Substrate-induced free radicals in prostaglandin H synthase

Area of Science:

• Biochemistry
• Enzymology
• Spectroscopy

Background:

• Prostaglandin H synthase (PGHS) catalyzes prostaglandin synthesis via cyclooxygenase and peroxidase activities.
• Tyrosine radicals are implicated as intermediates in PGHS catalysis, but their specific roles and identities remain debated.
• Electron paramagnetic resonance (EPR) spectroscopy is a key technique for detecting and characterizing free radicals.

Purpose of the Study:

• To characterize the tyrosine radical species produced during ovine PGHS reactions using EPR spectroscopy.
• To correlate the temporal sequence of EPR signals with heme optical changes and product formation.
• To elucidate the role of specific tyrosine radicals in the cyclooxygenase and peroxidase mechanisms of PGHS.

Main Methods:

• Reconstitution of ovine PGHS with either heme (Fe-PGHS) or manganese protoporphyrin IX (Mn-PGHS).
• Reaction of reconstituted PGHS with arachidonic acid or hydroperoxides.
• Monitoring of EPR signals, electronic absorbance spectra of the heme center, and prostaglandin product formation over time.

Main Results:

• Fe-PGHS reaction with arachidonic acid produced a transient wide doublet tyrosyl radical EPR signal, followed by a wide singlet signal.
• The temporal profile of the wide doublet EPR signal paralleled the accumulation of peroxidase Compound II and prostaglandin intermediates (PGG2, PGH2).
• Mn-PGHS also generated a distinct tyrosyl radical signal, with kinetics consistent with its involvement in cyclooxygenase catalysis.

Conclusions:

• The wide doublet tyrosyl radical observed with Fe-PGHS is the most likely oxidizing species responsible for hydrogen abstraction from arachidonic acid in cyclooxygenase catalysis.
• Distinct tyrosine radicals are generated depending on the PGHS active site cofactor (heme vs. Mn-protoporphyrin).
• EPR spectroscopy provides critical insights into the transient radical intermediates governing prostaglandin synthesis.