F(1)-ATPase is a rotary molecular machine with a subunit stoichiometry of α(3)β(3)γ(1)δ(1)ε(1). It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α(3)β(3) core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F(1)-ATPase have suggested that the α(3)β(3) core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F(1)-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ(o)) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F(1) complexes retained 21.7 to 44.6% of the native F(1)-ATPase activity. The γ-less F(1) subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ(o) conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α(3)β(3) subcomplex.