The high-resolution crystal structure of lobster hemocyanin shows its enzymatic capability as a phenoloxidase

Hemocyanin (Hc) and phenoloxidase (PO) are members of the type 3 copper protein family. Although arthropod Hc and PO exhibit similar three-dimensional structures of the copper-containing active site, Hc functions as an oxygen transport protein, showing minimal or no phenoloxidase activity. Here, we present the crystal structure of the oxy form of Hc from Panulirus japonicus (PjHc) at 1.58 Å resolution. The structure of the di-copper active site of PjHc was found to be almost identical to that of PO. Although conserved amino acids and the water molecule crucial for the enzymatic activity were observed in PjHc at almost the same positions as those in PO, PjHc showed no enzymatic activity under our experimental conditions. One striking difference between PjHc and arthropod PO was the presence of a “blocker residue” near the binuclear copper site of PjHc. This blocker residue comprised a phenylalanine residue tightly stacked with an imidazole ring of a CuA coordinated histidine and hindered substrates from accessing the active site. Our results suggest that the blocker residue is also a determining factor of the catalytic activity of type 3 copper proteins. •Hemocyanin and phenoloxidase are two major members of the type 3 copper proteins.•We determined the high-resolution crystal structure of hemocyanin from a crustacean.•The structure of its active site is almost identical to that of phenoloxidases.•However, hemocyanin has an active site blocker residue that is absent in phenoloxidase.•The blocker residue would cause the functional difference between these type 3 copper proteins.