Identification of a disulfide switch in BsSco, a member of the Sco family of cytochrome c oxidase assembly proteins.
BsSco is a membrane-associated protein from Bacillus subtilis characterized by the sequence CXXXCP, which is conserved in yeast and human mitochondrial Sco proteins, and their bacterial homologues. BsSco is involved in the assembly of the Cu(A) center in cytochrome c oxidase and may play a role in the transfer of copper to this site. We have characterized the soluble domain of BsSco by biochemical, spectroscopic, and structural approaches. Soluble BsSco is monomeric in solution, and the two conserved cysteines are involved in an intramolecular cystine bridge. The cystine bridge is easily reduced, and circular dichroism spectroscopy shows no large-scale changes in BsSco's secondary structure upon reduction. The crystal structure of soluble BsSco, determined at 1.7 A resolution, reveals typical elements of a thioredoxin fold. The CXXXCP motif, in which Cys45 and Cys49 are conserved, is located in a turn structure on the surface of the protein. In various native and His135Ala mutant structures, both disulfide-bonded and non-disulfide-bonded forms of CXXXCP are observed. However, despite extensive attempts, copper has not been found near or beyond the CXXXCP motif, a presumptive copper-binding site. Another potential copper binding residue, His135, is located in a highly flexible loop parallel to the CXXXCP loop but is more than 10 A from Cys45 and Cys49. If these three residues are to coordinate copper, a conformational change is necessary. The structural identification of a disulfide switch demonstrates that BsSco has the capability to fill a redox role in Cu(A) assembly.
Ye
Qilu
Q
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Imriskova-Sosova
Iveta
I
Hill
Bruce C
BC
Jia
Zongchao
Z
D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D004220:Disulfides; D008565:Membrane Proteins; C482909:Sco protein, Bacillus subtilis; D003576:Electron Transport Complex IV