Role of distal arginine residue in the mechanism of heme nitrite reductases

Heme nitrite reductases reduce NO2- by 1e-/2H+ to NO or by 6e-/8H+ to NH4+ which are key steps in the global nitrogen cycle. Second-sphere residues, such as arginine (with a guanidine head group), are proposed to play a key role in the reaction by assisting substrate binding and hydrogen bonding and by providing protons to the active site for the reaction. The reactivity of an iron porphyrin with a NO2- covalently attached to a guanidinium arm in its 2nd sphere was investigated to understand the role of arginine residues in the 2nd sphere of heme nitrite reductases. The presence of the guanidinium residue allows the synthetic ferrous porphyrin to reduce NO2- and produce a ferrous nitrosyl species ({FeNO}7), where the required protons are provided by the guanidinium group in the 2nd sphere. However, in the presence of additional proton sources in solution, the reaction of ferrous porphyrin with NO2- results in the formation of ferric porphyrin and the release of NO. Spectroscopic and kinetic data indicated that re-protonation of the guanidine group in the 2nd sphere by an external proton source causes NO to dissociate from a ferric nitrosyl species ({FeNO}6) at rates similar to those observed for enzymatic sites. This re-protonation of the guanidine group mimics the proton recharge mechanism in the active site of NiR. DFT calculations indicated that the lability of the Fe-NO bond in the {FeNO}6 species is derived from the greater binding affinity of anions (e.g. NO2-) to the ferric center relative to neutral NO due to hydrogen bonding and electrostatic interaction of these bound anions with the protonated guanidium group in the 2nd sphere. The reduced {FeNO}7 species, once formed, is not affected significantly by the re-protonation of the guanidine residue. These results provide direct insight into the role of the 2nd sphere arginine residue present in the active sites of heme-based NiRs in determining the fate of NO2- reduction. Specifically, the findings using the synthetic model suggest that rapid re-protonation of these arginine residues may trigger the dissociation of NO from the {FeNO}6, which may also be the case in the protein active site.