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Journal of Lipid Research, Vol. 47, 898-911, May 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology
* Department of Pathology and Laboratory Medicine, Center of Vascular Biology, Weill Medical College of Cornell University, New York, NY 10021
Department of Pharmacology, Weill Medical College of Cornell University, New York, NY 10021
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331
Published, JLR Papers in Press, February 9, 2006.
1 To whom correspondence should be addressed. e-mail: rsdeeb{at}med.cornell.edu
The mechanism by which the inflammatory enzyme prostaglandin H2 synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NOx). Purified PGHS-1 was stabilized when solubilized in ß-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO–) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO– exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO–, heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO–. Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO– only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO– and the demise of PGHS-1.
Supplementary key words cyclooxygenase • eicosanoids • heme reconstitution • peroxidase activity • proteolysis • mass spectrometry
Abbreviations: C10M, N-decyl-ß-D-maltopyranoside; DEDTC, diethyldithiocarbamate; 
, extinction coefficient; EtOH, ethanol; KI, potassium iodide; L-NAME, NG-nitro-L-arginine methyl ester; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; nLC-MS/MS, nanoflow liquid chromatography-tandem mass spectrometry; NOx, nitric oxide species; NOC-7, 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene; ßOG, N-octyl-ß-D-glucopyranoside; •OH, hydroxyl radical; ONOO–, peroxynitrite; PGHS-1, prostaglandin H2 synthase-1; SIN-1, 3-morpholinosydnonimine hydrochloride; TMPD, N,N,N',N'-tetramethylphenylenediamine; TNM, tetranitromethane; Tyr 385, tyrosine 385; UV-Vis, ultraviolet-visible
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