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 synth

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NOradical dot production in biological systems, controversy still remains concerning whether NOS directly produces NOradical dot. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO−), which is then converted to NOradical dot by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NOradical dot from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NOradical dot measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NOradical dot from NO− (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe2+-MGD, NOradical dot generated characteristic triplet NOradical dot-Fe2+-MGD signals (g = 2.04, aN = 12.7 G), whereas NO− from Angeli’s salt was EPR silent. Both NOradical dot and NO− gave rise to NOradical dot-Fe2+-MGD signals when Fe3+-MGD was used. Strong NOradical dot signals were measured from purified nNOS using the NOradical dot selective Fe2+-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NOradical dot and NO− and this depends on the redox status of the iron. The detection of NOradical dot from purified NOS by Fe2+-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NOradical dot but not NO−.