered up to 95% of its original value by addition of Ca2+ when lignin peroxidase was depleted of Ca2+ by incubation with EGTA. Although heme absorbance decreased when the enzyme was thermally inactivated, the amount of iron in the enzyme did not change. Changes in the heme environment of the inactivated enzyme were suggested by changes in the electronic absorption in which the Soret band shifted from 408 to 410 nm, the absorption at 502 nm shifted to 532 nm, and the absorption at 634 nm disappeared upon inactivation. Upon the addition of Ca2+, the bands returned to the original wavelength. Therefore, it is proposed that the inactivation mechanism of lignin peroxidase is that the loss of calcium causes heme environmental changes resulting in the loss of enzyme activity.

Basidiomycota↗

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↗