Biochemical and functional studies on CopA: The Escherichia coli copper ion translocating P-type ATPase

Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance. Expression of copA was induced by salts of copper or silver but not zinc or cobalt. Everted membrane vesicles from cells expressing copA exhibited ATP-coupled accumulation of copper, indicating that CopA is a Copper-translocating efflux pump. CopA formed a phosphorylated intermediate with γ-[32P]ATP. Phosphorylation was inhibited by ortho-vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523. Phosphorylation required a monovalent cation, either Cu(I) or Ag(I). Divalent cations Cu(II), Zn(II) or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II). CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a Km for ATP of 0.5 mM. CopA has two N-terminal Cys(X) ₂Cys sequences, GLSC₁₄GHC₁₇ and GMSC₁₁₀ASC ₁₁₃, and a Cys₄₇₉ProCys₄₈₁ motif in membrane spanning segment six. The requirement of these cysteine residues was investigated by the effect of mutations and deletions. Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys(X)₂Cys motif showed the similar resistance to copper as the wild type CopA and formed acylphosphate intermediates. From the copper dependence of phosphoenzyme formation, the mutants appear to have 2–3 fold higher affinity for Cu(I) than wild type CopA. In contrast, substitutions in Cys-479 or Cys-481 resulted in loss of copper resistance, transport and phosphoenzyme formation. These results imply that the cysteine residues of the CysProCys motif (but not the N-terminal cysteine residues) are required for CopA function.