Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite.
The mechanism by which the inflammatory enzyme prostaglandin H(2) 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 (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-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.
Department of Pathology and Laboratory Medicine, Center of Vascular Biology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Ruba S
RS
Deeb
Gang
G
Hao
Steven S
SS
Gross
Muriel
M
Lainé
Ju Hua
JH
Qiu
Brad
B
Resnick
Elisar J
EJ
Barbar
David P
DP
Hajjar
Rita K
RK
Upmacis
D000595:Amino Acid Sequence; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D051545:Cyclooxygenase 1 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016166:Free Radical Scavengers / Q000494:pharmacology; D006418:Heme / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism; D017665:Hydroxyl Radical / Q000494:pharmacology; D008297:Male; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D030421:Peroxynitrous Acid / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep; D014443:Tyrosine / Q000378:metabolism*
D001051:Apoenzymes; D016166:Free Radical Scavengers; D020035:Holoenzymes; D030421:Peroxynitrous Acid; D017665:Hydroxyl Radical; D014443:Tyrosine; D006418:Heme; C002385:linsidomine; D006427:Hemin; D008981:Molsidomine; D051545:Cyclooxygenase 1