Second coordination sphere controls structural flexibility of blue copper protein: A basis for entatic state formation.

Metalloproteins exhibit remarkable reactivity through the precise tuning of metal active sites embedded in the protein matrix. The concepts of the entatic state and rack-induced bonding describe how protein-imposed constraints optimize the metal-coordination structure for physiological functions. Blue copper proteins (BCPs) are prime examples of these concepts, where the protein modulates the balance between structural constraints (rigidity/flexibility) and Jahn-Teller distortion. However, the quantitative evaluation of protein flexibility and its interplay with the second coordination sphere remains unclear. This study investigated the dynamics of pseudoazurin (PAz) and its Met16 variants using quasi-elastic neutron scattering (QENS), elastic fixed window scan (EFWS), and differential scanning calorimetry (DSC). The results show that the Met16Phe variant exhibits increased rigidity, whereas Met16Ile displays enhanced structural dynamics and reduced thermal stability, correlating with altered noncovalent interactions in the second coordination sphere. These findings suggest that subtle changes in the second coordination sphere significantly impact mean protein flexibility, thereby supporting the entatic state hypothesis and highlighting the critical role of protein dynamics in metalloprotein function and design.