Nitro-arachidonic acid inhibits prostaglandin endoperoxide H synthase 2 through histidine-mediated heme-binding conformational perturbation.

Prostaglandin endoperoxide H synthase (PGHS) is a heme-containing enzyme that catalyzes the conversion of arachidonic acid (AA) into prostaglandin H2. The anti-inflammatory mediator nitro-arachidonic acid (NO2AA) has been identified as a potent inhibitor of both PGHS1 and PGHS2 isoforms. While this inhibition involves the displacement of the essential heme prosthetic group from the active site, the underlying molecular mechanism remains incompletely elucidated. Herein, we investigated the inhibitory action of NO2AA against murine PGHS2 (mPGHS2) using site-directed mutagenesis targeting the peroxidase (POX) active sites, combined with kinetic analysis and mass spectrometry. NO2AA dose-dependently inactivates both COX and POX activities. Mutations of key histidine residues -H386A, H207A, and H388F- abrogated this inactivation in WT-mPGHS2. The apoenzyme incubated with NO2AA and then reconstituted with heme, as well as the holoenzyme inhibited by the addition of NO2AA and then reconstituted with heme, did not recover their enzymatic activity. Despite the potential for NO2AA to modify residues that coordinate the heme group, we were unable to detect covalent nitroalkylation at these sites, suggesting that NO2AA induces heme release through a non-covalent, irreversible mechanism. Nano-scale differential scanning fluorimetry (nanoDSF) confirmed that NO2AA triggers a selective disruption of heme stabilization within the protein structure, a process hindered by mutations at His-207, His-386, and His-388. These results support a model in which NO2AA interacts with the POX active site, with histidine residues playing a crucial role in the interaction that triggers heme displacement. These insights enhance our understanding of the NO2AA mechanism of action and underscore its potential as a scaffold for targeted anti-inflammatory drug development.