Nanoscale spatial confinement of proton flux by cardiolipin drives high-speed lateral proton transport in mitochondria

Abstract

Cellular respiration depends on the rapid, lateral flow of protons along the inner mitochondrial membrane to drive ATP synthesis. The precise nanoscale thermodynamic forces confining protons to this “local circuit” remain highly debated. Previous attempts to model macroscopic interfacial proton diffusion have been hindered by parameter equifinality and geometric artifacts, preventing the deconvolution of structural water networks from lipid electrostatics. Here, we resolve this ambiguity using a constrained, high-resolution two-dimensional continuum model. By incorporating experimentally validated buffer proton consumption rates as strict biological priors, we break mathematical degeneracy and isolate the specific thermodynamic components of planar lipid bilayers. Calibrating our model against time-resolved DOPG fluorescence kinetics, we decouple a universal structural water barrier (5.7 kBT) from the specific −1e electrostatic trap (4.3kBT). Extrapolating these first principles, we predict the confinement architecture of cardiolipin, the signature −2e dimeric lipid of mitochondria. Our simulations reveal a deep 14.3 kBT thermodynamic well. Crucially, this massive barrier confines protons within 1 to 2 nanometres of the membrane surface, virtually abolishing vertical leakage into the bulk aqueous phase of the inter-membrane space. We demonstrate that this spatial confinement triggers “dimensional squeezing”, preserving a robust lateral concentration gradient that actively accelerates radial proton wave propagation. Biologically, these findings reveal that cardiolipin does not merely prevent proton dissipation, it functions as a highly efficient, quasi-two-dimensional nanoscale antenna that captures and rapidly channels protons directly to ATP synthase, ensuring the kinetic viability of eukaryotic energy production. Statement of significance Cellular respiration and ATP synthesis rely on the rapid, lateral flow of protons across mitochondrial membranes, forming a “local proton circuit” that avoids dissipation into the bulk fluid of the inter-membrane space. While it is known that protons migrate along the membrane-water interface, the exact physical forces that confine them there have remained highly debated. By combining time-resolved fluorescence kinetics with high-resolution, two-dimensional spatial modelling, we successfully deconvoluted the thermodynamic trap confining these protons. We isolated a universal ∼5.7 kBT structural barrier governed by interfacial water, which is amplified by lipid electrostatics. Crucially, we demonstrate that cardiolipin – the signature lipid of mitochondria – generates a deep ∼14.3 kBT thermodynamic well. This spatial confinement triggers “dimensional squeezing”, which preserves the lateral concentration gradient and actively accelerates radial proton transport. By restricting vertical leakage, cardiolipin functions as a high-speed, quasi-two-dimensional nanoscale antenna that rapidly channels protons directly to ATP synthase.