γM23K mutation enhances ADP inhibition of mitochondrial F-ATPase yet has a minor effect on yeast growth rate

ADP inhibition of ATP hydrolysis is a conserved regulatory mechanism found in virtually all F-type ATP synthases. It occurs when ADP binds tightly to a catalytic site in the absence of phosphate, locking the enzyme in an inactive conformation. This inhibition is thought to prevent wasteful ATP hydrolysis, particularly under conditions where the proton-motive force is insufficient to drive ATP synthesis. However, the physiological significance of this mechanism remains unclear. To explore this further, we introduced the γM23K mutation-previously shown to enhance ADP inhibition in bacterial ATP synthases-into the yeast mitochondrial ATP synthase by engineering the corresponding ATP3M23K substitution in Saccharomyces cerevisiae. In isolated mitochondria, this mutation did not impair ATP synthesis but significantly increased the sensitivity of ATPase activity to ADP and azide, indicating enhanced ADP inhibition. In vivo, the mutation had no effect on growth under fermentable conditions and only mildly reduced the growth rate of rho⁺ cells on a non-fermentable carbon source. Contrary to expectations, the ATP3M23K mutation did not impair the growth rate or viability of rho⁰ cells, which rely on ATP hydrolysis in the matrix to maintain mitochondrial membrane potential via ATP/ADP exchange with the cytoplasm. Furthermore, no significant differences were observed in lag phase duration following prolonged starvation, nor in ATP content in stationary-phase cultures. These findings confirm that ADP inhibition can be selectively enhanced in yeast F_OF₁-ATPase without disrupting ATP synthesis, but also suggest that this regulatory mechanism plays a limited role in shaping cellular physiology under standard laboratory conditions.