The Selenocysteine-Substituted Blue Copper Center: Spectroscopic Investigations of Cys112SeCys Pseudomonas aeruginosa Azurin

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Abstract
Azurin is a small electron-transfer protein belonging to the cupredoxin family. The Cu atom is located within a trigonal plane coordinated by two histidines (His46 and His117) and a cysteine (Cys112) with two more distant ligands (Gly45 and Met121) providing axial interactions. A Cys112SeCys derivative has been prepared by expressed protein ligation, and detailed UV/vis, EPR and EXAFS studies at the Cu and Se K-edges have been carried out. Marked changes are observed between the EPR parameters of the Cys112SeCys and WT azurin derivatives, which include a 2-fold increase in A||, a decrease in g-values, and a large increase in rhombicity of the g-tensor. The Cu−Se and Se−Cu bond lengths obtained from analysis of the Cu and Se K-EXAFS of the oxidized protein were found to be 2.30 and 2.31 Å, respectively, 0.14 Å longer than the Cu−S distance of the WT protein. Unexpectedly, the Cu−Se bond lengths were found to undergo only minor changes during reduction, suggesting a very similar structure in both redox states and extending the “rack” hypothesis to the Se-substituted protein.