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Recycling of the high valence States of heme proteins by cysteine residues of THIMET-oligopeptidase.

Ferreira JC, Icimoto MY, Marcondes MF, Oliveira V, Nascimento OR, Nantes IL - PLoS ONE (2013)

Bottom Line: The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme.The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging.These findings are discussed towards a possible occurrence of these reactions in cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brasil ; Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brasil.

ABSTRACT
The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H2O2. The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging. These findings are discussed towards a possible occurrence of these reactions in cells.

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EPR spectra of Mb.A) Absence of TOP. Line a = 5 µM Fe3+Mb, lines b, c and d are the respective EPR spectra obtained immediately, 30 min, and 180 min after the addition of 50 µM H202. B) Presence of 20 µM TOP: Line a = 5 µM Fe3+Mb, lines b, c, d and e are the respective EPR spectra obtained immediately, 30, 60, and 90 min after the addition of 50 µM H2O2. The expanded field view shows the signal of a free radical signal overlapped on the g2 component of EPR spectrum of heme iron obtained 30 min after the addition of H2O2. When present, TOP was previously treated with 1 mM TCEP. The concentration of DMPO = 20 mM. EPR conditions were: microwave frequency = 9.47177 GHz, central field, 240 mT, scanning field, 400 mT, number of points, 2048, modulation amplitude, 1 mT, gain, 45 dB, temperature, 4.30 K, time constant, 20.5 ms, conversion time, 81.9 ms, microwave power, 5 mW. The reactions were carried out in 20 mM Tris buffer pH 7.4 treated with Chelex-100®.
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pone-0079102-g003: EPR spectra of Mb.A) Absence of TOP. Line a = 5 µM Fe3+Mb, lines b, c and d are the respective EPR spectra obtained immediately, 30 min, and 180 min after the addition of 50 µM H202. B) Presence of 20 µM TOP: Line a = 5 µM Fe3+Mb, lines b, c, d and e are the respective EPR spectra obtained immediately, 30, 60, and 90 min after the addition of 50 µM H2O2. The expanded field view shows the signal of a free radical signal overlapped on the g2 component of EPR spectrum of heme iron obtained 30 min after the addition of H2O2. When present, TOP was previously treated with 1 mM TCEP. The concentration of DMPO = 20 mM. EPR conditions were: microwave frequency = 9.47177 GHz, central field, 240 mT, scanning field, 400 mT, number of points, 2048, modulation amplitude, 1 mT, gain, 45 dB, temperature, 4.30 K, time constant, 20.5 ms, conversion time, 81.9 ms, microwave power, 5 mW. The reactions were carried out in 20 mM Tris buffer pH 7.4 treated with Chelex-100®.

Mentions: To detect and characterize any radical species formed during the Mb peroxidase cycle in the absence (Figure 3A) and in the presence of TOP (Figure 3B), the reaction of Mb with H2O2 was accompanied by EPR spectroscopy at helium liquid temperature. The heme iron EPR spectra were obtained at different reaction time intervals with samples frozen at defined times and run at the indicated temperature. The EPR spectrum of native Mb obtained in 20 mM Tris buffer (line a), pH 7.4, reveals, at the low field region, the presence of Fe3+ heme iron in the high spin form (g┴ = 5.85 and g// = 2.00) with spin 5/2 and axial symmetry. The addition of H2O2 led to the disappearance of the Fe3+ EPR signal, which was in accordance to the formation of compound II, an EPR-silent species (Figure 3A) [40,42]. The g = 4.3 signal could be assigned to an oxidized form of the porphyrin ring and is normally associated with Soret band bleaching. Recycling of high valence states of Mb was not detected up to 180 min after the addition of H2O2, (Figure 3A, line d). In the presence of TOP, immediately after the addition of H2O2 (line b), a reminiscent Mb heme iron signal was observed. Significant recycling of Mb Compound II to the resting form was observed 90 min after the addition of H2O2, (Figure 3B). The recycling of the Mb high valence state by the presence of TOP led to a high yield of free radical generation.


