Part:BBa_K2346002
Nitrous oxide reductase (nosZ)
The enzyme contains two copper center, namely CuA and Cuz, which can transfer the electron. The assay: We prepared a standard measuring mixture(2ml) consisting of 50mM Tris-HCL,pH9, 0.5mM methyl viologen, a proper amount of NosZ and 1mM sodium dithionite. Methyl viologen and sodium dithionite will go on to react and form a blue intermediate product, with a relativelly stable absorbance at 600nm. The blue interdiate product actually serves as an electron donor for the reduction of nitrous oxide. Therefore, as the reaction goes on, the blue substance starts to lose electrons and its color dims, resulting in a lower 600nm absorbance. Thus, by directly measuring the change of color in the system, we are able to track the processing of the reaction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1
Illegal XhoI site found at 1918 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 939
- 1000COMPATIBLE WITH RFC[1000]
Background
This enzyme catalyzes the reduction of nitrous oxide(N2O) to nitrogen(N2) and oxygen(O2). The enzyme comes from Agrobacterium tumefaciens. The enzyme contains two copper center, namely CuA and Cuz, which can transfer the electron.
Assay Method
We prepared a standard measuring mixture(2ml) consisting of 50mM Tris-HCL(pH9), 0.5mM methyl viologen, a proper amount of NosZ and 1mM sodium dithionite and 160uL of nitrous oxide solution.
Methyl viologen and sodium dithionite will go on to react and form a blue intermediate product, with a relativelly stable absorbance at 600nm. The blue intermediate product serves as an electron donor for the reduction process of nitrous oxide. Therefore, as the reaction goes on, the blue substance starts to lose electrons and its color dims, resulting in a lower 600nm absorbance. Thus, by directly measuring the change of 600nm absorbance of the system, we are able to track the processing of the reaction.
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