Reconstitution of the second 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 subunits. Trypsinolysis of iNOS inactivates its NO synthesis activity and cleaves 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 synthesis activity utilizing the isolated oxygenase and reductase domains of iNOS. Mixing the domains at various ratios showed that NO was not produced from L-arginine but could be formed from the reaction intermediate N omega-hydroxy-L-arginine (L-NOHA). The apparent Km with L-NOHA in the reconstituted system was 100 microM versus 19 microM for native iNOS. D-NOHA was not a substrate. Maximum specific activity (per heme) occurred at an oxygenase to reductase molar ratio of 4:1, with higher ratios causing some inhibition. Reconstitution of activity was associated with electron transfer between the domain fragments and led to an incomplete reduction of the oxygenase domain heme iron. L-NOHA, but not L-arginine, increased NADPH consumption in the reconstituted system. Between 2.5 and 3.0 NADPH were consumed per NO formed from L-NOHA, considerably higher than the stoichiometry obtained with native iNOS (0.5 NADPH oxidized per NO formed), indicating an uncoupled electron transfer between the domain fragments. Thus, the isolated iNOS reductase and oxygenase domains each retain their separate catalytic functions but interact to catalyze only the second step of NO synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Ghosh
D K
DK
Department of Immunology, Cleveland Clinic Research Institute, Ohio 44195, USA.
Abu-Soud
H M
HM
Stuehr
D J
DJ
D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D002956:Citrulline / Q000096:biosynthesis; D004579:Electron Transport; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D013379:Substrate Specificity
D002956:Citrulline; D009569:Nitric Oxide; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D010088:Oxidoreductases; D010105:Oxygenases; D019001:Nitric Oxide Synthase