Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.
The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.
van der Oost
J
J
European Molecular Biology Laboratory, Heidelberg, Germany.
Lappalainen
P
P
Musacchio
A
A
Warne
A
A
Lemieux
L
L
Rumbley
J
J
Gennis
R B
RB
Aasa
R
R
Pascher
T
T
Malmström
B G
BG
D000595:Amino Acid Sequence; D001400:Azurin / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005818:Genetic Engineering; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; C050107:cupredoxin; D001400:Azurin; D003300:Copper; C017168:cytochrome bo, E coli; D010088:Oxidoreductases; C020094:duroquinol oxidase; D003576:Electron Transport Complex IV