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Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

Ferreira JC, Marcondes MF, Icimoto MY, Cardoso TH, Tofanello A, Pessoto FS, Miranda EG, Prieto T, Nascimento OR, Oliveira V, Nantes IL - PLoS ONE (2015)

Bottom Line: This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species.Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils.This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

ABSTRACT
We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.

No MeSH data available.


Related in: MedlinePlus

EPR spectra of tyrosyl DBNBS-adduct immobilized in Cygb challenged by hydrogen peroxide.The upper panel shows the EPR spectrum of tyrosyl DBNBS adduct in the native protein and the lower panel shows EPR spectrum of tyrosyl DBNBS adduct after digestion by proteinase K. The simulation of the EPR spectra of DBNBS adduct was performed as described in materials and methods. The simulation parameters obtained for this spectrum were: AN = 13.5 Gauss, g = 2.0061, L/G = 0.2 and LW = 2.40 Gauss, considering the tumbling effect (LW = a + b x m + c x m2 and a = 2.3 b = -0.2 and c = 0.3128). The EPR parameters are consistent with tyrosyl-DBNBS adduct. The inset of upper panel shows a zoom of heme region in the tertiary Cygb structure. The residue represented in red corresponds to Tyr59. Tyr131 and Trp123 are represented in yellow. The inset of the lower panel shows the structure of a C-1-centered radical of tyrosine phenoxyl ring. The EPR spectra were measured using 20 μmol.L-1 protein solution, 1 mmol.L-1 hydrogen peroxide and 20 mmol.L-1 DBNBS. These results are representative of three independent replicates.
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pone.0136554.g008: EPR spectra of tyrosyl DBNBS-adduct immobilized in Cygb challenged by hydrogen peroxide.The upper panel shows the EPR spectrum of tyrosyl DBNBS adduct in the native protein and the lower panel shows EPR spectrum of tyrosyl DBNBS adduct after digestion by proteinase K. The simulation of the EPR spectra of DBNBS adduct was performed as described in materials and methods. The simulation parameters obtained for this spectrum were: AN = 13.5 Gauss, g = 2.0061, L/G = 0.2 and LW = 2.40 Gauss, considering the tumbling effect (LW = a + b x m + c x m2 and a = 2.3 b = -0.2 and c = 0.3128). The EPR parameters are consistent with tyrosyl-DBNBS adduct. The inset of upper panel shows a zoom of heme region in the tertiary Cygb structure. The residue represented in red corresponds to Tyr59. Tyr131 and Trp123 are represented in yellow. The inset of the lower panel shows the structure of a C-1-centered radical of tyrosine phenoxyl ring. The EPR spectra were measured using 20 μmol.L-1 protein solution, 1 mmol.L-1 hydrogen peroxide and 20 mmol.L-1 DBNBS. These results are representative of three independent replicates.

