400 500 600 70C
Fig. 2. Absorption spectra of eNOS (4.0 ^M) in the presence of nitrite (500 ^M). Thin solid line: before injection of NADPH. Dotted line: t = 30 s after injection of 100 ^M of NADPH. Dashed line: t = 2 min. Dash-dotted line: t = 5 min. Fine dots: t = 11 min. Thick solid line: t = 25 min. The arrows indicate the direction of time. The reaction proceeded at room temperature in 50 mM Tris buffer (pH = 7.5) containing 150 mM NaCl and 5% glycerol, 1 mM arginine, 40 ^M BH4, 10 ^M calmodulin and 1 mM Ca2+.
Fig. 3. EPR spectra at 77 K from trapping of traces of 15NO carried by the argon purge of the reaction cuvette. This cuvette contained 15N-labeled nitrite anions (500 ^M) and eNOS = 5 ^M. The hyperfine doublet near g = 2.035 is the 15N-labeled ferrous MNIC adduct. Trapping was for 15 min. Curve a: Reference EPR spectrum from 2.7 nmol 15NO-Fe2+ -(MGD)2 in frozen solution. The intensity is scaled down by a factor of 20. Curve b: 130 ± 13 pmol 15N-MNIC adducts after trapping of 15NO carried by the argon purge of the cuvette containing reduced full-length eNOS. Curve c: Absence of adducts after trapping of 15NO from the argon purge when the cuvette did not contain any eNOS.
The site of this nitrite reduction is presumably localized in the oxygenase domain of eNOS, since eNOSoxy proved capable of nitrite reduction as well, though at a slower rate. The reaction utilizes the flavins from the reductase domain to channel electrons from NADPH towards the heme. Arginine was found to play an interesting role in this reaction. In the absence of arginine, optical spectroscopy showed extensive nitrosylation of the ferrous heme, but no release of free NO could be detected electrochemically and the NADPH consumption came to a halt by nitrosylation of the heme moiety.
The main interest in this new pathway comes from its potential relevance for physiology. Since the conventional arginine pathway is blocked under conditions of low oxygen tension , we speculated that the newly discovered anoxic nitrite reductase pathway might provide a significant alternative source of NO for tissues under acute hypoxia. We therefore proceeded to test this hypothesis in a more complex model, namely endothelial cell cultures.
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