Structural perturbation of the a3-CuB site in mitochondrial cytochrome c oxidase by alcohol solvents

Abstract

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 a3Fe and of CuB formed by low-temperature photodissociation of the a3FeCO complex. Carbon monoxide binds to reduced cytochrome oxidase in two major structural forms, alpha and beta, 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 beta-forms. Ethanol addition caused a concentration-dependent elimination of the beta-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 beta-forms were detectable. The alpha-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 degrees C, while higher concentrations of ethanol caused no further inhibition. This is the first demonstration that alpha- and beta-forms of cytochrome c oxidase can be modified by an externally added reagent. Changes in the spectra of alpha-CuBCO in the presence of 50% (v/v) ethylene glycol were quite striking, but variable.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate-Induced Carbon Monoxide Reactivity Suggests Multiple Enzyme Conformations at the Catalytic Copper M-Center of Peptidylglycine Monooxygenase

“…56, 57 Cytochrome c oxidase (CcO) is the terminal respiratory enzyme in the mitochondrial electron transfer chain responsible for reduction of molecular oxygen to water. In strongly visible light, CO is dissociated from the heme a3 pocket and rebinds to the Cu B with at least two CO stretching frequencies between 2060 cm −1 and 2035 cm −1 .…”

Modulation of the active site conformation by site-directed mutagenesis in cytochrome c oxidase from Paracoccus denitrificans

“…The discovery of the α and β-forms of the CO-complex of C c O was first made by Alben and coworkers in low temperature photolysis studies of bovine mitochondria with FTIR [18, 24-26]. The presence of the two conformers of C c O was later confirmed in several different preparations of mammalian oxidases, including rat heart myocytes and opossum heart tissue slices [24].…”

Additive Effects of Mitochondrion-targeted Cytochrome CYP2E1 and Alcohol Toxicity on Cytochrome c Oxidase Function and Stability of Respirosome Complexes

“…In one of the earliest studies, Lieber and co-workers (20) showed that alcohol treat-ment in baboons resulted in markedly altered hepatic mitochondrial CcO activity. Similarly, exposure of mitochondrial membrane fractions with alcohol caused the structural perturbation of the a3-CuB site, affecting CcO activity (21). Another study showed that hepatic mitochondrial NO levels markedly increased under chronic alcohol treatment (22,23), which covalently modified the heme moiety leading to reduced CcO activity.…”