SUBSTITUTION OF ASPARAGINE FOR ASPARTATE-135 IN SUBUNIT-I OF THE CYTOCHROME-BO UBIQUINOL OXIDASE OF ESCHERICHIA-COLI ELIMINATES PROTON-PUMPING ACTIVITY

The terminal quinol oxidase, cytochrome bo, of Escherichia coli is a m ember of the large terminal oxidase family, which includes cytochrome aa3-type terminal oxidases from bacteria, plants, and animals. These e nzymes conserve energy by linking electron transfer to vectorial proto n translocation across mitochondrial or bacterial cell membranes. Site -directed mutagenesis of the five most highly conserved acidic amino a cids in subunit I of cytochrome bo was performed to study their role i n proton transfer. Mutation of only one of these sites, Asp135, to the corresponding amide, results in a dramatic decrease in proton pumping but with little change in electron-transfer activity. However, the co nservative mutation Asp135Glu is active in proton translocation. It is proposed that an acidic residue at position 135 in subunit I may be i mportant to form a functional proton input channel of the proton pump.