STRUCTURAL PERTURBATION OF THE ALPHA-3-CUB SITE IN MITOCHONDRIAL CYTOCHROME-C-OXIDASE BY ALCOHOL SOLVENTS

Ethanol has been observed to cause a perturbation of the catalytic center of the major respiratory protein cytochrome c oxidase. These effects were examined by Fourier transform infrared spectroscopy of carbon monoxide complexes of cytochrome α3Fe and of CuB formed by low-temperature photodissociation of the α3FeCO complex. Carbon monoxide binds to reduced cytochrome oxidase in two major structural forms, α and ß, both of which are altered by ethanol. In the absence of ethanol, 15–22% of the total cytochrome oxidase in beef heart mitochondria was observed as ß-forms. Ethanol addition caused a concentration-dependent elimination of the 0-forms with 40% disappearing at 0.05 M (0.23%) ethanol, a concentration that can readily be achieved in the blood of intoxicated individuals. At 0.5 M (2.3%) ethanol and above, almost no ß-forms were detectable. The α-CuBCO absorption normally splits into two bands at temperatures below 40 K. This effect was decreased in the presence of ethanol and eliminated by high ethanol concentrations. It appears that ethanol increases the structural fluctuations at the active site of the enzyme, analogous to the effects of increased temperature. There was an 8–10% decrease in the maximum rate of oxygen reduction by mitochondrial cytochrome oxidase in 0.05 M ethanol at 24 °C, while higher concentrations of ethanol caused no further inhibition. This is the first demonstration that α- and ß-forms of cytochrome c oxidase can be modified by an externally added reagent. Changes in the spectra of α-CuBCO in the presence of 50% (v/v) ethylene glycol were quite striking, but variable. In contrast, the effect of glycerol appears to be limited to extraction of water, as samples prepared with glycerol had spectra similar to those of aqueous preparations. Low-temperature splitting of the α-CuBCO band is consistent with an exchange process due to H-bond formation between an ionizable ligand to α-CuB and an adjacent ionizable group from the protein. Ethanol disrupts this interaction. A model is presented. We suggest that these data require the presence of a mobile proton adjacent to the coordinated CuB complex. Such a proton may participate in dioxygen reduction to water or in proton pumping mechanisms. A corresponding exchange process is not observed in ß-forms of cytochrome c oxidase. © 1990, American Chemical Society. All rights reserved.