FOURIER-TRANSFORM INFRARED STUDY OF CYANIDE BINDING TO THE FE(A3)-CU(B) BINUCLEAR SITE OF BOVINE HEART CYTOCHROME-C-OXIDASE - IMPLICATION OF THE REDOX-LINKED CONFORMATIONAL CHANGE AT THE BINUCLEAR SITE

Cyanide binding to the Fe(a3)-Cu(B) binuclear center of cytochrome c oxidase purified from bovine heart mitochondria was examined by Fourier-transform infrared spectroscopy. In the fully oxidized state, cyanide binding caused an appearance of a sharp infrared C-N stretching band at 2152 cm-1. This 2152-cm-1 band was assigned to a bridging structure, Fe(a3)3+-C-N-Cu(B2+) on the basis of the isotope replacement experiments. The bound cyanide giving the 2152-cm-1 band was hardly exchangeable with an exogenous ligand added afterward in the fully oxidized state and, upon partial reduction, was converted specifically to the 2132-cm-1 band species assignable to Fe(a3)3+-C-N. The reduction of the Fe(a3) center resulted in appearances of two new infrared bands at 2058 and 2045 cm-1 concomitantly. At higher concentration of cyanide (>5 mM) an additional two infrared bands appeared at 2093 and 2037 cm-1. The former two bands are assignable to the Fe(a3)2+-bound cyanides, whereas the latter two bands are possibly due to the Cu(B)1+-bound cyanides on the basis of the competition experiments using carbon monoxide. These observations suggest that there are three kinds of conformational change to occur at the Fe(a3)-Cu(B) binuclear site upon reduction of the metal centers. The first one occurs upon reduction of the Cu(B) center, and the second one occurs upon reduction of the Fe(a) and/or Cu(A) centers. These are associated with the ''closed' to ''open'' conformational transition characterized by the disappearance of the 2152-cm-1 band and the appearance of the 2132- and 2093-cm-1 bands. The third one can be induced upon reduction of the Fe(a3) center, and this enables the binding of a second cyanide to the Cu(B)1+-CN center, at a higher concentration of cyanide, being oriented toward the Fe(a3)2+ center to produce the 2037-cm-1 band. These structural changes at the Fe(a3)-Cu(B) binuclear site controlled by the redox levels of the metal centers may provide a functional role(s) for the cytochrome c oxidase-catalyzed reactions, such as the reduction of dioxygen to water and the vectorial proton pumping across the mitochondrial inner membrane.