Assignment and functional roles of the cyoABCDE gene products required for the Escherichia coli bo-type quinol oxidase.
Cytochrome bo from Escherichia coli belongs to the heme-copper terminal oxidase superfamily and functions as a redox-driven proton pump. In the present study, we examined the functional roles of the cyoABCDE genes, which encode cytochrome bo. We expressed the cyoABCDE genes in minicells using pTTQ18 derivatives and identified subunits II, I, III, and IV of the oxidase complex and heme O synthase as polypeptides with molecular weights of 33,500, 75,000, 20,500, 12,000, and 28,000, respectively. The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just tightly enough for the incorporation of heme O into the oxidase complex. To facilitate functional analysis of the gene products, we developed a single copy expression vector pHNF2, a derivative of the F-sex factor. Genetic complementation tests showed that deletions in each gene resulted in nonfunctional enzymes. Western blotting analysis indicated that the expression levels of subunits I and II were not affected by the deletions in the other cyo gene products. However, spectroscopic analyses of the mutant membranes revealed that all the deletions perturbed or eliminated the redox metal centers in subunit I. Present findings suggest that subunits II, III, and IV of the oxidase complex are required for the assembly of the metal centers in subunit I.
Nakamura
H
H
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo.
Saiki
K
K
Mogi
T
T
Anraku
Y
Y
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group; D003580:Cytochromes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000502:physiology*; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics; D008670:Metals; D008970:Molecular Weight; D009876:Operon / Q000235:genetics; D010084:Oxidation-Reduction; D017384:Sequence Deletion
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D008670:Metals; C017168:cytochrome bo, E coli; D019883:Alkyl and Aryl Transferases; C078356:CyoE protein, Bacteria; C489727:CyoE protein, E coli