The activities of biomolecules are affected by the proton concentrations at biological membranes. Here, we succeeded in evaluating the interface proton concentration (-log[H⁺] defined as pH') of cardiolipin (CL)-enriched membrane models of the inner mitochondrial membrane (IMM) using a spiro-rhodamine-glucose molecule (RHG). According to fluorescence microscopy and ¹H-NMR studies, RHG interacted with the Stern layer of the membrane. The acid/base equilibrium of RHG between its protonated open form (o-RHG) and deprotonated closed spiro-form (c-RHG) at the membrane interface was monitored with UV-vis absorption and fluorescence spectra. The interface pH' of 25% cardiolipin (CL)-containing large unilamellar vesicles (LUVs), which possess similar lipid properties to those of the IMM, was estimated to be ∼3.9, when the bulk pH was similar to the mitochondrial intermembrane space pH (6.8). However, for the membranes containing mono-anionic lipids, the interface pH' was estimated to be ∼5.3 at bulk pH 6.8, indicating that the local negative charges of the lipid headgroups in the lipid membranes are responsible for the deviation of the interface pH' from the bulk pH. The peroxidase activity of cyt c increased 5-7 fold upon lowering the pH to 3.9-4.3 or adding CL-containing (10-25% of total lipids) LUVs compared to that at bulk pH 6.8, indicating that the pH' decrease at the IMM interface from the bulk pH enhances the peroxidase activity of cyt c. The peroxidase activity of cyt c at the membrane interface of tetraoleoyl CL (TOCL)-enriched (50% of total lipids) LUVs was higher than that estimated from the interface pH', while the peroxidase activity was similar to that estimated from the interface pH' for tetramyristoyl CL (TMCL)-enriched LUVs, supporting the hypothesis that when interacting with TOCL (not TMCL), cyt c opens the heme crevice to substrates. The present simple methodology allows us to estimate the interface proton concentrations of complex biological membranes.

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