Spectroscopic characterization of the interaction of azide and thiocyanate with the binuclear center of cytochrome oxidase: evidence for multiple ligand sites.

The interactions of azide and thiocyanate with the binuclear center of oxidized cytochrome c oxidase have been characterized by Fourier transform infrared and UV-vis spectroscopy, electron paramagnetic resonance and magnetic and natural circular dichroism. Azide binds in two phases, a high-affinity phase (Kd = 64 microM) in which it is bound as a bridge to the binuclear center and a low-affinity phase (Kd = 20 mM) in which it displaces one of the axial ligands to cytochrome a. Thiocyanate also binds in two phases. The high-affinity phase (Kd = 2.7 mM) involves binding in a terminal mode to CuB; the low-affinity phase is complex and involves both CuA and cytochrome a. In contrast to the recent proposal of Yoshikawa and Caughey [(1990) J. Biol. Chem. 265, 7945-7958], we conclude that cyanide also functions as a bridge between cytochrome a3 and CuB. In the presence of cyanide, azide does not bind to its high-affinity site but thiocyanate does bind to its high-affinity site.