Effects of cadmium on manganese peroxidase (original) (raw)
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Bacterial Mn 2+ Oxidizing Systems and Multicopper Oxidases: An Overview of Mechanisms and Functions
Geomicrobiology Journal, 2000
Manganese is oxidized by a wide variety of bacteria. The current state of knowledge on mechanisms and functions of Mn 2+ oxidation in two strains of Pseudomonas putida, in Leptothrix discophora SS-1, and in Bacillus sp. strain SG-1 is reviewed. In all three species, proteins bearing resemblance to multicopper oxidases appear to be involved in the oxidation process. A short description of the classi cation of Cu centers is followed by a more detailed review of properties and postulated functions of some well-known multicopper oxidases. Finally, suggestions are made for future research to assess the potential role of multicopper oxidases in bacterial Mn 2+ oxidation.
Heterogeneous inhibition of horseradish peroxidase activity by cadmium
Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd 2 + for various periods of time. The H 2 O 2-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd 2 + was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd 2 + concentration. Furthermore, for the same inhibition type, K i values dropped as incubation time increased. These results suggested that Cd 2 + would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd 2 + altered the heme Soret absorption band on binding HRP and exhibited a K d which decreased as the incubation time of HRP with Cd 2 + increased. Hill plots suggested a cooperative binding of up to three Cd 2 + ions per molecule of HRP. Thus, Cd 2 + binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd 2 + ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.
Biochemistry, 2001
Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of the extracellular lignin degrading system in these organisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn II and efficiently release Mn III. Initial site-directed mutagenesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-type Mn binding and rate of oxidation, calling into question the role of E39 as an Mn ligand. To investigate this hypothesis, we performed computer modeling studies which indicated metal-ligand bond distances in the E39D variant and in an E35D-E39D-D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D variants used in the previous study, as well as an E39A single variant and the E35D-E39D-D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn II binding (K m increases 20-30-fold) and Mn II oxidation (k cat decreases 50-400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The catalytic efficiency of the E39A variants decreased ∼10 4-fold, while that of the E39D variant decreased ∼10 3-fold. Contrary to initial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of the triple variant decreased only 10 4-fold, which is ∼10 2-fold better than that reported for the E35Q-D179N double variant. These combined studies indicate that precise geometry of the Mn ligands within the Mn binding site of MnP is essential for the efficient binding, oxidation, and release of Mn by this enzyme. The results clearly indicate that E39 is a Mn ligand and that mutation of this ligand decreases both Mn binding and the rate of Mn oxidation.
Cadmium-Dependent Enzyme Activity Alteration Is Not Imputable to Lipid Peroxidation
Archives of Biochemistry and Biophysics, 2000
The effect of cadmium on the liver-specific activities of NADPH-cytochrome P450 reductase (CPR), malic dehydrogenase (MDH), glyceraldehyde-3-phosphate dehydrogenase (GADPH), and sorbitol dehydrogenase (SDH) was assessed 6, 24, and 48 h after administration of the metal to rats (2.5 mg/kg of body weight, as CdCl 2 , single ip injection). CPR specific activity increased after 6 h and afterward decreased significantly, while MDH specific activity increased up to 24 h and then remained unchanged. Both SDH and GADPH specific activities reduced after 6 h, the former only a little but the latter much more, and after 24 and 48 h were strongly inhibited. In vitro experiments, by incubating rat liver microsomes, mitochondria, or cytosol with CdCl 2 in the pH range 6.0 -8.0, excluded cadmium-induced lipid peroxidation as the cause of the reduction in enzyme activity. In addition, from these experiments, we obtained indications on the type of interactions between cadmium and the enzymes studied. In the case of CPR, the inhibitory effect is probably due to Cd 2؉ binding to the histidine residue of the apoenzyme, which, at physiological pH, acts as a nucleophilic group. In vitro, mitochondrial MDH was not significantly affected by cadmium at any pH, indicating that this enzyme is probably not involved in the decrease in mitochondrial respiration caused by this metal. As for GADPH specific activity, its inhibition at pH 7.4 and above is imputable to the binding of cadmium to the SH groups present in the enzyme active site, since in the presence of dithiothreitol this inhibition was removed. SDH was subjected to a dual effect when cytosol was exposed to cadmium. At pH 6.0 and 6.5, its activity was strongly stimulated up to 75 M CdCl 2 while at higher metal concentrations it was reduced. At pH 7.4 and 8.0, a stimulation up to 50 M CdCl 2 occurred but above this concentration, a reduc-tion was found. These data seem to indicate that cadmium can bind to different enzyme sites. One, at low cadmium concentration, stimulates the SDH activity while the other, at higher metal concentrations, substitutes for zinc, thus causing inhibition. This last possibility seems to occur in vivo essentially at least 24 h after intoxication. The cadmium-induced alterations of the investigated enzymes are discussed in terms of the metabolic disorders produced which are responsible for several pathological conditions.
