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.
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.
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
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Tsubaki
D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000494:pharmacology; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D003486:Cyanides / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism*; D005583:Fourier Analysis; D008929:Mitochondria, Heart / Q000201:enzymology; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared
D003486:Cyanides; D005290:Ferric Compounds; D003300:Copper; D002248:Carbon Monoxide; D004492:Edetic Acid; D003576:Electron Transport Complex IV