The incorporation of nitrosocyanin copper binding loop into azurin.

Abstract

Metalloprotein design and engineering can be used to probe our understanding of active site structure and function. Loop-directed mutagenesis has been used in the metalloprotein field to change the copper binding loops from a number of members of the cupredoxin family into other protein scaffolds. We report the replacement of a ten amino acid loop that supports the copper binding site in the blue copper protein azurin with the red copper binding loop from the protein nitrosocyanin. Azurin is an electron transfer protein while the role of nitrosocyanin is unknown, yet believed to be catalytic. In addition to the loop, we added a carboxylic acid residue into the copper binding site which fully models the site of nitrosocyanin. Synthesis, expression, and UV-visible absorption and EPR spectra for this series of azurin variants will be reported.