PROTON-TRANSFER IN CYTOCHROME BO(3) UBIQUINOL OXIDASE OF ESCHERICHIA-COLI - 2ND-SITE MUTATIONS IN SUBUNIT-I THAT RESTORE PROTON-PUMPING IN THE MUTANT ASP135-]ASN

The ubiquinol oxidase, cytochrome bo(3), of Escherichia coli is a memb er of the respiratory heme-copper oxidase family and conserves energy from the reduction of dioxygen to water by translocation of protons ac ross the bacterial membrane. Mutation of an aspartic acid residue (Asp 135) to asparagine in subunit I of this enzyme was previously found to impair proton translocation [Thomas et al. (1993) Biochemistry 32, 10 923-10928]. This residue is located in an interhelical ''loop'' betwee n transmembranous helices II and III, which contains six well-conserve d residues (Asn124, Pro128, G1y132, Asp135, Pro139, and Asn142). Site- directed mutagenesis was performed to study the function of this entir e domain. Nonconservative mutations of Asn124 and Asn142 also resulted in a loss of proton translocation, whereas their conservative substit ution to glutamine had no effect. Mutations in eight other positions w ithin this domain did not affect proton translocation. Introduction of an acidic group at positions 139 or 142, but not at eight other teste d positions, restored proton pumping in the Asp135--> Asn mutated prot ein. These results suggest that the C-terminal part of the domain may be alpha-helical and that the entire ''loop'' plays an important struc tural and functional role as part of an input channel of the proton tr anslocation machinery.