A mechanism by which nitric oxide (NO) enhances prostaglandin-H synthase-1 (PCHS-1) activity is described. Under aerobic conditions, NO stimulates 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 are located in the catalytic domain. Our data demonstrate that NO activated 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 relative to the addition of heme alone. PGHS-1 activity was also inhibited by H2O2. NO also activated PGHS-1 independently of Tyr 385, since tetranitromethane treatment of PGHS-1 did not block the enhancement of PGHS-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 measurements of PGHS-1 activity. The formation of nitrosothiols occurred concomitantly 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, with subsequent alterations in secondary structure.