NITRIC-OXIDE ENHANCES PROSTAGLANDIN-H SYNTHASE-1 ACTIVITY BY A HEME-INDEPENDENT MECHANISM - EVIDENCE IMPLICATING NITROSOTHIOLS

A mechanism by which nitric oxide (NO) enhances prostaglandin-H syntha se-1 (PCHS-1) activity is described. Under aerobic conditions, NO stim ulates the conversion of arachidonic acid to prostaglandin E(2) (PGE(2 )) and PGD(2) in a dose- (5-100 mu M) and time- (over 5 min) dependent manner. PGHS-1 possesses several potential targets for NO interaction ; heme and Tyr 385 (both of which are necessary for catalytic function ) and three reduced cysteines (Cys 313, 512, and 540), all of which ar e located in the catalytic domain. Our data demonstrate that NO activa ted PGHS-1 independently of heme. The Soret band absorbance of PGHS-1 was unaffected by NO in air but was increased by carbon monoxide (GO). Heme-NO conjugates added to apo-PGHS-1 inhibited PGHS-1 activity rela tive to the addition of heme alone. PGHS-1 activity was also inhibited by H2O2. NO also activated PGHS-1 independently of Tyr 385, since tet ranitromethane treatment of PGHS-1 did not block the enhancement of PG HS-1 by NO. However, NO promoted formation of nitrosothiols in a dose- dependent manner which plateaued at 3 mol nitrosothiol/mol PGHS-1. The kinetics of nitrosothiol formation directly correlated with measureme nts of PGHS-1 activity. The formation of nitrosothiols occurred concom itantly with a significant change in the secondary structure of PGHS-1 . Our data suggest that enhancement of PGHS-1 activity by NO occurs by S-nitrosation of cysteine residues located in the catalytic domain, w ith subsequent alterations in secondary structure.