Roles of Three Conserved Active Site Residues of Cytochrome c Nitrite Reductases During the Early Steps of Nitrite...

Cytochrome c nitrite reductases (ccNiRs) catalyze reduction of nitrite to ammonium in a six-electron, eight-proton process. This report explores how the behavior of three active site ccNiR variants differs from that of the wild type enzyme (ccNiR_wt) in the presence of nitrite and a variety of electron donors. While the nitrite-loaded active site of Shewanella oneidensis ccNiR_wt could be 2-electron reduced within seconds to yield a 6-coordinate ferrous nitrosyl ({Fe(L)NO}⁷) at high applied potentials, quantitative reduction of the nitrite-loaded variants R103Q, H257Q, or Y206F required substantially stronger reductants and yielded a different product over several hours. Thus, nitrite-loaded variant reduction by a large excess of hexaammineruthenium(II) yielded quantitatively, within 2 h, a species with UV-visible spectroscopic characteristics distinct from those obtained from nitrite-loaded ccNiR_wt reduction. The same species was generated when the nitrite-loaded variants were treated with reduced indigo tetrasulfonate (I4S_red), but under these conditions, the species gradually disappeared unless the nitric oxide generator 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEANO) was also added to the reaction mixture. Characterization by electron paramagnetic resonance (EPR) spectroscopy of the variant species generated by I4S_red reduction showed them to have a 5-coordinate {FeNO}⁷ active site heme, in which the ligand L trans to the NO^• had dissociated from the iron center. By contrast, L did not dissociate in the 2-electron reduced wild type ccNiR. Thus, the ccNiR active site residues R103, H257, and Y206 are all needed to facilitate fast {Fe(L)NO}⁷ formation at high applied potentials, and to prevent dissociation of the trans ligand from {Fe(L)NO}⁷.