Functional importance of a pair of conserved glutamic acid residues and of Ca²⁺ binding in the cbb ₃-type oxygen reductases from rhodobacter sphaeroides and vibrio cholerae

The cbb₃-type cytochrome c oxidases are members of the family of heme-copper proton pumping respiratory oxygen reductases. The structure of the cbb₃-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 Ca²⁺. The calcium bridges two propionate carboxyls at the interface between the low-spin heme b and the active-site heme b₃ and, in addition, is ligated to a serine in subunit CcoO and by a glutamate in subunit CcoN. The glutamate that is ligated to Ca²⁺ 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 cbb₃-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 Ca²⁺ 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 Ca²⁺. 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 Ca²⁺. It is concluded that the Ca²⁺ 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 cbb₃-type oxygen reductases. A more likely role for these glutamates is the maintenance of the structural integrity of the active conformation of the enzyme.