Fourier-transform infrared study of azide binding to the Fea3-CuB binuclear site of bovine heart cytochrome c oxidase: new evidence for a redox-linked conformational change at the binuclear site.

Azide binding to the Fea3-CuB site of cytochrome c oxidase purified from bovine heart mitochondria was investigated in various redox levels by Fourier-transform infrared spectroscopy. In the fully oxidized state there were two azide species bound to the binuclear center, one (major) exhibiting an azide (14N3) antisymmetric stretching band at 2051 cm-1, and the other (minor) at 2039.5 cm-1. The former is considered to be in a bridging structure between Fea3(3+) and CuB2+, whereas in the latter species a bridging structure may not exist on the basis of the azide isotope substitution technique. Upon addition of cyanide to the preformed fully oxidized cytochrome c oxidase-azide complex, a new azide species exhibiting a sharp antisymmetric stretching band at 2032.5 cm-1 was formed. The cyanide is considered to be coordinated to the CuB2+ center (not observable by infrared spectroscopy), whereas the azide presumably still in a bridging structure. This cytochrome c oxidase-azide-cyanide ternary complex is relatively stable, and cyanide ion replaces the 2032.5-cm-1 azide species very slowly, resulting in the formation of the Fea3(3+)-C-N-CuB2+ bridging structure characterized by the 2152-cm-1 band. Upon the introduction of 1 electron equivalent to the fully oxidized cytochrome c oxidase-azide complex, an azide band at 2003.5 cm-1 developed. Upon further introduction of electron equivalents, the 2003.5-cm-1 band disappeared and a new azide band at 2015.5 cm-1 appeared. In these partially reduced states azide coordinates to the Fea3(3+) center presumably in an end-on fashion and cannot form a bridging structure any more.(ABSTRACT TRUNCATED AT 250 WORDS)