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 aa(3)-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 Ca(2+) 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 Ca(2+) (K(d) = 1 microM) with only one sodium ion (K(d) = 4 mM). The second binding site for Na(+) in bovine oxidase is proposed to involve D442, homologous to D477 in P. denitrificans oxidase. A putative place for Na(+)((2)) in subunit I of bovine oxidase has been found with the aid of structure modeling located 7.4 A from the bound Na(+)((1)) . Na(+)((2)) interacts with a cluster of residues forming an exit part of the so-called H-proton channel, including D51 and S441.
Kirichenko
Anna V
AV
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia.
Pfitzner
Ute
U
Ludwig
Bernd
B
Soares
Claudio M
CM
Vygodina
T V
TV
Konstantinov
Alexander A
AA
D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002412:Cations; D002417:Cattle; D017124:Conserved Sequence; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology; D017433:Protein Structure, Secondary; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D013053:Spectrophotometry
D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002412:Cations; D003576:Electron Transport Complex IV