Cytochrome c Nitrite Reductase (ccNiR) is a soluble periplasmic, homodimericenzyme containing five hemes per protomer, of which the active site is five coordinated with unusual axial ligand lysine on it. Other hemes are six coordinated. CcNiR catalyzes the six-electron reduction of nitrite to ammonia (ammonification). Under physiological conditions ccNiRcatalyzes the process without release of intermediates. However, in vitro we have found,by controlling the electrochemical potential, it’s possible to trap intermediates between nitrite and ammonia. Such experiments provide valuable insights regarding ccNiR-catalyzed ammonification of nitrite. When the weak reductant ferrocyanide is used as the electron source, S. oneidensisccNiR catalyzes the one-electron reduction of nitrite to nitric oxide.In steady-state kinetic studies the reaction rate has hyperbolic dependence on nitrite and ferrocyanide concentrations, and linear dependence on ccNiR concentration. These 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.More recently we have followed up the steady-state studies with stopped-flow and laser flash photolysis ones. The mechanistic implications of the steady-state and pre-steady-state results will be discussed in light of earlier spectropotentiometric studies from our laboratories.

A mechanistic view of Cytochrome c nitrite reductase (ccNiR)-catalyzed reduction of nitrite to nitric oxide- a partial reduction of six-electron reduction of nitrite to ammonia (ammonification)- by weak reducing agent

255th National Meeting (2018)

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