Mentions: To corroborate the identity of the protein-centered free radical detected by direct CW-EPR measurements at low temperature, we also analyzed the free radical using an EPR spin trapping technique. The formation of the tyrosyl radical during the reaction of Cygb with hydrogen peroxide is related to the mechanism of the peroxide cleavage. It is expected that the reaction of Cygb with hydrogen peroxide converts the heme group to oxo-ferryl π cation species. However, considering the similarity of the Cygb structure with that of Mb, it is possible that the oxidizing equivalent of the porphyrin ring is transferred to a globin amino acid residue. In this case, it is more likely that the oxidation of tyrosine 59, which is positioned close to the vinyl, methyl edge of the heme group, occurs. The tyrosine residue 123 must not be discarded because it is also in the proximity of the edge of the heme group. Tryptophan 171 is farther from the heme group and could be a secondary location of the radical, more likely by the transfer of one oxidant equivalent of a tyrosyl radical. DBNBS added to a Cygb solution challenged by hydrogen peroxide provided an EPR spectrum consistent with that of a partially immobilized nitroxide (Fig 8, upper panel). EPR signals were not detected when any of the reactants was absent (not shown). The EPR spectra of the DBNBS adduct was simulated, as described in the Materials and Methods section. The simulation resulted in a large Gaussian line width (3.77 G). For this tyrosyl-DBNBS adduct, the large linewidth results from the super hyperfine interactions in a highly heterogeneous microenvironment around nitroxide radical; these interactions were not resolved by the CW EPR spectrum [47]. The oxidative potential of tyrosine and tryptophan are similar; when these residues are neighbors in a protein structure, the unpaired electron density can be found in these amino acids in a population of hemeproteins treated with peroxides. However, considering the EPR parameters of the direct EPR signal and the location of tyrosine 59 and 123 at the vicinity of heme group edge (inset of Fig 8, upper panel), it is reasonable to assign the location of the unpaired electron to a tyrosine residue [49,67]. When DBNBS was added to the Cygb solution challenged by hydrogen peroxide and the sample underwent nonspecific proteolysis by proteinase K, an isotropic ESR spectrum consisting primarily of three lines was detected (Fig 8, lower panel). The simulation parameters obtained for this spectrum were: AN = 13.5 G, g = 2.0061, L/G = 0.2 and LW = 2.40 G, considering the tumbling effect (LW = a + b x m + c x m2 and a = 2.3, b = -0.2 and c = 0.3128). The EPR parameters are consistent with a tyrosyl-DBNBS adduct. The EPR spectrum does not exhibit additional hyperfine structure that is consistent with a tertiary carbon-centered radical adduct establishing no bonds with atoms with nuclear spin. Such a structural feature is consistent with the C-1-centered radical of a tyrosine phenoxyl ring (inset Fig 8, lower panel).


Intermediate Tyrosyl Radical and Amyloid Structure in Peroxide-Activated Cytoglobin.

Ferreira JC, Marcondes MF, Icimoto MY, Cardoso TH, Tofanello A, Pessoto FS, Miranda EG, Prieto T, Nascimento OR, Oliveira V, Nantes IL - PLoS ONE (2015)

EPR spectra of tyrosyl DBNBS-adduct immobilized in Cygb challenged by hydrogen peroxide.The upper panel shows the EPR spectrum of tyrosyl DBNBS adduct in the native protein and the lower panel shows EPR spectrum of tyrosyl DBNBS adduct after digestion by proteinase K. The simulation of the EPR spectra of DBNBS adduct was performed as described in materials and methods. The simulation parameters obtained for this spectrum were: AN = 13.5 Gauss, g = 2.0061, L/G = 0.2 and LW = 2.40 Gauss, considering the tumbling effect (LW = a + b x m + c x m2 and a = 2.3 b = -0.2 and c = 0.3128). The EPR parameters are consistent with tyrosyl-DBNBS adduct. The inset of upper panel shows a zoom of heme region in the tertiary Cygb structure. The residue represented in red corresponds to Tyr59. Tyr131 and Trp123 are represented in yellow. The inset of the lower panel shows the structure of a C-1-centered radical of tyrosine phenoxyl ring. The EPR spectra were measured using 20 μmol.L-1 protein solution, 1 mmol.L-1 hydrogen peroxide and 20 mmol.L-1 DBNBS. These results are representative of three independent replicates.
© Copyright Policy
Related In: Results  -  Collection

