Fourier-transform infrared study of cyanide binding to the Fea3-CuB binuclear site of bovine heart cytochrome c oxidase: implication of the redox-linked conformational change at the binuclear site

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Abstract
Cyanide binding to the Fea3-CuB 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, Fea3(3+)-C-N-CuB2+, 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 Fea3(3+)-C-N. The reduction of the Fea3 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 Fea3(2+)-bound cyanides, whereas the latter two bands are possibly due to the CuB(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 Fea3-CuB binuclear site upon reduction of the metal centers. The first one occurs upon reduction of the CuB center, and the second one occurs upon reduction of the Fea and/or CuA 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 Fea3 center, and this enables the binding of a second cyanide to the CuB(1+)-CN center, at a higher concentration of cyanide, being oriented toward the Fea3(2+) center to produce the 2037-cm-1 band. These structural changes at the Fea3-CuB 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.

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