Recycling of the high valence States of heme proteins by cysteine residues of THIMET-oligopeptidase.

Ferreira JC, Icimoto MY, Marcondes MF, Oliveira V, Nascimento OR, Nantes IL - PLoS ONE (2013)

EPR spectra of Mb.A) Absence of TOP. Line a = 5 µM Fe3+Mb, lines b, c and d are the respective EPR spectra obtained immediately, 30 min, and 180 min after the addition of 50 µM H202. B) Presence of 20 µM TOP: Line a = 5 µM Fe3+Mb, lines b, c, d and e are the respective EPR spectra obtained immediately, 30, 60, and 90 min after the addition of 50 µM H2O2. The expanded field view shows the signal of a free radical signal overlapped on the g2 component of EPR spectrum of heme iron obtained 30 min after the addition of H2O2. When present, TOP was previously treated with 1 mM TCEP. The concentration of DMPO = 20 mM. EPR conditions were: microwave frequency = 9.47177 GHz, central field, 240 mT, scanning field, 400 mT, number of points, 2048, modulation amplitude, 1 mT, gain, 45 dB, temperature, 4.30 K, time constant, 20.5 ms, conversion time, 81.9 ms, microwave power, 5 mW. The reactions were carried out in 20 mM Tris buffer pH 7.4 treated with Chelex-100®.
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Related In: Results  -  Collection

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pone-0079102-g003: EPR spectra of Mb.A) Absence of TOP. Line a = 5 µM Fe3+Mb, lines b, c and d are the respective EPR spectra obtained immediately, 30 min, and 180 min after the addition of 50 µM H202. B) Presence of 20 µM TOP: Line a = 5 µM Fe3+Mb, lines b, c, d and e are the respective EPR spectra obtained immediately, 30, 60, and 90 min after the addition of 50 µM H2O2. The expanded field view shows the signal of a free radical signal overlapped on the g2 component of EPR spectrum of heme iron obtained 30 min after the addition of H2O2. When present, TOP was previously treated with 1 mM TCEP. The concentration of DMPO = 20 mM. EPR conditions were: microwave frequency = 9.47177 GHz, central field, 240 mT, scanning field, 400 mT, number of points, 2048, modulation amplitude, 1 mT, gain, 45 dB, temperature, 4.30 K, time constant, 20.5 ms, conversion time, 81.9 ms, microwave power, 5 mW. The reactions were carried out in 20 mM Tris buffer pH 7.4 treated with Chelex-100®.
Mentions: To detect and characterize any radical species formed during the Mb peroxidase cycle in the absence (Figure 3A) and in the presence of TOP (Figure 3B), the reaction of Mb with H2O2 was accompanied by EPR spectroscopy at helium liquid temperature. The heme iron EPR spectra were obtained at different reaction time intervals with samples frozen at defined times and run at the indicated temperature. The EPR spectrum of native Mb obtained in 20 mM Tris buffer (line a), pH 7.4, reveals, at the low field region, the presence of Fe3+ heme iron in the high spin form (g┴ = 5.85 and g// = 2.00) with spin 5/2 and axial symmetry. The addition of H2O2 led to the disappearance of the Fe3+ EPR signal, which was in accordance to the formation of compound II, an EPR-silent species (Figure 3A) [40,42]. The g = 4.3 signal could be assigned to an oxidized form of the porphyrin ring and is normally associated with Soret band bleaching. Recycling of high valence states of Mb was not detected up to 180 min after the addition of H2O2, (Figure 3A, line d). In the presence of TOP, immediately after the addition of H2O2 (line b), a reminiscent Mb heme iron signal was observed. Significant recycling of Mb Compound II to the resting form was observed 90 min after the addition of H2O2, (Figure 3B). The recycling of the Mb high valence state by the presence of TOP led to a high yield of free radical generation.

Bottom Line: The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme.The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging.These findings are discussed towards a possible occurrence of these reactions in cells.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brasil ; Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brasil.

ABSTRACT
The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H2O2. The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging. These findings are discussed towards a possible occurrence of these reactions in cells.

Show MeSH
Related in: MedlinePlus