Functional Importance of a Pair of Conserved Glutamic Acid Residues and of Ca²⁺ Binding in the cbb₃-Type Oxygen Reductases from Rhodobacter sphaeroides and Vibrio cholerae

Abstract

The cbb3-type cytochrome c oxidases are members of the 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 hemes b, three hemes c and CuB), 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 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 the current work, mutants in these glutamates are investigated in the cbb3-type oxidases from Vibrio cholerae and from Rhodobacter sphaeroides. Metal analysis shows that each of these wild type enzymes contains Ca2+. Mutations of the glutamate expected to ligate the Ca2+ in each of these enzymes (E126 in V. cholerae; E180 in R. sphaeroides) result in the loss of activity as well as loss of Ca2+. Mutations in the nearby glutamate (E129 in V. cholerae; E183 in R. sphaeroides) also resulted in loss of oxidase activity and 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.

Conformational coupling between the active site and residues within the K C -channel of the Vibrio cholerae cbb 3 -type (C-family) oxygen reductase

“…This is demonstrated by the perturbations of the UV-visible spectra, as well as by the kinetics of CO recombination after flash photolysis of the CO adduct of the fully reduced enzyme. A similar phenotype was previously observed in studies of mutations of residues that are hydrogen-bonded to propionates of heme b or heme b 3 (26). We cannot state with certainty that these mutations also block proton transfer through the K C -channel, because this cannot be evaluated owing to a lack of CO photolysis; however, the simplest explanation is that perturbations at the active site result in a lack of catalytic function.…”

Direct Regulation of Cytochrome c Oxidase by Calcium Ions

“…The same role of locking securely the non-operative proton pathway can be proposed for the tightly-bound Ca 2+ in the cbb 3 oxidases, where the cation binds with the critical residues gating connection to the exit of the D-channel-associated proton pumping pathway that works in the A-type oxidases but is non-operative in the C-type cbb 3 oxidases [58], [59].…”

Characterization of the nitric oxide reductase from Thermus thermophilus

“…There is a single Ca 2+ in the same location in the homologous qNORs (9) and also in the C-family oxygen reductases (cbb 3 oxygen reductases) (8). Site-directed mutagenesis experiments have also concluded that the pair of glutamic acids corresponding to E134 and E137 in TtcNOR is required for structural stability of both the qNORs (9) and the cbb 3 oxygen reductases (34). The A and B family of oxygen reductases do not have Ca 2+ at the equivalent position but, rather, have a pair of arginine residues that appear to have a similar function (35)(36)(37)(38).…”