Abstract
Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane. Neither the redox center(s) associated with proton pumping nor the pumping mechanism presumably common to all heme-copper oxidases has been established. A possible conformational coupling between the catalytic center (Fe(a3)(3+)-Cu(B)(2+)) and a protein site has been identified earlier from ligand binding studies, whereas a structural change initiated by azide binding to the protein has been proposed to facilitate the access of cyanide to the catalytic center of the oxidized bovine enzyme. Here we show that cytochrome oxidase pretreated with a low concentration of azide exhibits a significant increase in the apparent rate of cyanide binding relative to that of free enzyme. However, this increase in rate does not reflect a conformational change enhancing the rapid formation of a Fe(a3)(3+)-CN-Cu(B)(2+) complex. Instead the cyanide-induced transition of a preformed Fe(a3)(3+)-N(3)-Cu(B)(2+) to the ternary complex of Fe(a3)(3+)-N(3) Cu(B)(2+)-CN is the most likely reason for the observed acceleration. Significantly, the slow rate of azide release from the ternary complex indicates that cyanide ligated to Cu(B) blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at Cu(B) and that, during catalysis, ligands present at this copper center control access to the iron of heme a(3) from the bulk medium.
Highlights
Cellular respiration involves membrane-bound complexes that catalyze the coupling of electron transfer to formation of a proton gradient
Cytochrome c oxidase is a member of the heme-copper family of oxygen reductases in which electron transfer is linked to the pumping of protons across the membrane
Cytochrome c oxidases, terminal enzymes in mitochondria and many prokaryotes, are members of a family of heme-copper oxygen reductases sharing a common structure at the heme a3–CuB catalytic site
Summary
CcO, cytochrome c oxidase; Fea3ϩ, oxidized iron of heme a; Fea33ϩ, oxidized iron of heme a3; CuB2ϩ, oxidized copper at the catalytic site; FTIR, Fourier-transform infrared spectroscopy; DM, n-dodecyl--D-maltoside. It is shown that the presumed conformational interaction between the distal azide binding site and the heme a3–CuB center is not very likely Rather it appears that the acceleration of absorbance changes observed in the presence of azide results from cyanide binding to CuB in the preformed complex of Fea33ϩ–N3–CuB2ϩ. The data imply that alteration of the ligation of CuB can be a gating mechanism controlling the opening of a passage between heme a3 and the solvent during turnover
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