Fluorescence Studies of Oxygen Binding in Limulus Hemocyanin

Limulus hemocyanin, a weakly fluorescent copper protein when oxygenated, is highly fluorescent in the deoxygenated forms. A large enhancement of the fluorescence yield, 8.8-fold at neutral pH, was observed on complete deoxygenation, and a 9.4-fold increase was observed on removal of copper from oxyhemocyanin. The weak fluorescence of oxyhemocyanin is attributed to radiationless energy transfer from the tryptophan residues to the Cu⋯O group. The average distance between these two moieties was 21.4 Å. In oxyhemocyanin, the active site containing copper and oxygen is strongly hydrophobic, and a change in oxygen binding may profoundly alter the hydrophobic structure of the protein. This alteration is indicated by the binding of tetracycline to the protein where the distance between tryptophans and the tetracycline binding site is significantly increased when the protein is deoxygenated or when copper is removed. From the fluorescence results, it was found that the Cu⋯O group in Limulus, an arthropod hemocyanin, is nearer and interacts more strongly with the tryptophan residues than these groups in the mollusk (Leuentina hierosolima and Murex trunculus) hemocyanins. Deoxygenation of oxyhemocyanin by sodium sulfite was catalyzed by trace amounts of certain metal ions. The reaction rate was increased significantly by Co(II), Cu(II), and Mn(II), in decreasing order, but not by Zn(II).