Beyond Bioenergetics: Moonlighting Functions of F₁F_O-ATPase From ATP Synthesis to an ATP-Wasting Death Switch

Mitochondrial F₁F_O-ATPase is classically viewed as the "splendid" rotary nanomachine that sustains life by converting the proton-motive force (pmf) into ATP. Yet this same complex can adopt a second, context-dependent function that links bioenergetics to cell fate. When oxygen becomes limiting during ischemia, stroke, hypoxia, or anoxia, respiratory chain activity declines, pmf collapses, and the enzyme reverses direction: ATP hydrolysis drives rotor turnover and pumps H⁺ across the inner mitochondrial membrane to partially restore pmf. This reverse mode can be protective by preserving membrane potential and basic mitochondrial homeostasis, but it also imposes a severe energetic burden on the cell. A key determinant of whether F₁F_O-ATPase acts as an energy-conserving or energy-dissipating machine is the identity of the catalytic divalent cation. Under physiological conditions, Mg²⁺ supports efficient, reversible ATP synthesis/hydrolysis. In pathology, mitochondrial Ca²⁺ overload may replace Mg²⁺ at catalytic sites, shifting catalysis toward ATP hydrolysis and promoting an ATP-wasting state that triggers mitochondrial permeability transition pore (mPTP) formation and regulated cell death. Thus, ATP hydrolysis by F₁F_O-ATPase beyond regulation by pmf and inhibitory proteins such as IF1, cation cofactor selection emerges as a decisive switch that repurposes the enzyme from "enzyme of life" to "enzyme of death." Here, this cofactor-dependent reversibility is framed as a moonlighting role of F₁F_O-ATPase, integrating energy conversion with death signaling. Conceptual questions are outlined to resolve current debate and to exploit this switch for therapeutic insight.