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Published on: November 26, 2014
Escherichia coli cytochrome c nitrite reductase NrfA
Thomas A Clarke1, Paul C Mills, Susie R Poock
1Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
Methods in Enzymology
|April 25, 2008
Summary
Escherichia coli
Area of Science:
- Microbiology
- Biochemistry
- Enzyme kinetics
Background:
- The periplasmic cytochrome c nitrite reductase (Nrf) system in Escherichia coli facilitates anaerobic respiration.
- This system reduces nitrite to ammonium, potentially via a nitric oxide (NO) intermediate.
- The NrfA enzyme component is known to interact with exogenous NO.
Purpose of the Study:
- To detail methods for assaying Nrf-catalyzed nitric oxide reduction in whole Escherichia coli cells.
- To outline procedures for obtaining purified NrfA for further biochemical analysis.
Main Methods:
- Whole-cell assays to measure Nrf-catalyzed NO reduction.
- Purification protocols for the NrfA enzyme.
- Preparation of highly purified NrfA for kinetic, spectroscopic, voltammetric, and crystallization studies.
Main Results:
- Established a method to quantify Nrf-mediated NO reduction in vivo.
- Successfully purified NrfA to a high degree.
- Prepared NrfA suitable for diverse downstream biochemical and biophysical investigations.
Conclusions:
- The described methods enable robust study of the Nrf system's role in NO metabolism.
- Availability of purified NrfA facilitates detailed characterization of its enzymatic mechanisms and structure.
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