Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Ah Lim Tsai, Gang Wu, Corina E. Rogge, Jian Ming Lü, Sheng Peng, Wilfred A. Van Der Donk, Graham Palmer, Gary J. Gerfen, Richard J. Kulmacz

Research output: Contribution to journalArticle

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Abstract

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O2. The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ∼ 90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.

Original languageEnglish (US)
Pages (from-to)366-374
Number of pages9
JournalJournal of Inorganic Biochemistry
Volume105
Issue number3
DOIs
StatePublished - Mar 2011

Fingerprint

Cyclooxygenase 1
Cyclooxygenase 2
Arachidonic Acid
Paramagnetic resonance
Peroxides
Prostaglandin-Endoperoxide Synthases
Carbon
Oxygen
Catalysis
Spectrum Analysis
Kinetics

Keywords

  • EPR
  • High-field EPR
  • PGHS radical
  • Radical dynamics

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

Tsai, A. L., Wu, G., Rogge, C. E., Lü, J. M., Peng, S., Van Der Donk, W. A., ... Kulmacz, R. J. (2011). Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2. Journal of Inorganic Biochemistry, 105(3), 366-374. https://doi.org/10.1016/j.jinorgbio.2010.11.012

Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2. / Tsai, Ah Lim; Wu, Gang; Rogge, Corina E.; Lü, Jian Ming; Peng, Sheng; Van Der Donk, Wilfred A.; Palmer, Graham; Gerfen, Gary J.; Kulmacz, Richard J.

In: Journal of Inorganic Biochemistry, Vol. 105, No. 3, 03.2011, p. 366-374.

Research output: Contribution to journalArticle

Tsai, AL, Wu, G, Rogge, CE, Lü, JM, Peng, S, Van Der Donk, WA, Palmer, G, Gerfen, GJ & Kulmacz, RJ 2011, 'Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2', Journal of Inorganic Biochemistry, vol. 105, no. 3, pp. 366-374. https://doi.org/10.1016/j.jinorgbio.2010.11.012
Tsai, Ah Lim ; Wu, Gang ; Rogge, Corina E. ; Lü, Jian Ming ; Peng, Sheng ; Van Der Donk, Wilfred A. ; Palmer, Graham ; Gerfen, Gary J. ; Kulmacz, Richard J. / Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2. In: Journal of Inorganic Biochemistry. 2011 ; Vol. 105, No. 3. pp. 366-374.
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abstract = "Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O2. The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ∼ 90{\%} of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.",
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AU - Rogge, Corina E.

AU - Lü, Jian Ming

AU - Peng, Sheng

AU - Van Der Donk, Wilfred A.

AU - Palmer, Graham

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