Mutations on the N-terminal edge of the DELSEED loop in either the α or β subunit of the mitochondrial F₁-Atpase enhance ATP hydrolysis in the absence of the central γ rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃ β₃ γ₁ δ₁ ε₁. 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 confioormatnal changes to the catalytic sites in the α₃ β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ 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 F₁-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°) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-typeγ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. Theγ-less F₁ 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° conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.