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

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, s uggesting that nitroxyl (NO-) or a related molecule is the principal reacti on product of NOS, which is SOD-dependently converted to NO. This hypothesi s was questioned by experiments using electron paramagnetic resonance spect roscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap fo r NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produc ed an electron paramagnetic resonance signal, the NO- donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, a nd important positive control experiments showing that AS was intact were l acking. 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 stoi chiometric 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 inva lid. Thus, NOS may form NO- or related N-oxides instead of NO. (C) 2000 Els evier Science Inc.