S-Nitrosation and regulation of inducible nitric oxide synthase.
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.
Mitchell
Douglas A
DA
Department of Chemistry, University of California, Berkeley, Berkeley, California 94720-1460, USA.
Erwin
Phillip A
PA
Michel
Thomas
T
Marletta
Michael A
MA
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
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