Introduction of an Artificial Cu Binding Site at the Surface of CA II: Pitfalls of Rational Design Finally Scooped by Serendipity

The aim of this study was the introduction of an artificial copper center at the surface of carbonic anhydrase II (CA II) which subsequently might be used by azide and alkyne building blocks to catalyze triazole formation following 2+3 dipolar cycloaddition. Several mutants were produced to introduce favorable copper coordinating residues taking model copper-proteins from nature as reference templates. Unfortunately, the embarked rational design strategy remained unsuccessful which underlines that our still rather limited understanding of amino acid exchanges on the architecture of proteins might provoke unpredictable effects while tampering with complex biological systems such as a highly functionalized proteins. Finally, more by serendipity than design the formation of an artificial Cu center at the surface of CA II could be achieved. The metal ion is coordinated by two histidines and a serine residue in square planar coordination geometry.