title: S-Nitrosation and regulation of inducible nitric oxide synthase.
abstract: The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 micromol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 micromol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 microM DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in approximately 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 microM DEA/NO compared to a control sample. Using the biotin switch method under the same conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDI-TOF/TOF MS. Immunoprecipitation of iNOS from the mouse macrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endogenous S-nitrosation of iNOS. The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release from the dimer interface and formation of inactive monomers, suggesting that this mode of inhibition might occur in vivo.
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, California 94720-1460, USA.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Mitchell
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Erwin
authors.forename: Thomas
authors.initials: T
authors.lastname: Michel
authors.forename: Michael A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D000818:Animals; D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D002863:Chromogenic Compounds / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D015538:Nitrosation; D011485:Protein Binding / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012118:Resorcinols / Q000378:metabolism; D026403:S-Nitrosothiols / Q000378:metabolism; D012680:Sensitivity and Specificity; D015032:Zinc / Q000378:metabolism
chemical_list: D002863:Chromogenic Compounds; D007527:Isoenzymes; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D012118:Resorcinols; D026403:S-Nitrosothiols; C406802:diethylammonium-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; C093483:neutravidin; D001360:Avidin; D009569:Nitric Oxide; D006418:Heme; D006735:Horseradish Peroxidase; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D015032:Zinc; D003545:Cysteine; C028398:4-(2-pyridylazo)resorcinol