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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 t-BuOOH.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of t-BuOOH. 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.g007: Changes in the EPR spectrum of resting Cygb during the reaction with t-BuOOH.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of t-BuOOH. 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: CW-EPR of Cygb heme iron during the reaction with t-BuOOH (Fig 7) showed a progressive decrease in the low spin signal, with g values of 3.228, 2.033 and 1.385. Interestingly, the t-butylperoxyl free radical that had been previously observed during the reaction of cytochrome c with t-BuOOH was detected at the early times of the reaction (30 sec) in which almost 80% of the low-spin signal was yet present [65,66]. The t-butylperoxyl radical and the heme iron low spin signals disappeared at 60 sec, concomitantly with a significant increase of the g = 4.3 signal and the appearance of the same signal of a protein-centered radical that was detected during the reaction with hydrogen peroxide. The intensity of the protein radical signal increased at 210 sec. Very similar EA and EPR results were obtained for the reaction of Cygb with CumOOH. In this condition, the Soret band blueshift was observed in the EA spectrum, as well as a protein-centered free radical in the EPR spectrum (not shown).


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 t-BuOOH.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of t-BuOOH. 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

License
Show All Figures
getmorefigures.php?uid=PMC4552303&req=5

pone.0136554.g007: Changes in the EPR spectrum of resting Cygb during the reaction with t-BuOOH.The spectra marked as a, b and c were obtained at 30, 60 and 210 s after the addition of t-BuOOH. 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: CW-EPR of Cygb heme iron during the reaction with t-BuOOH (Fig 7) showed a progressive decrease in the low spin signal, with g values of 3.228, 2.033 and 1.385. Interestingly, the t-butylperoxyl free radical that had been previously observed during the reaction of cytochrome c with t-BuOOH was detected at the early times of the reaction (30 sec) in which almost 80% of the low-spin signal was yet present [65,66]. The t-butylperoxyl radical and the heme iron low spin signals disappeared at 60 sec, concomitantly with a significant increase of the g = 4.3 signal and the appearance of the same signal of a protein-centered radical that was detected during the reaction with hydrogen peroxide. The intensity of the protein radical signal increased at 210 sec. Very similar EA and EPR results were obtained for the reaction of Cygb with CumOOH. In this condition, the Soret band blueshift was observed in the EA spectrum, as well as a protein-centered free radical in the EPR spectrum (not shown).

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