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pone.0136554.g008: EPR spectra of tyrosyl DBNBS-adduct immobilized in Cygb challenged by hydrogen peroxide.The upper panel shows the EPR spectrum of tyrosyl DBNBS adduct in the native protein and the lower panel shows EPR spectrum of tyrosyl DBNBS adduct after digestion by proteinase K. The simulation of the EPR spectra of DBNBS adduct was performed as described in materials and methods. The simulation parameters obtained for this spectrum were: AN = 13.5 Gauss, g = 2.0061, L/G = 0.2 and LW = 2.40 Gauss, considering the tumbling effect (LW = a + b x m + c x m2 and a = 2.3 b = -0.2 and c = 0.3128). The EPR parameters are consistent with tyrosyl-DBNBS adduct. The inset of upper panel shows a zoom of heme region in the tertiary Cygb structure. The residue represented in red corresponds to Tyr59. Tyr131 and Trp123 are represented in yellow. The inset of the lower panel shows the structure of a C-1-centered radical of tyrosine phenoxyl ring. The EPR spectra were measured using 20 μmol.L-1 protein solution, 1 mmol.L-1 hydrogen peroxide and 20 mmol.L-1 DBNBS. These results are representative of three independent replicates.
Mentions: To corroborate the identity of the protein-centered free radical detected by direct CW-EPR measurements at low temperature, we also analyzed the free radical using an EPR spin trapping technique. The formation of the tyrosyl radical during the reaction of Cygb with hydrogen peroxide is related to the mechanism of the peroxide cleavage. It is expected that the reaction of Cygb with hydrogen peroxide converts the heme group to oxo-ferryl π cation species. However, considering the similarity of the Cygb structure with that of Mb, it is possible that the oxidizing equivalent of the porphyrin ring is transferred to a globin amino acid residue. In this case, it is more likely that the oxidation of tyrosine 59, which is positioned close to the vinyl, methyl edge of the heme group, occurs. The tyrosine residue 123 must not be discarded because it is also in the proximity of the edge of the heme group. Tryptophan 171 is farther from the heme group and could be a secondary location of the radical, more likely by the transfer of one oxidant equivalent of a tyrosyl radical. DBNBS added to a Cygb solution challenged by hydrogen peroxide provided an EPR spectrum consistent with that of a partially immobilized nitroxide (Fig 8, upper panel). EPR signals were not detected when any of the reactants was absent (not shown). The EPR spectra of the DBNBS adduct was simulated, as described in the Materials and Methods section. The simulation resulted in a large Gaussian line width (3.77 G). For this tyrosyl-DBNBS adduct, the large linewidth results from the super hyperfine interactions in a highly heterogeneous microenvironment around nitroxide radical; these interactions were not resolved by the CW EPR spectrum [47]. The oxidative potential of tyrosine and tryptophan are similar; when these residues are neighbors in a protein structure, the unpaired electron density can be found in these amino acids in a population of hemeproteins treated with peroxides. However, considering the EPR parameters of the direct EPR signal and the location of tyrosine 59 and 123 at the vicinity of heme group edge (inset of Fig 8, upper panel), it is reasonable to assign the location of the unpaired electron to a tyrosine residue [49,67]. When DBNBS was added to the Cygb solution challenged by hydrogen peroxide and the sample underwent nonspecific proteolysis by proteinase K, an isotropic ESR spectrum consisting primarily of three lines was detected (Fig 8, lower panel). The simulation parameters obtained for this spectrum were: AN = 13.5 G, g = 2.0061, L/G = 0.2 and LW = 2.40 G, considering the tumbling effect (LW = a + b x m + c x m2 and a = 2.3, b = -0.2 and c = 0.3128). The EPR parameters are consistent with a tyrosyl-DBNBS adduct. The EPR spectrum does not exhibit additional hyperfine structure that is consistent with a tertiary carbon-centered radical adduct establishing no bonds with atoms with nuclear spin. Such a structural feature is consistent with the C-1-centered radical of a tyrosine phenoxyl ring (inset Fig 8, lower panel).

Bottom Line: This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species.Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils.This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

ABSTRACT
We characterized the peroxidase mechanism of recombinant rat brain cytoglobin (Cygb) challenged by hydrogen peroxide, tert-butylhydroperoxide and by cumene hydroperoxide. The peroxidase mechanism of Cygb is similar to that of myoglobin. Cygb challenged by hydrogen peroxide is converted to a Fe4+ oxoferryl π cation, which is converted to Fe4+ oxoferryl and tyrosyl radical detected by direct continuous wave-electron paramagnetic resonance and by 3,5-dibromo-4-nitrosobenzene sulfonate spin trapping. When organic peroxides are used as substrates at initial reaction times, and given an excess of peroxide present, the EPR signals of the corresponding peroxyl radicals precede those of the direct tyrosyl radical. This result is consistent with the use of peroxide as a reducing agent for the recycling of Cygb high-valence species. Furthermore, we found that the Cygb oxidation by peroxides leads to the formation of amyloid fibrils. This result suggests that Cygb possibly participates in the development of degenerative diseases; our findings also support the possible biological role of Cygb related to peroxidase activity.

No MeSH data available.


Related in: MedlinePlus