Characterization of the peroxidase mechanism upon reaction of prostacyclin synthase with peracetic acid. Identification of a tyrosyl radical intermediate

Hui Chun Yeh, Gary J. Gerfen, Jinn Shyan Wang, Ah Lim Tsai, Lee Ho Wang

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H 2, an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo - ferryl (Fe(IV)=O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P 1/2, of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g x, g y, g z) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P 1/2 value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration (∼0.2 s -1) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.

Original languageEnglish (US)
Pages (from-to)917-928
Number of pages12
JournalBiochemistry
Volume48
Issue number5
DOIs
StatePublished - Feb 10 2009

Fingerprint

Peracetic Acid
Peroxidase
Cytochrome P-450 Enzyme System
Paramagnetic resonance
Prostaglandins H
Electrons
Guaiacol
Reducing Agents
Peroxides
Epoprostenol
Hydrogen Bonding
Bleaching
Isomerization
Heme
Linewidth
Tensors
Absorption spectra
Hydrogen bonds
Membranes
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of the peroxidase mechanism upon reaction of prostacyclin synthase with peracetic acid. Identification of a tyrosyl radical intermediate. / Yeh, Hui Chun; Gerfen, Gary J.; Wang, Jinn Shyan; Tsai, Ah Lim; Wang, Lee Ho.

In: Biochemistry, Vol. 48, No. 5, 10.02.2009, p. 917-928.

Research output: Contribution to journalArticle

@article{6495d31e6c23457b847d08074281ddea,
title = "Characterization of the peroxidase mechanism upon reaction of prostacyclin synthase with peracetic acid. Identification of a tyrosyl radical intermediate",
abstract = "Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H 2, an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo - ferryl (Fe(IV)=O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P 1/2, of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g x, g y, g z) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P 1/2 value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration (∼0.2 s -1) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.",
author = "Yeh, {Hui Chun} and Gerfen, {Gary J.} and Wang, {Jinn Shyan} and Tsai, {Ah Lim} and Wang, {Lee Ho}",
year = "2009",
month = "2",
day = "10",
doi = "10.1021/bi801382v",
language = "English (US)",
volume = "48",
pages = "917--928",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "5",

}

TY - JOUR

T1 - Characterization of the peroxidase mechanism upon reaction of prostacyclin synthase with peracetic acid. Identification of a tyrosyl radical intermediate

AU - Yeh, Hui Chun

AU - Gerfen, Gary J.

AU - Wang, Jinn Shyan

AU - Tsai, Ah Lim

AU - Wang, Lee Ho

PY - 2009/2/10

Y1 - 2009/2/10

N2 - Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H 2, an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo - ferryl (Fe(IV)=O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P 1/2, of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g x, g y, g z) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P 1/2 value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration (∼0.2 s -1) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.

AB - Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H 2, an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo - ferryl (Fe(IV)=O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P 1/2, of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g x, g y, g z) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P 1/2 value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration (∼0.2 s -1) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.

UR - http://www.scopus.com/inward/record.url?scp=61449159908&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=61449159908&partnerID=8YFLogxK

U2 - 10.1021/bi801382v

DO - 10.1021/bi801382v

M3 - Article

VL - 48

SP - 917

EP - 928

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 5

ER -