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The effect of copper on the mitochondrial carnitine/acylcarnitine carrier (CAC) was studied. Transport function was assayed as [³H]carnitine/carnitine antiport in proteoliposomes reconstituted with the native protein extracted from rat liver mitochondria or with the recombinant CAC over-expressed in <i>E. coli</i>. Cu²⁺ (as well as Cu⁺) strongly inhibited the native transporter. The inhibition was reversed by GSH (reduced glutathione) or by DTE (dithioerythritol). Dose-response analysis of the inhibition of the native protein was performed from which an IC₅₀ of 1.6 &#181;M for Cu²⁺ was derived. The mechanism of inhibition was studied by using the recombinant WT or Cys site-directed mutants of CAC. From the dose-response curve of the effect of Cu²⁺ on the recombinant protein, an IC₅₀ of 0.28 &#181;M was derived. Inhibition kinetics revealed a non-competitive type of inhibition by Cu²⁺. However, a substrate protection experiment indicated that the interaction of Cu²⁺ with the protein occurred in the vicinity of the substrate-binding site. Dose-response analysis on Cys mutants led to much higher IC₅₀ values for the mutants C136S or C155S. The highest value was obtained for the C136/155S double mutant, indicating the involvement of both Cys residues in the interaction with Cu²⁺. Computational analysis performed on the WT CAC and on Cys mutants showed a pattern of the binding energy mostly overlapping the binding affinity derived from the dose-response analysis. All the data concur with bridging of Cu²⁺ with the two Cys residues, which blocks the conformational changes required for transport cycle.