Displacement of O2 and CO by cyanide from the active site of helix pomatia β-hemocyanin. Formation of a mixed-liganded species

Abstract

Abstract Addition of KCN to Helix pomatia β-hemocyanin fully saturated with either O 2 or CO results in a decrease of the spectroscopic properties of the protein (absorbance at 340 nm and luminescence at 550 nm) due to the displacement of the gaseous ligands (O 2 or CO) from the active site. The anionic form of cyanide (CN − ) is supposed to bind to the active site; its intrinsic affinity for the protein, as calculated from independent O 2 and CO displacement experiments, is between 2 and 6 × 10 6 M −1 . The replacement of O 2 or CO shows some differences which may be correlated with the different modes of binding at the active site. Thus, while displacement of oxygen by cyanide is hyperbolic, addition of cyanide to carbonylated hemocyanin shows a lag phase. This finding suggests the formation of a mixed liganded complex at the active site. The simultaneous presence of CO and CN − at the active site of hemocyanin is also supported by the experiment in which addition of small amounts of KCN to hemocyanin partially saturated with O 2 and CO gives rise to an increase of emission intensity and a concomitant decrease of the O 2 absorption band. The mixed-liganded species displays luminescence properties similar to those of CO-saturated hemocyanin, and the formation of the complex is reversible on dialysis or oxygenation.