Mutagenic analysis of Cox11 of Rhodobacter sphaeroides: insights into the assembly of Cu(B) of cytochrome c oxidase.
The Cu(I) chaperone Cox11 is required for the insertion of Cu(B) into cytochrome c oxidase (CcO) of mitochondria and many bacteria, including Rhodobacter sphaeroides. Exploration of the copper binding stoichiometry of R. sphaeroides Cox11 led to the finding that an apparent tetramer of both mitochondrial and bacterial Cox11 binds more copper than the sum of the dimers, providing another example of the flexibility of copper binding by Cu(I)-S clusters. Site-directed mutagenesis has been used to identify components of Cox11 that are not required for copper binding but are absolutely required for the assembly of Cu(B), including conserved Cys-35 and Lys-123. In contrast to earlier proposals, Cys-35 is not required for dimerization of Cox11 or for copper binding. These findings, and the location of Cys-35 at the C-terminus of the predicted transmembrane helix and thereby close to the surface of the membrane, allow a proposal that Cys-35 is involved in the transfer of copper from the Cu(I) cluster of Cox11 to the Cu(B) ligands His-333 and His-334 during the folding of CcO subunit I. Lys-123 is located near the Cu(I) cluster of Cox11, in an area otherwise devoid of charged residues. From the analysis of several Cox11 mutants, including K123E, -L, and -R, we conclude that a previous proposal that Lys-123 provides charge balance for the stabilization of the Cu(I) cluster is unlikely to account for its absolute requirement for Cox11 function. Rather, consideration of the properties of Lys-123 and the apparent specificity of Cox11 suggest that Lys-123 plays a role in the interaction of Cox11 with its target.
Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Audie K
AK
Thompson
Daniel
D
Smith
Jimmy
J
Gray
Heather S
HS
Carr
Aimin
A
Liu
Dennis R
DR
Winge
Jonathan P
JP
Hosler
D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009153:Mutagens; D017433:Protein Structure, Secondary / Q000235:genetics; D012242:Rhodobacter sphaeroides / Q000235:genetics* / Q000378:metabolism
D008024:Ligands; D018832:Molecular Chaperones; D009153:Mutagens; D003300:Copper; D003576:Electron Transport Complex IV; D003545:Cysteine