Characterization of the copper-binding site of NosL of the nitrous oxide reductase system

CHED 795

Craig T. Gutman, cgutman@pacificu.edu and Jeannine M. Chan, chanj@pacificu.edu. Department of Chemistry, Pacific University, 2043 College Way, Forest Grove, OR 97116
The reduction of nitrous oxide to dinitrogen is an important step in the global nitrogen cycle and is catalyzed by the enzyme nitrous oxide reductase. This enzyme contains a unique tetranuclear copper center that requires accessory proteins for assembly, including the protein NosL. Little is known about the function of NosL, but it has been shown to selectively bind Cu(I) in a suspected Cu(O/N)S2 binding motif. Here we detail our work in determining the Cu binding ligands in NosL. Using site-directed mutagenesis an altered (NosL C24S) form of the protein was produced and was recombinantly expressed in E. coli on copper supplemented media. A copper-binding assay will be used to determine the level of copper in the purified protein and a comparison between the altered form and the wild type will be made.