Event Title

Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Nitrite by Ferrocyanide

Presenter Information

Kaleb Keefe

Mentor 1

Arsenio Pacheco

Location

Union Wisconsin Room

Start Date

27-4-2018 1:00 PM

Description

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia (ammonification). Under physiological conditions ccNiR catalyzes the process without release of intermediates. However, in vitro we have found it possible to trap putative intermediates, or to release partially reduced nitrogen species such as nitric oxide, by controlling the electrochemical potential at which reduction takes place. Such experiments provide valuable insights regarding ccNiR-catalyzed ammonification of nitrite. 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 and ferrocyanide concentrations, and linear dependence on ccNiR concentration. Presence of Ferricyaniude serves to reduce the dissociability of Ferrocyanide which improves the accuracy of the methods used. 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. The mechanistic implications of these findings will be discussed in the context of earlier investigations of ccNiR.

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Apr 27th, 1:00 PM

Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Nitrite by Ferrocyanide

Union Wisconsin Room

Cytochrome c Nitrite Reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia (ammonification). Under physiological conditions ccNiR catalyzes the process without release of intermediates. However, in vitro we have found it possible to trap putative intermediates, or to release partially reduced nitrogen species such as nitric oxide, by controlling the electrochemical potential at which reduction takes place. Such experiments provide valuable insights regarding ccNiR-catalyzed ammonification of nitrite. 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 and ferrocyanide concentrations, and linear dependence on ccNiR concentration. Presence of Ferricyaniude serves to reduce the dissociability of Ferrocyanide which improves the accuracy of the methods used. 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. The mechanistic implications of these findings will be discussed in the context of earlier investigations of ccNiR.