Fig. 6. The mechanism of nitrite reduction at the di heme of NIR-cdi. (With permission from Ref. [35].)

The reduction step is a dehydration reaction where the proton is provided by one of the pairs of nearby distal histidines and the NO remains liganded to the ferric iron. In Enemark-Feltham notation the electronic configuration is that of an {FeNO}6 species. The final step is the subsequent replacement of the axial NO ligand on the ferric heme by a tyrosine residue, followed by the release of free NO from the enzyme. Its heme is left in ferric d5 configuration. The next cycle is initiated by reduction of the di heme to ferrous state after receiving an electron from the nearby c-heme that acts as the electron entry into the enzyme. For this particular enzyme, the binding of nitrite to the heme does not depend only on the charge state of the iron and the geometry of the heme pocket. Theoretical considerations suggest that protonation of these nearby histidines greatly enhances the binding of nitrite to di heme [56].

Possibly, anoxic nitrite reduction by eNOS may proceed in an analogous manner, via a reaction like Eq. (3) that takes place at the heme of the oxygenase domain. The first step is binding of the nitrite anion at the axial position of the heme, with the nitrogen atom coordinating to the iron, followed by reduction of the heme to ferrous state by an electron channeled from the flavins of the reductase domain in the usual way. The subsequent step is acidic reduction of the nitrite according to Eq. (3), with the proton presumably being provided by a nearby amino acid. The final step is the release of the NO ligand from the enzyme, and is a complex kinetic process depending on factors like the charge state of the heme and the presence of cofactors like arginine and tetrahydrobiopterin. We tried to confirm direct binding of nitrite to the heme by optical spectroscopy. However, we failed to detect any spectroscopic changes from such binding at the relevant nitrite concentrations in the submillimolar range.

In our enzymatic assay, arginine was found to play a decisive role in the release of NO from the complex. In the absence of arginine, optical spectroscopy showed extensive heme nitrosylation by the characteristic shoulder around 440 nm. It is direct proof of formation of NO. However, this NO was not released from the heme since free NO levels remained below the detection threshold of the NO electrode. In the presence of arginine, copious quantities of free NO were detected. The binding site of arginine is in close proximity to the heme, and kinetic studies [57,58] have shown that binding of arginine affects the geminate recombination of NO to heme. The kinetic constants show that the presence of arginine facilitates the release of the NO ligand from the heme. When eNOS functions as nitrite reductase, arginine itself is not consumed. Rather, its binding to the nearby site promotes the release of free NO from the enzyme. EPR spectroscopy on nitrosylated eNOS heme has shown that the binding of L-arginine significantly reduces the motional freedom of the NO ligand with respect to the heme plane [59]. The effect was attributed to a weakening of the Fe—NO bond due to the electrostatic attraction between negatively charged oxygen of the NO ligand and the delocalized positive charge on the protonated guanidine group of the L-arginine.


In an anaerobic 50 mM Tris buffer containing BH4, arginine and 0.5 mM nitrite at pH = 7.6, eNOS is able to release NO after the administration of NADPH. The NO-specific electrode

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