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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: 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.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

Changes in the EPR spectrum of resting Cygb during the reaction with hydrogen peroxide.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of hydrogen peroxide. The inset shows a zoom in the spectra of the free radical produced concomitantly with the formation of high valence species. For EPR experiments, the protein concentration was of 1.2 mmol.L-1and when present, the peroxide concentration was of 12 mmol.L-1. These results are representative of three independent replicates.
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pone.0136554.g004: Changes in the EPR spectrum of resting Cygb during the reaction with hydrogen peroxide.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of hydrogen peroxide. The inset shows a zoom in the spectra of the free radical produced concomitantly with the formation of high valence species. For EPR experiments, the protein concentration was of 1.2 mmol.L-1and when present, the peroxide concentration was of 12 mmol.L-1. These results are representative of three independent replicates.

Mentions: In the following, the formation high valence states of Cygb during the reaction with hydrogen peroxide was investigated by CW EPR of Cygb heme iron (Fig 4). Fig 4 shows the changes in the Cygb EPR spectrum run during the reaction of hydrogen peroxide with Cygb. The addition of hydrogen peroxide promotes a decrease in the EPR signal of the resting form of Cygb. This result is consistent with the formation of the EPR silent oxoferryl form of heme iron (Fe4+ = O). In addition, we observed a progressive increase in the signal with g = 4.3 that might be due to oxidative damage of heme iron [59], also supported by the occurrence of Soret band bleaching in the EA spectrum (Fig 3A). In the course of the reaction of Cygb with hydrogen peroxide, we also observed the signal of a free radical with g1 = 2.0053, g2 = 2.0053 and g3 = 2.0017. The EPR parameters are consistent with a tyrosyl radical, as has also been described for other hemeproteins such as cytochrome c oxidase [61,62]. The gy and gz values that were determined for the protein-centered radical are according to the known tyrosyl radicals. These values are in general equal to or larger than 2.0042 and 2.0020, respectively. The gx-gy and gx-gz values are equal to 0.0036, which is close to the range described for tyrosyl radicals (0.007–0.004) and close to the value described for Micobacterium tuberculosis catalase-peroxidase (0.00364) [63].


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)

Changes in the EPR spectrum of resting Cygb during the reaction with hydrogen peroxide.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of hydrogen peroxide. The inset shows a zoom in the spectra of the free radical produced concomitantly with the formation of high valence species. For EPR experiments, the protein concentration was of 1.2 mmol.L-1and when present, the peroxide concentration was of 12 mmol.L-1. These results are representative of three independent replicates.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4552303&req=5

pone.0136554.g004: Changes in the EPR spectrum of resting Cygb during the reaction with hydrogen peroxide.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of hydrogen peroxide. The inset shows a zoom in the spectra of the free radical produced concomitantly with the formation of high valence species. For EPR experiments, the protein concentration was of 1.2 mmol.L-1and when present, the peroxide concentration was of 12 mmol.L-1. These results are representative of three independent replicates.
Mentions: In the following, the formation high valence states of Cygb during the reaction with hydrogen peroxide was investigated by CW EPR of Cygb heme iron (Fig 4). Fig 4 shows the changes in the Cygb EPR spectrum run during the reaction of hydrogen peroxide with Cygb. The addition of hydrogen peroxide promotes a decrease in the EPR signal of the resting form of Cygb. This result is consistent with the formation of the EPR silent oxoferryl form of heme iron (Fe4+ = O). In addition, we observed a progressive increase in the signal with g = 4.3 that might be due to oxidative damage of heme iron [59], also supported by the occurrence of Soret band bleaching in the EA spectrum (Fig 3A). In the course of the reaction of Cygb with hydrogen peroxide, we also observed the signal of a free radical with g1 = 2.0053, g2 = 2.0053 and g3 = 2.0017. The EPR parameters are consistent with a tyrosyl radical, as has also been described for other hemeproteins such as cytochrome c oxidase [61,62]. The gy and gz values that were determined for the protein-centered radical are according to the known tyrosyl radicals. These values are in general equal to or larger than 2.0042 and 2.0020, respectively. The gx-gy and gx-gz values are equal to 0.0036, which is close to the range described for tyrosyl radicals (0.007–0.004) and close to the value described for Micobacterium tuberculosis catalase-peroxidase (0.00364) [63].

Bottom Line: 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.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