Effects of side-loop flexibility on the redox potential and stability of the blue copper protein, pseudoazurin

Abstract Blue copper proteins are a well-studied group of metalloproteins characterized by a type 1 copper site. The properties of the active site are influenced by the primary coordination sphere and the second coordination sphere. In this study, we investigated the effects of Met16Gly and Met16Ala variants of pseudoazurin, which were designed to remove the S–π noncovalent interaction with a Cu-coordinating His81. Both variants were spectroscopically similar to the wild type (WT), indicating minimal changes in the electronic structure of the active site. However, the redox potential of Met16Gly was ca. 50 and 40 mV higher than that of WT and Met16Ala, respectively. The stability of the variants followed the order WT > Met16Ala > Met16Gly, as determined by electrospray-mass spectrometry. X-ray crystal structure analyses revealed that the Met16Gly adopts both “closed” and “open” conformations of the “side-loop,” while Met16Ala and WT only show a closed conformation. The open conformation in Met16Gly exposes the Cu-coordinating His81 to the solvent, likely facilitating copper ion dissociation and protein unfolding. The positive shift of redox potential can be rationalized in terms of the entatic state or rack-induced bonding model, in which increased side-loop flexibility relaxes the protein-imposed constraints on the Cu-coordination.