Our experiments have shown that the nitrite reductase pathway of eNOS utilizes a significant stretch of the normal electron transport chain of the enzyme. The electrons are received from NADPH at the flavins of the reductase domain and optical spectroscopy shows that the electrons are channeled towards the heme just like with the normal arginine pathway. Therefore, both oxic and anoxic pathways should be similarly affected by the binding of calmodulin. Binding of this coenzyme facilitates the passage of electrons from the reductase to the oxygenase domain. The binding of calmodulin is controlled by the local Ca2+ concentration and this binding/unbinding of calmodulin represents one of the dominant regulatory mechanisms of the enzymatic activity of eNOS .
The site of the nitrite reduction is clearly located in the oxygenase domain, since the latter domain is by itself capable of reducing nitrite if electrons are provided to the heme, in our case by administration of dithionite. The significant inhibition of nitrite reduction by heme inhibitors like imidazole shows that the heme moiety of the oxygenase domain is crucial. Additionally, the effect of the arginine analogs NLA and l-NAME proved that steric hindrance of access to the axial heme position is strongly inhibiting the nitrite reduction. Finally, we observed that anoxic nitrite reduction leads to nitrosylation of the heme without the release of any free NO if no arginine is added to the solution. These observations suggest that the anoxic reduction may take place at the heme itself. It is known that ferrous heme may reduce nitrite in a proton-consuming reaction
A prominent and well-studied example of this reaction is afforded by deoxy-hemoglobin . The ferrous heme reduces nitrite to nitric oxide and ferric methemoglobin, consuming a proton in the process. A similar reaction is documented for the so-called NIR-cdi bacterial nitrite reductases [35,56]. The functional enzyme is a homodimer containing one heme c and one heme di per subunit. The catalytic action of the nitrite reductase takes place at this di heme and entails three successive stages: First, binding of the nitrite anion to the heme, with the nitrogen atom coordinating to the iron. Second, the reduction to NO under consumption of a proton and dehydration, with the nitrosyl remaining bound to the heme. The final step is the release of NO from the enzyme. In NIR-cd1, the two final stages are mediated by nearby amino acid residues (cf Fig. 6 of Ref. ).
Tyr25v H,0 O
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