FOURIER-TRANSFORM INFRARED STUDY OF AZIDE BINDING TO THE FE(A3)-CU(B) BINUCLEAR SITE OF BOVINE HEART CYTOCHROME-C-OXIDASE - NEW EVIDENCE FOR A REDOX-LINKED CONFORMATIONAL CHANGE AT THE BINUCLEAR SITE

Azide binding to the Fe(a3)-Cu(B) 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 (N-14(3)) 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 Fe(a3)3+ and Cu(B)2+, 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 Cu(B)2+ 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 Fe(a3)3+-C-N-Cu(B)2+ 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 Fe(a3)3+ center presumably in an end-on fashion and cannot form a bridging structure any more. These observations are consistent with our proposal that two kinds of conformational changes can occur at the binuclear site on the basis of cyanide binding to the partially reduced cytochrome c oxidase; the first one occurs upon introduction of the first electron presumably to the Cu(B) center, and the second one occurs upon introduction of the second or third electron presumably to the Fe(a) or Cu(A) Center (Tsubaki & Yoshikawa, 1993; preceding paper in this issue).