Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation

Abstract

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains 5 hemes per protomer, were investigated by UV/Visible and EPR spectropotentiometries. Global analysis of the UV/Vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-Ray diffraction studies) by comparing the EPR and UV/Vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site’s midpoint potential, as the cyanide bound to the initially 5-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR silent. At high applied potentials interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three out of five hemes in each protomer. At lower applied potentials the spectra obtained in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed from UV/Vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme’s histidine axial ligands was replaced with a methionine.

A quasi-reagentless point-of-care test for nitrite and unaffected by oxygen and cyanide

“…In the presence of CN − , the cathodic peak current obtained for the c c NiR-modified PGE showed a drastic decrease bellow the non-catalytic current, accompanied by a + 90 mV shift in the peak potential. A similar shift (+70 mV) was observed in non-turnover conditions for the c c NiR from Shewanella oneidensis , which was attributed to the binding of the ligand to the penta-coordinated catalytic heme 35 . It was also reported that the binding event of CN − to the c c NiR from Escherichia coli resulted in a decrease in catalytic current and a displacement of the peak potential towards more positive values 26 .…”

Trapping of a Putative Intermediate in the Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite: Implications for the ccNiR Reaction Mechanism

“…This feature, together with the peak at g = 3.33 and trough at g = 2.83, has been attributed to the S = 5 / 2 heme 1, weakly exchange-coupled with one or more of the nearby low-spin hemes (S = 1 / 2 ). 11,16,43,47,48 The high-spin signal provides an excellent spectroscopic handle for the active site, and titration of ccNiR by nitrite was followed by monitoring its disappearance as nitrite was added. A spectrum was obtained at each nitrite concentration, and in each case the spectrum obtained in the presence of 6 mM nitrite (Figure 2b) was subtracted from it, to generate a difference spectrum analogous to that of Figure 2c.…”

“…This is much higher than the midpoint potential obtained for the first reduction step of S. oneidensis ccNiR in the absence of strongfield ligands and much higher even than that obtained in the presence of the strong-field ligand cyanide (20 mV vs NHE). 16 Singular value decomposition (SVD) analysis 16,51,52 and the presence of tight isosbestic points at 341 and 415.5 nm in Figure 4a reveal only a single spectral component attributable to c-heme reduction.…”

Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

“…The SO , acted as weak inhibitors in the activity assay [156]. Azide, N 3 − , was expected to bind to the active site heme iron as a competitive inhibitor [181,182] just like SO 4 2− . However, the structure of the NrfAws-N 3 − complex (2.0 Å resolution) revealed a water bound to Fe at 2.05 Å, and in close proximity the N 3 − anion, bound to residues lining Fig.…”

“…As expected both SO 4 2− , and structurally related PO 4 3− , acted as weak inhibitors in the activity assay [ 156 ]. Azide, N 3 − , was expected to bind to the active site heme iron as a competitive inhibitor [ 181 , 182 ] just like SO 4 2− . However, the structure of the NrfAws-N 3 − complex (2.0 Å resolution) revealed a water bound to Fe at 2.05 Å, and in close proximity the N 3 − anion, bound to residues lining the active site entrance, with hydrogen bonds to Gln 276 (3.0 Å), Tyr 218 (2.8 Å) and two hydrogen bonds to Arg 114.…”