ASSIGNMENT AND FUNCTIONAL ROLES OF THE CYOABCDE GENE-PRODUCTS REQUIRED FOR THE ESCHERICHIA-COLI BO-TYPE QUINOL OXIDASE

Cytochrome be from Escherichia cell belongs to the heme-copper termina l oxidase superfamily and functions as a redox-driven proton pump, In the present study, we examined the functional roles of the cyoABCDE ge nes, which encode cytochrome be, We expressed the cyoABCDE genes in mi nicells using pTTQ18 derivatives and identified subunits II, I, III, a nd 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, respe ctively, The expression level of heme O synthase (CyoE) was much lower than those of the oxidase subunits and seems to be controlled just ti ghtly 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 facto r, Genetic complementation tests showed that deletions in each gene re sulted in nonfunctional enzymes, Western blotting analysis indicated t hat the expression levels of subunits I and II were not affected by th e deletions in the other cyo gene products, However, spectroscopic ana lyses of the mutant membranes revealed that all the deletions perturbe d or eliminated the redox metal centers in subunit I. Present findings suggest that subunits II, III, and IV of the oxidase complex are requ ired for the assembly of the metal centers in subunit I.