Peroxide-induced spectral perturbations of the 280-nm absorption band of cytochrome c oxidase

Abstract

It is now widely believed that the first two electrons transferred to the dioxygen reduction site in cytochrome c oxidase (CcO) are not coupled to proton translocation. The activation of the pump cycle correlates with the binding of dioxygen to the binuclear center. In order to investigate conformational changes in CcO associated with the formation of dixoygen intermediates during the catalytic cycle of CcO, the effects of hydrogen peroxide binding to CcO have been examined using UV optical absorption and second derivative techniques. Our data indicates that in the presence low concentrations of H 2O 2 (2:1 molar ratio) an initial CcO-peroxide species is formed in which the 280-nm absorption band is red shifted. This red shift occurs prior to spectral changes associated with H 2O 2 binding to cytochrome a 3. Upon addition of higher concentrations of H 2O 2 (⪢10 equivalents of H 2O 2 per equivalent of CcO) oxidized CcO is converted to F-state enzyme with no corresponding shift at 280 nm. It is suggested that H 2O 2 initially binds to Cu 2+ B resulting in a conformational change in the enzyme giving rise to a red-shifted 280 nm band. The absence of any conformational changes in F-state enzyme is consistent with the lack of bridging interactions with Cu 2+ B in this intermediate.