Intersubunit bridging by Na+ ions as a rationale for the unusual stability of the c-rings of Na+-translocating F1F0 ATP synthases

The oligomeric c-rings of Na⁺-translocating F₁F₀ ATP synthases exhibit unusual stability, resisting even boiling in SDS. Here, we show that the molecular basis for this remarkable property is intersubunit crossbridging by Na⁺ or Li⁺ ions. The heat stability of c₁₁ was dependent on the presence of Na⁺ or Li⁺ ions. For equal stability, 10 times higher Li⁺ than Na⁺ concentrations were required, reflecting the 10 times lower binding affinity for Li⁺ than for Na⁺. In a recent structural model of c₁₁, the Na⁺ or Li⁺ binding ligands are located on neighboring c-subunits, which thus become crossbridged by the binding of either alkali ion with a concomitant increase in the stability of the ring. Site-directed mutagenesis strengthens the essential role of glutamate 65 in the crossbridging of the subunits and also corroborates the proposed stabilizing effect of an ion bridge including aspartate 2.