Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: Applications in identifying nitrogen monoxide products from nitric oxide synthase

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NO. production in biological systems, controversy still remains concerning whether NOS directly produces NO.. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO-), which is then converted to NO. by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarba mate iron (Fe-MGD), we directly detected NO. from purified NOS in the absen ce of SOD (Xia et al., PNAS 93:12705, 1997), We also showed that the requir ement for SOD in the previous NO. measurements appeared to be due to the hi gh levels of exogenous superoxide production in their reaction system becau se of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NO. from NO- (Komarov et al., FRBM 28:739-742, 200 0). In this study we examined the trapping specificity of different odor fo rms of Fe-MGD. With Fe2+-MGD. NO. generated characteristic triplet NO.-Fe2-MGD signals (g = 2.04, a(N) = 12.7 G), whereas NO- from Angeli's salt was EPR silent. Both NO. and NO- gave rise to NO.-Fe2+-MGD signals when Fe3+-MG D was used. Strong NO. signals were measured from purified nNOS using the N O. selective Fe2+-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NO. and NO- and this depends on the redox stat us of the iron. The detection of NO. from purified NOS by Fe2+-MGD unambigu ously reconfirms our previous report that NOS directly synthesizes NO . but not NO-. (C) 2000 Elsevier Science Inc.