Hemocyanin (Hc) and phenoloxidase (PO) are the members of 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.

中文翻译：

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龙虾血蓝蛋白的高分辨率晶体结构显示出其作为酚氧化酶的酶促能力。

血蓝蛋白（Hc）和酚氧化酶（PO）是3型铜蛋白家族的成员。尽管节肢动物的Hc和PO表现出相似的三维结构的含铜活性位点，但Hc却起着氧转运蛋白的作用，显示出极少或没有酚氧化酶活性。在这里，我们介绍了以1.58Å分辨率分离的来自Panulirus japonicus（PjHc）的Hc的氧形式的晶体结构。发现PjHc的双铜活性位点的结构与PO几乎相同。尽管在PjHc中观察到与PO中几乎相同的氨基酸和酶活性至关重要的水分子，但在我们的实验条件下，PjHc没有酶活性。PjHc和节肢动物PO之间的一个显着差异是存在“阻滞剂残基” PjHc的双核铜位附近。该阻滞剂残基包含与丙氨酸配位的组氨酸的咪唑环紧密堆叠的苯丙氨酸残基，阻碍了底物进入活性位点。我们的结果表明，阻滞剂残基也是3型铜蛋白催化活性的决定因素。