Displacement of O2 and CO by Cyanide from the Active Site of Helix pomatia &Hemocyanin. Formation of a Mixed-Liganded Species Lello Zolla, Harry A. Kuiper, Alessandro Finazzi Agrb, and Maurizio Brunori Institutes of ChemWy and Biochetntitry, Faculties of Medicine and Pharmacy, University of Rome “La Sapienza’, and CNR Center for Molecular Biology, Rome, Italy ABSTRACT Addition of KCN to Helix pomatia &hemocyanJn fully saturated with either 0s 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 (Or 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 Or and CO displacement experiments, is between 2 and 6 x lo6 M-t. The replacement of 0s 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 Jag phase. This finding suggests the formation of a mixed Jiganded 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 smaJJ amounts of KCN to hemocyanin partially saturated with 0s and CO gives rise to an increase of emission intensity and a concomitant decrease of the 0s absorption band. The mixed-Jiganded species displays JumJnescence properties similar to those of CO-saturated hemocyanin, and the formation of the complex is reversible on dialysis or oxygenation. INTRODUCTION Hemocyanin reversibly binds dioxygen, as well as carbon monoxide, with a stoichiometry of one ligand per two copper atoms [l-3]. Binding of O2 to deoxy- Address reprint requests to Dr. L. Zolla, Institutes of Chemistry and Biochemistry, Faculties of Medicine and Pharmacy, University of Rome “La Sapienza,” and CNR Center for Molecular Biology, Rome, Italy. Journal of Inorganic Biochemktry 22. 143-153 (1984) 0 1984 Hsevier science PublisJring Co., Inc. 52 Vanderbilt Ave., New York, NY 10017 143 0162-0134/84/$3.00