Role of the copper ion in pseudoazurin during the mechanical unfolding process

Metalloproteins require the corresponding metal cofactors to exert their proper function. The presence of metal cofactors in the metalloprotein makes it more difficult to investigate its folding and unfolding process. In this study, we employed atomic-force-microscopy-based single-molecule force spectroscopy to reveal the unfolding process of pseudoazurin (PAZ) that belongs to blue copper proteins. Our study shows that holo-PAZ requires a higher rupture force for mechanical unfolding comparing with the apo-PAZ. This result demonstrates that the copper atom not only enables PAZ access to transfer electron, but should also have an influence on its stability. The results also suggest that the electronic configuration of the metal cofactors has a striking effect on the strength of the organometallic bonds. Moreover, the results also reveal that there is an intermediate state during the unfolding process of PAZ. This study provides insight into the characteristics of metalloproteins and leads to a better knowledge of their interaction at the individual molecule level.