Oxy and Met States of Type 3 Copper in a Single Functional Unit Revealed by the Crystal Structure of Abalone Hemocyanin.

Hemocyanin is a giant oxygen transporter widely distributed in invertebrates, belonging to the type 3 copper protein family. However, its met state form with the detailed structural information and other functions has not yet been determined. In this study, we established a new purification method to isolate the purple functional unit of type 1 hemocyanin from abalone (Haliotis discushannai) (HtH1-h). The molecular-level structure of HtH1-h was determined by X-ray crystallography at resolution of 2.0 Å. Two distinct oxygen-binding states have been captured within the HtH1-h homodimer (Oxygenation state and Met state), which provides the first structural case of the met state of hemocyanin. Additionally, enzymatic characterization revealed detectable diphenolase activity in HtH1-h, whereas monophenolase activity was not observed. These findings suggest that hemocyanin might be an active protein with multiple functions.