Date of Award

December 2019

Degree Type


Degree Name

Doctor of Philosophy



First Advisor

Arsenio A Pacheco

Committee Members

Nicholas R Silvaggi, Marius Schmidt, Brian Bennett, Graham Moran


Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it’s been assumed that this is also true in vivo. However, this report demonstrates that in vitro it’s possible to trap putative intermediates by controlling the electrochemical potential at which reduction takes place. Such experiments provide valuable insights regarding ccNiR-catalyzed nitrite ammonification.

UV/Vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted 2-electron step to generate an {FeNO}7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of 20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO}7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO}7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least -120 mV, well below the midpoint potential for {FeNO}7 formation. Thus, it appears that an {FeNO}7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and doesn’t accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

When the weak reductant ferrocyanide is used as the electron source, S. oneidensis ccNiR catalyzes the one-electron reduction of nitrite to nitric oxide. The reaction rate has hyperbolic dependence on nitrite concentration and linear dependence on ccNiR concentration. NO release is minimal compared to the rate of ammonia formation at lower applied potentials. Kinetic studies also show that the rate of NO production is pH-dependent, and that an amino acid with pKa of 6.9, probably His268, needs to be protonated for the enzyme to be active.

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