Functional importance of a pair of conserved glutamic acid residues and of Ca(2+) binding in the cbb(3)-type oxygen reductases from Rhodobacter sphaeroides and Vibrio cholerae.

The cbb(3)-type cytochrome c oxidases are members of the family of heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb(3)-type oxidase from Pseudomonas stutzeri reveals that, in addition to the six redox-active metal centers (two b-type hemes, three c-type hemes, and Cu(B)), the enzyme also contains at least one Ca(2+). The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b(3) and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in subunit CcoN. The glutamate that is ligated to Ca(2+) is one of a pair of glutamic acid residues that has previously been suggested to be part of a proton exit pathway for pumped protons. In this work, mutations of these glutamates are investigated in the cbb(3)-type oxidases from Vibrio cholerae and Rhodobacter sphaeroides. Metal analysis shows that each of these wild-type enzymes contains Ca(2+). Mutations of the glutamate expected to ligate the Ca(2+) in each of these enzymes (E126 in V. cholerae and E180 in R. sphaeroides) result in a loss of activity as well as a loss of Ca(2+). Mutations of the nearby glutamate (E129 in V. cholerae and E183 in R. sphaeroides) also resulted in a loss of oxidase activity and a loss of Ca(2+). It is concluded that the Ca(2+) is essential for assembly of the fully functional enzyme and that neither of the glutamates is likely to be part of a pathway for pumped protons within the cbb(3)-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme.