The alpha-like subunits of F1F0 ATP synthases share primary structural homology in two segments near their carboxyl termini. However, the amino acids at the functionally important positions occupied by alpha Gly-218 and alpha His-245 in Escherichia coli vary depending upon organism and organelle. The alpha G218-->D,H245-G and alpha G218-->K,H245-->G double mutations were constructed in the E. coli uncB(alpha) gene to model the chloroplast ATPase IV subunit and alkaliphilic bacterial alpha subunit, respectively. Strains carrying each of the single mutations, alpha G218-->D, alpha G218-->K, and alpha H245-->G, had marked reductions in F1F0 ATP synthase function. The alpha G218-->K mutation was alone sufficient to virtually eliminate enzyme function. Membranes prepared from the alpha G218-->D mutant exhibited increased levels of ATP hydrolysis activity without a corresponding increase in active proton transport, suggesting a mechanistic uncoupling of catalytic activity and proton translocation. However, much of the lost F1F0 ATP synthase activity was restored in the alpha G218-->D,H245-->G and alpha G218-->K,H245-->G double mutant strains demonstrating that these mutations act as mutual intragenic second-site suppressors. The evidence is consistent with a close spatial interaction between alpha Gly-218 and alpha His-245.