An improved assay for the enzyme cytochrome c nitrite reductase
Mentor 1
Arsenio Pachecho
Location
Union Wisconsin Room
Start Date
5-4-2019 1:30 PM
End Date
5-4-2019 3:30 PM
Description
Cytochrome c Nitrite Reductase (ccNiR) is an enzyme that catalyzes the six-electron reduction of nitrite (NO2-) to ammonium (NH4+). This process plays an important role in the nitrogen cycle for microorganisms during anaerobic respiration. In vitro ccNiR’s activity is assessed with an assay in which the electron donor is the methyl viologen monocation radical (MVred). The deep blue MVred oxidizes to a colorless species, which makes it very easy to monitor reaction progress using UV/Visible spectroscopy. One problem with this assay is that MVred actually exists in equilibrium with a dimeric species MVred2, a fact that is typically ignored by users of the assay. While such an oversight is acceptable for routine work, it may have led to some misinterpretations of more exacting analyses in the published literature. Herein we report a study of ccNiR’s activity in which the MVred - MVred2 equilibrium is explicitly accounted for. Using the improved assay, the dependence of ccNiR’s activity on NO2-, ccNiR and MVtot concentrations was determined for two batches of ccNiR that were prepared using different methods.
An improved assay for the enzyme cytochrome c nitrite reductase
Union Wisconsin Room
Cytochrome c Nitrite Reductase (ccNiR) is an enzyme that catalyzes the six-electron reduction of nitrite (NO2-) to ammonium (NH4+). This process plays an important role in the nitrogen cycle for microorganisms during anaerobic respiration. In vitro ccNiR’s activity is assessed with an assay in which the electron donor is the methyl viologen monocation radical (MVred). The deep blue MVred oxidizes to a colorless species, which makes it very easy to monitor reaction progress using UV/Visible spectroscopy. One problem with this assay is that MVred actually exists in equilibrium with a dimeric species MVred2, a fact that is typically ignored by users of the assay. While such an oversight is acceptable for routine work, it may have led to some misinterpretations of more exacting analyses in the published literature. Herein we report a study of ccNiR’s activity in which the MVred - MVred2 equilibrium is explicitly accounted for. Using the improved assay, the dependence of ccNiR’s activity on NO2-, ccNiR and MVtot concentrations was determined for two batches of ccNiR that were prepared using different methods.
