Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product.
Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)
Hill
J
J
School of Chemical Sciences, University of Illinois, Urbana 61801.
Goswitz
V C
VC
Calhoun
M
M
Garcia-Horsman
J A
JA
Lemieux
L
L
Alben
J O
JO
Gennis
R B
RB
D019883:Alkyl and Aryl Transferases; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000032:analysis*; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005583:Fourier Analysis; D006418:Heme / Q000032:analysis*; D010088:Oxidoreductases / Q000737:chemistry*; D010957:Plasmids; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006418:Heme; D003300:Copper; D010088:Oxidoreductases; D003576:Electron Transport Complex IV; D019883:Alkyl and Aryl Transferases; C078356:CyoE protein, Bacteria; C489727:CyoE protein, E coli