RECONSTITUTION OF THE 2ND STEP IN NO SYNTHESIS USING THE ISOLATED OXYGENASE AND REDUCTASE DOMAINS OF MACROPHAGE NO SYNTHASE

Inducible macrophage NO synthase (iNOS) is a homodimer of 130 kDa subu nits. Trypsinolysis of NOS inactivates its NO synthesis activity and c leaves the enzyme into a dimeric oxygenase fragment that contains heme , tetrahydrobiopterin, and the substrate binding site and a monomeric reductase fragment that contains FAD, FMN, calmodulin, and the binding site for NADPH [Ghosh, D. I., & Stuehr, D. H. (1995) Biochemistry 34, 801-807]. In this paper, we describe the reconstitution of NO synthes is activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not prod uced from L-arginine but could be formed from the reaction intermediat e N-omega-hydroxy-L-arginine (L-NOHA). The apparent K-m with L-NOHA in the resonstituted system was 100 mu M versus 19 mu M for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occu rred at an oxygenase to reductase molar ratio of 4:1, with higher rati os causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an inco mplete reduction of the oxygenase domain heme iron. L-NOHA, but not L- arginine, increased NADPH consumption in the reconstituted system. Bet ween 2.5 and 3.0 NADPH were consumed per NO formed from L-NOHA, consid erably higher than the stoichiometry obtained with native iNOS (0.5 NA DPH oxidized per NO formed), indicating an uncoupled electron transfer between the domain fragments. Thus, the isolated iNOS reductase and o xygenase domains each retain their separate catalytic functions but in teract to catalyze only the second step of NO synthesis. In this way, they form a monooxygenase system similar to those of eukaryotic cytoch romes P-450.