2016
This study investigated the steady-state kinetics of engineered wild-type and manganese (II) binding site mutants of recombinant Phlebia radiata manganese peroxidase 3(rPr-MnP3). The effect (activation or inhibition) of some metal ions (Co2+, Zn2+ Cu2+ and Na+) on the activity of rPr-MnP3 enzymes was also studied. The results obtained showed that the rPr-MnP3 mutants in which the metal binding functionality has been largely lost have been created. Na+ (mono-valent ion) and Co2+showed similar characteristics by exhibiting stimulatory effects on the activity of wild-type rPr-MnP3. However, Cu2+ and Zn2+ had mixed inhibitory effects on wild-type and mutants (E40H, E44H, E40H/E44H). It was observed that Cu2+ was by far the strongest inhibitor of engineered rPr-MnP3 enzymes while Co2+ exhibited a non-competitive inhibitory effect on the double mutant (E40H/E44H) and D186H activities. In addition, Zn2+ and Cu2+also had non-competitive inhibitory effect on D186H mutant enzyme activity. The...
Biochemistry, 2001
Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of the extracellular lignin degrading system in these organisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn II and efficiently release Mn III. Initial site-directed mutagenesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-type Mn binding and rate of oxidation, calling into question the role of E39 as an Mn ligand. To investigate this hypothesis, we performed computer modeling studies which indicated metal-ligand bond distances in the E39D variant and in an E35D-E39D-D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D variants used in the previous study, as well as an E39A single variant and the E35D-E39D-D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn II binding (K m increases 20-30-fold) and Mn II oxidation (k cat decreases 50-400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The catalytic efficiency of the E39A variants decreased ∼10 4-fold, while that of the E39D variant decreased ∼10 3-fold. Contrary to initial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of the triple variant decreased only 10 4-fold, which is ∼10 2-fold better than that reported for the E35Q-D179N double variant. These combined studies indicate that precise geometry of the Mn ligands within the Mn binding site of MnP is essential for the efficient binding, oxidation, and release of Mn by this enzyme. The results clearly indicate that E39 is a Mn ligand and that mutation of this ligand decreases both Mn binding and the rate of Mn oxidation.
Manganese pre-treatment attenuates cadmium induced hepatotoxicity in Swiss albino mice
Journal of Trace Elements in Medicine and Biology, 2015
Cadmium (Cd) is a soft, malleable bluish-white metal with low melting point, a ubiquitous heavy metal and an environmental pollutant, found in soil, water and air. The presence of Cd in the components of the environment such as air, soil and groundwater is to a large part due to human activity, and the general population is exposed mainly by contaminated drinking water or food. Manganese (Mn) is a component in many enzymes, which play an important role in counteracting oxidative stress. In vitro experiments have revealed the ability of Mn to scavenge oxygen free radicals generated in differently mediated lipid peroxidation (LPO) conditions. The aim of the present study was to investigate the in vivo preventive effect of Mn 2+ pre-treatment on acute Cd-intoxication with regard to oxidative stress biomarker and antioxidant defense system in liver of Swiss albino mice. On exposure to Cd a significant increase in LPO levels, decrease in thiol content and induction in glutathione metabolizing enzyme were observed. Mn pre-treatment attenuated the modulation caused in the above-mentioned parameters due to acute Cd exposure in mice. In conclusion, the results from this study demonstrate that the protective effect of Mn in Cd-induced systemic toxicity in mice. Further investigations are required on the relation between Mn accumulation and resistance to oxidative stress and on the factors influencing Mn/Cd transport in rodents are needed to elucidate the molecular basis of this protective effect.
