Ligand Trapping by Cytochrome c Oxidase IMPLICATIONS FOR GATING AT THE CATALYTIC CENTER

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 (Fea33+–CuB2+) 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 Fea33+–CN–CuB2+ complex. Instead the cyanide-induced transition of a preformed Fea33+–N3–CuB2+ to the ternary complex of Fea33+–N3 CuB2+–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 CuB blocks a channel between the catalytic site and the solvent. The results suggest that there is a pathway that originates at CuB and that, during catalysis, ligands present at this copper center control access to the iron of heme a3 from the bulk medium.