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Heme catalyzes tyrosine nitration and inactivation of cyclooxygenase‐1 by peroxynitrite
Author(s) -
Deeb Ruba S.,
Laine Muriel,
Hao Gang,
Gross Steven S.,
Barbar Elisar J.,
Hajjar David P.,
Upmacis Rita K.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1083-b
Subject(s) - peroxynitrite , heme , nitration , chemistry , hemin , peroxynitrous acid , biochemistry , tetranitromethane , enzyme , organic chemistry , superoxide
The mechanism by which the inflammatory enzyme cyclooxygenase‐1 (COX‐1) deactivates remains undefined. This study aimed to determine stabilizing parameters of COX‐1 and identify factors leading to COX‐1 modification and loss of function by peroxynitrite (ONOO − ). Purified COX‐1 activity was improved when solubilized in β‐octylglucoside (rather than Tween‐20 or CHAPS) and reconstituted with hemin chloride (rather than hematin). Following ONOO − exposure, holoCOX‐1 (containing heme) could not metabolize arachidonic acid, whereas apoCOX‐1 (without heme) pre‐treated with ONOO − was fully active following heme reconstitution. ONOO − ‐deactivation of holoCOX‐1 was manifested as heme loss, protein unfolding, aggregation and nitration. ONOO − compromised the structure in holoCOX‐1 rather than apoCOX‐1, since limited proteolysis exposed a peptide containing Tyr385 and proximal His388 only when heme was present. Tandem mass spectrometric analysis of denatured and fully digested COX‐1 following ONOO ‐ treatment revealed nitration of Tyr385 in holoCOX‐1 but not in apoCOX‐1. Since nitration was targeted to Tyr385 and levels of nitration and multimer formation were increased in holoCOX‐1 compared to apoCOX‐1, we conclude that the heme plays a role in catalyzing these processes and in the demise of COX‐1. This research was supported by the NIH, Pfizer Inc., Philip Morris USA Inc. and Philip Morris International.