American Journal of Molecular Biology
The goal of this study was to determine whether mutation of the Mn-binding site of wild-type recombinant Phlebia radiata manganese peroxidase 3 affected the pH-dependence kinetic parameters. pH range investigated was 2.5-12.0. The catalytic efficiency of the mutant enzymes at high and low pH in comparison to the wild-type was investigated using standard rPr-MnP3 protocol. Wild-type recombinant Phlebia radiata MnP3 enzyme showed optimal activity with Mn (II) as substrate at pH 5.0 and remained moderately active (approximately 40%) in the pH range of 6.0-9.0. The rPr-MnP3 mutants' maximum activity ranged between 5.5 and 8.0. Wild-type and mutants rPr-MnP3 enzymes exhibited a similar pH profile with optimum pH of 3.0 for ABTS oxidation. Mutation has severely decreased the catalytic efficiency for Mn (II) oxidation at pH 5.0. The rPr-MnP3 enzymes showed enhanced affinity for Mn (II) at alkaline pH and a more alkaline range for catalysis than ever reported for any Manganese Peroxidase. This study reveals that at higher pH, rPr-MnP3 can function with alternative ligands in the Mn (II) site and does not have an absolutely obligate requirement for an all carboxylate ligand set. These results further strongly confirm that Mn 2+ binding site is the only productive catalytic site for Mn (II) oxidation.
Inorganic Chemistry, 2009
Herein, we report reactivity studies of the mononuclear water-soluble complex [Mn(II)(HPClNOL)(η 1 -NO 3 )(η 2 -NO 3 )] 1, where HPClNOL ) 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, toward peroxides (H 2 O 2 and tertbutylhydroperoxide). Both the catalase (in aqueous solution) and peroxidase (in CH 3 CN) activities of 1 were evaluated using a range of techniques including electronic absorption spectroscopy, volumetry (kinetic studies), pH monitoring during H 2 O 2 disproportionation, electron paramagnetic resonance (EPR), electrospray ionization mass spectrometry in the positive ion mode [ESI(+)-MS], and gas chromatography (GC). Electrochemical studies showed that 1 can be oxidized to Mn(III) and Mn(IV). The catalase-like activity of 1 was evaluated with and without pH control. The results show that the pH decreases when the reaction is performed in unbuffered media. Furthermore, the activity of 1 is greater in buffered than in unbuffered media, demonstrating that pH influences the activity of 1 toward H 2 O 2 . For the reaction of 1 with H 2 O 2 , EPR and ESI(+)-MS have led to the identification of the intermediate [Mn(III)Mn(IV)(µ-O) 2 (PClNOL) 2 ] + . The peroxidase activity of 1 was also evaluated by monitoring cyclohexane oxidation, using H 2 O 2 or tert-butylhydroperoxide as the terminal oxidants. Low yields (<7%) were obtained for H 2 O 2 , probably because it competes with 1 for the catalase-like activity. In contrast, using tert-butylhydroperoxide, up to 29% of cyclohexane conversion was obtained. A mechanistic model for the catalase activity of 1 that incorporates the observed lag phase in O 2 production, the pH variation, and the formation of a Mn(III)-(µ-O) 2 -Mn(IV) intermediate is proposed. (1) (a) Sies, H. Angew. Chem. 1986, 25, 1058-1071. (b) Doctrow, S. R.; Huffman, K.; Marcus, C. B.; Tocco, G.; Malfroy, E.; Adinolfi, C. A.; Kruk, H.; Baker, K.; Lazarowych, N.; Mascarenhas, J.; Malfroy, B.
Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium
Toxicology, 2002
Catalase, Mn-superoxide dismutase (MnSOD) and Cu,Zn-superoxide dismutase (CuZnSOD) activities were studied in rat liver and kidney 6-48 h after CdCl 2 intraperitoneal administration or 10 -30 days daily oral CdCl 2 intake in drinking water. This approach provided some indications as to the sensitivity of each enzyme to cadmium toxicity. These experiments showed that the formation of thiobarbituric acid reactive substance (TBARS) did not strictly depend on how well the antioxidant enzyme worked. From in vitro experiments it appeared that TBARS removal by vitamin E did not restore the three enzyme activities at all. As for cadmium's inhibitory mechanism on catalase activity, our data, obtained in the pH range 6.0-8.0, are a preliminary indication that the negative effect of this metal is probably due to imidazole residue binding of His-74 which is essential in the decomposition of hydrogen peroxide. Cadmium inhibition of liver mitochondrial MnSOD activity was completely removed by Mn 2 + ions, suggesting that the reducing effect on this enzyme is probably due to the substitution of cadmium for manganese. We also observed the antioxidant capacity of Mn 2 + ions, since they were able to normalize the increased TBARS levels occurring when liver mitochondria were exposed to cadmium. The reduced activity of CuZnSOD does not seem to be due to the replacement of Zn by Cd, nor to the peroxides formed. As this enzyme activity was almost completely recovered after 48 h, we hypothesize that the momentary inhibition is imputable to a cadmium/enzyme interaction. This causes some perturbation in the enzyme topography which is critical for its catalytic activity. The pathological implications linked to antioxidant enzyme disorders induced by cadmium toxicity are discussed.