title: Domains of macrophage N(O) synthase have divergent roles in forming and stabilizing the active dimeric enzyme.
abstract: The cytokine-inducible NO synthase (iNOS) is a flavin-containing hemeprotein that must dimerize to generate NO. Trypsin cleaves the dimeric enzyme into an oxygenase domain fragment that remains dimeric, contains heme and H4biopterin, and binds L-arginine and a reductase domain fragment that is monomeric, binds NADPH, FAD, FMN, and catalyzes the reduction of cytochrome c [Ghosh, D. K. & Stuehr, D. J. (1995) Biochemistry 34, 801-807]. The current study investigates the isolated oxygenase and reductase domains of iNOS to understand how they form and stabilize the active dimeric enzyme. The dimeric oxygenase domain dissociated into folded, heme-containing monomers when incubated with 2-5 M urea, whereas the reductase domain unfolded under these conditions and lost its ability to catalyze NADPH-dependent cytochrome c reduction. Spectral analysis of the dissociation reaction showed that it caused structural changes within the oxygenase domain and exposed the distal side of the heme to solvent, enabling it to bind dithiothreitol as a sixth ligand. Importantly, the oxygenase domain monomers could reassociate into a dimeric form even in the absence of the reductase domain. The reaction required L-arginine and H4biopterin and completely reversed the structural changes in heme pocket and protein structure that occurred upon dissociating the original dimer. Together, this confirms that the oxygenase domain contains all of the determinants needed for subunit dimerization and indicates that the dimeric structure greatly affects the heme and protein environment in the oxygenase domain.
authors.affiliation: Department of Immunology, Cleveland Clinic Research Institute, Ohio 44195, USA.
authors.forename: D K
authors.initials: DK
authors.lastname: Ghosh
authors.forename: H M
authors.initials: HM
authors.lastname: Abu-Soud
authors.forename: D J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D004795:Enzyme Stability; D008264:Macrophages / Q000201:enzymology*; D008954:Models, Biological; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D010446:Peptide Fragments / Q000737:chemistry* / Q000187:drug effects; D011487:Protein Conformation; D011489:Protein Denaturation; D014508:Urea / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D014508:Urea; D019001:Nitric Oxide Synthase