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.
Department of Chemistry, University of Illinois, Urbana, Illinois 61801, United States.
Hanlin
H
Ouyang
Huazhi
H
Han
Jung H
JH
Roh
James
J
Hemp
Jonathan P
JP
Hosler
Robert B
RB
Gennis
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011485:Protein Binding; D021122:Protein Subunits; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D021122:Protein Subunits; D018698:Glutamic Acid; D006418:Heme; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV; D002118:Calcium