Cytochrome c Oxidase as a Calcium Binding Protein. Studies on the Role of a Conserved Aspartate in Helices XI−XII Cytoplasmic Loop in Cation Binding†

The aa3-type cytochrome c oxidases from mitochondria and bacteria contain a cation-binding site located in subunit I near heme a. In the oxidases from Paracoccus denitrificans or Rhodobacter sphaeroides, the site is occupied by tightly bound calcium, whereas the mitochondrial oxidase binds reversibly calcium or sodium that compete with each other. The functional role of the site has not yet been established. D477A mutation in subunit I of P. denitrificans oxidase converts the cation-binding site to a mitochondrial-type form that binds reversibly calcium and sodium ions [Pfitzner, U., Kirichenko, A., et al. (1999) FEBS Lett. 456, 365−369]. We have studied reversible cation binding with P. denitrificans D477A oxidase and compared it with that in bovine enzyme. In bovine oxidase, one Ca2+ competes with two Na+ for the binding, indicating the presence of two Na+-binding sites in the enzyme, Na+(1) and Na+(2). In contrast, the D477A mutant of COX from P. denitrificans reveals competition of Ca2+ (Kd = 1 μM) wi...