title: Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase.
abstract: Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.
authors.affiliation: George Washington University Medical Center, Department of Physiology and Experimental Medicine, Washington, DC, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Komarov
authors.forename: D A
authors.initials: DA
authors.lastname: Wink
authors.forename: M
authors.initials: M
authors.lastname: Feelisch
authors.forename: H H
authors.initials: HH
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000032:analysis* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D009589:Nitrogen Oxides / Q000032:analysis* / Q000378:metabolism; D013012:Sorbitol / Q000031:analogs & derivatives; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000378:metabolism; D013859:Thiocarbamates
chemical_list: D005609:Free Radicals; D009573:Nitrites; D009589:Nitrogen Oxides; D013113:Spin Labels; D013859:Thiocarbamates; C021229:oxyhyponitrite; D009569:Nitric Oxide; D013012:Sorbitol; C041262:N-methyl-D-glucamine dithiocarbamate; D019001:Nitric Oxide Synthase; D013482:Superoxide Dismutase; C039900:nitroxyl