ARGININE-41 OF SUBUNIT-C OF ESCHERICHIA-COLI H-ATP SYNTHASE IS ESSENTIAL IN BINDING AND COUPLING OF F1 TO F-0()

Two substitutions were made for Arg(41) in the polar loop of subunit c of the Escherichia coli F1F0 H+-transporting ATP synthase. The R41K a nd R41H mutants were initially studied by use of a plasmid carrying th e complete c R41K or c R41H unc (F1F0) operon in a chromosomal strain deleted for the unc operon. The extent of F-0 incorporation into membr anes of these cells was quite variable, and the system was concluded t o be unsuitable for biochemical characterization. Ultimately, the muta nt genes were recombined into the chromosome using a novel method for the unc system. The biochemical phenotype of the chromosomally express ed mutants proved to be reproducible. The c R41H mutation causes a spe cific defect in assembly of F-0, i.e. subunit alpha was not incorporat ed into the membrane despite near normal incorporation of subunits b a nd c. On the other hand, c R41K mutant F-0 assembled normally in one o f two background strains studied. (In the second genetic background, s ubunit alpha was inefficiently incorporated into the c R41K membrane.) In membranes prepared from a c R41K strain assembling a complete F-0, R41K F-0 was found to bind F-1 with near normal affinity and to trans port H+ at near normal rates. Although R41K F-0 binds F-1, F-1-ATPase activity and H+ transport remained uncoupled. The uncoupling was indic ated by a lack of ATP-driven H+ translocation and by the high proton p ermeability of membranes with F-1 bound to F-0. The uncoupled phenotyp e of the R41K mutant closely resembles that previously reported for th e c Q42E mutant.