Oxyferryl Heme and Not Tyrosyl Radical Is the Likely Culprit in Prostaglandin H Synthase-1 Peroxidase Inactivation

Wu, Gang; Rogge, Corina E.; Wang, Jinn Shyan; Kulmacz, Richard J.; Palmer, Graham; Tsai, Alan C.

doi:10.1021/bi061859h

Cited by 19 publications

(21 citation statements)

References 34 publications

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“…Therefore, directly comparing it with appearance and disappearance of the amino acid radical may simply be due to a combination of catalytic events and of heme destruction proceeding simultaneously. In sharp contrast to a recent study on prostaglandin H synthase, loss of peroxidase activity correlates well with the disappearance of the oxyferryl heme and is at least an order of magnitude faster than the tyrosyl radical decay (76). …”

Section: Discussioncontrasting

confidence: 99%

Characterization of the Peroxidase Mechanism upon Reaction of Prostacyclin Synthase with Peracetic Acid. Identification of a Tyrosyl Radical Intermediate

et al. 2009

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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.Cytochromes P450 (P450) 1 play important roles in physiological processes, pharmaceutical metabolism, and catalysis of a variety of reactions, such as hydroxylations, epoxidations, Nand O-dealkylations, and isomerizations. P450 enzymes contain a low-spin ferric heme with a cysteinate residue as the proximal ligand (1). Binding of the substrate often induces the heme to convert to high spin with detachment of the distal ligand and an increase of the redox

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Section: Discussioncontrasting

confidence: 99%

Characterization of the Peroxidase Mechanism upon Reaction of Prostacyclin Synthase with Peracetic Acid. Identification of a Tyrosyl Radical Intermediate

et al. 2009

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“…Further, rapid-freeze quench EPR kinetic measurements and parallel stopped-flow measurements for heme redox changes (A 410 for Intermediate I disappearance and A 580 for Intermediate II formation) in reactions with peroxide demonstrated that oxidized peroxidase intermediates and tyrosyl radicals could be generated very quickly in oPGHS-1, hPGHS-2, and even Mn-PGHS-1 (Fig. 14) [57, 73]. These findings offered some reassurance that at least two tyrosyl radicals, WD1 in PGHS-1 and WD2 in PGHS-2, were formed from peroxidase reaction intermediates on a time scale consistent with participation in cyclooxygenase catalysis.…”

Section: Sorting Out Structure and Function For The Many Tyrosyl Radimentioning

confidence: 99%

“…To address this, the kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of various hydroperoxides with oPGHS-1 reconstituted with heme or MnPPIX (Fig. 25) [73]. Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels.…”

Section: Self-inactivation Of Pghsmentioning

confidence: 99%

“…25). Computer modeling to a minimal mechanism indicated that the oxyferryl heme could account for peroxidase inactivation without contribution from tyrosyl radical [73]. The lack of correlation between tyrosyl radical intensity and peroxidase inactivation was also observed for the NS1a tyrosyl radical formed in the indomethacin-treated oPGHS-1 [73].…”

Section: Self-inactivation Of Pghsmentioning

confidence: 99%

“…Computer modeling to a minimal mechanism indicated that the oxyferryl heme could account for peroxidase inactivation without contribution from tyrosyl radical [73]. The lack of correlation between tyrosyl radical intensity and peroxidase inactivation was also observed for the NS1a tyrosyl radical formed in the indomethacin-treated oPGHS-1 [73]. These results have led to a working hypothesis that the culprit for peroxidase inactivation is the high-oxidation state heme, whereas the WD1/WS1 tyrosyl radical may be either directly or indirectly involved in damage at the cyclooxygenase site.…”

Section: Self-inactivation Of Pghsmentioning

confidence: 99%

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Prostaglandin H synthase: Resolved and unresolved mechanistic issues

Tsai

Kulmacz

2010

Archives of Biochemistry and Biophysics

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The cyclooxygenase and peroxidase activities of prostaglandin H synthase (PGHS)-1 and -2 have complex kinetics, with the cyclooxygenase exhibiting feedback activation by product peroxide and irreversible self-inactivation, and the peroxidase undergoing an independent self-inactivation process. The mechanistic bases for these complex, non-linear steady-state kinetics have been gradually elucidated by a combination of structure/function, spectroscopic and transient kinetic analyses. It is now apparent that most aspects of PGHS-1 and -2 catalysis can be accounted for by a branched chain mechanism involving a classic heme-based peroxidase cycle and a radical-based cyclooxygenase cycle. The two cycles are linked by the Tyr385 radical, which originates from an oxidized peroxidase intermediate and begins the cyclooxygenase cycle by abstracting a hydrogen atom from the fatty acid substrate. Peroxidase cycle intermediates have been well characterized, and peroxidase self-inactivation has been kinetically linked to a damaging side reaction involving the oxyferryl heme oxidant in an intermediate that also contains the Tyr385 radical. The cyclooxygenase cycle intermediates are poorly characterized, with the exception of the Tyr385 radical and the initial arachidonate radical, which has a pentadiene structure involving C11-C15 of the fatty acid. Oxygen isotope effect studies suggest that formation of the arachidonate radical is reversible, a conclusion consistent with electron paramagnetic resonance spectroscopic observations, radical trapping by NO, and thermodynamic calculations, although moderate isotope selectivity was found for the Habstraction step as well. Reaction with peroxide also produces an alternate radical at Tyr504 that is linked to cyclooxygenase activation efficiency and may serve as a reservoir of oxidizing equivalent. The interconversions among radicals on Tyr385, on Tyr504, and on arachidonate, and their relationships to regulation and inactivation of the cyclooxygenase, are still under active investigation for both PGHS isozymes.Prostaglandin H synthase (PGHS) is a key enzyme in prostanoid biosynthesis. Mammalian systems have two distinct PGHS isozymes sharing ~60% sequence identity. The constitutive isozyme, PGHS-1, is thought to function usually as a housekeeping enzyme, whereas the inducible isozyme, PGHS-2, is associated with cytokine and mitogen dependent processes, such as inflammation and cell proliferation [1,2]. Both PGHS isozymes catalyze the same two reactions: dioxygenation of arachidonic acid (AA) to yield prostaglandin G 2 (PGG 2 ), containing both a 9-11 endoperoxide and a 15-peroxide group; and a peroxidase reaction, which converts PGG 2 to prostaglandin H 2 (PGH 2 ) where the 15-peroxide is reduced to an alcohol © 2009 Elsevier Inc. All rights reserved. *CORRESPONDING AUTHOR: FAX: 713 500-6810; Ah-Lim.Tsai@uth.tmc.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this e...

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