title: Control of nitric oxide synthase dimer assembly by a heme-NO-dependent mechanism.
abstract: Homodimer formation is a key step that follows heme incorporation during assembly of an active inducible nitric oxide synthase (iNOS). In cells, heme incorporation into iNOS becomes limited due to interaction between self-generated NO and cellular heme [Albakri, Q., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 5414-5421]. Here we investigated if NO can regulate at points downstream in the process by inhibiting dimerization of heme-containing iNOS monomer. Heme-containing monomers were generated by treating iNOS dimer or iNOS oxygenase domain dimer (iNOSoxy) with urea. Both monomers dimerized when incubated with Arg and 6R-tetrahydrobiopterin (H4B), as shown previously [Abu-Soud, H. M., Loftus, M., and Stuehr, D. J. (1995) Biochemistry 34, 11167-11175]. The NO-releasing drug S-nitrosyl-N-acetyl-D,L-penicillamine (SNAP; 0-0.5 mM) inhibited dimerization of iNOS monomer in a dose- and time-dependent manner, without causing heme release. SNAP-pretreated monomer also did not dimerize in response to H4B plus Arg. SNAP converted Arg- and H4B-free iNOS dimer into monomer that could not redimerize, but had no effect on iNOS dimer preincubated with Arg and H4B. Anaerobic spectral analysis showed that NO from SNAP bound to the ferric heme of iNOSoxy monomer or dimer. Adding imidazole as an alternative heme ligand prevented SNAP from inhibiting iNOS monomer dimerization. We conclude that NO and related species can block iNOS dimerization at points downstream from heme incorporation. The damage to heme-containing monomer results from a reaction with the protein and appears irreversible. Although dimeric structure alone does not protect, it does enable Arg and H4B to bind and protect. Inhibition appears mediated by NO coordinating to the ferric heme iron of the monomer.
authors.affiliation: Department of Immunology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
authors.forename: Yishi
authors.initials: Y
authors.lastname: Chen
authors.forename: Koustubh
authors.initials: K
authors.lastname: Panda
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000693:Anaerobiosis; D019281:Dimerization; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D013053:Spectrophotometry; D014508:Urea
chemical_list: D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D026423:S-Nitroso-N-Acetylpenicillamine; D014508:Urea; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II