Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

Ruba S Deeb,Gang Hao,Steven S Gross,Muriel Laineé,Ju Hua Qiu,Brad Resnick,Elisar J Barbar,David P Hajjar,Rita K Upmacis

doi:10.1194/jlr.m500384-jlr200

Ruba S Deeb, Gang Hao + Show 7 more

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https://doi.org/10.1194/jlr.m500384-jlr200

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Abstract

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.

Highlights

The mechanism by which the inflammatory enzyme prostaglandin H2 synthase-1 (PGHS-1) deactivates remains undefined
By Western blot analysis with a anti-nitrotyrosine antibody, we examined the extent of PGHS-1 Tyr nitration by ONOO2 as a function of time in the presence and absence of hemin and arachidonic acid (Fig. 8A, B)
PGHS-1 proteolysis was limited when solubilized in bOG compared with Tween20 (Fig. 1C), suggesting that PGHS-1 possesses a more ordered structure in bOG

Summary

Introduction

The mechanism by which the inflammatory enzyme prostaglandin H2 synthase-1 (PGHS-1) deactivates remains undefined. After ONOO2 exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. Others have suggested that a cluster of aromatic amino acids in the vicinity of the heme group in addition to Tyr 385 can form radicals involved in enzyme catalysis and inactivation [11]. It was shown that the process of PGHS-1 oxidant-driven inactivation is altered by inhibitor Eicosanoids such as the prostaglandins, thromboxanes, and leukotrienes, which are formed from arachidonic acid metabolism, regulate vascular tone and platelet aggregation [1]. 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; bOG, N-octyl-b-D-glucopyranoside; .OH, hydroxyl radical; ONOO2, peroxynitrite; PGHS-1, prostaglandin H2 synthase-1; SIN-1, 3-morpholinosydnonimine hydrochloride; TMPD, N,N,N 9,N 9-tetramethylphenylenediamine; TNM, tetranitromethane; Tyr 385, tyrosine 385; UV-Vis, ultraviolet-visible. 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; bOG, N-octyl-b-D-glucopyranoside; .OH, hydroxyl radical; ONOO2, peroxynitrite; PGHS-1, prostaglandin H2 synthase-1; SIN-1, 3-morpholinosydnonimine hydrochloride; TMPD, N,N,N 9,N 9-tetramethylphenylenediamine; TNM, tetranitromethane; Tyr 385, tyrosine 385; UV-Vis, ultraviolet-visible. 1 To whom correspondence should be addressed

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