Magic angle spinning 31P NMR spectroscopy reveals two essentially identical ionization states for the cardiolipin phosphates in phospholipid liposomes

Abstract

Specific membrane lipid composition is crucial for optimized structural and functional organization of biological membranes. Cardiolipin is a unique phospholipid and important component of the inner mitochondrial membrane. It is involved in energy metabolism, inner mitochondrial membrane transport, regulation of multiple metabolic reactions and apoptotic cell death. The physico-chemical properties of cardiolipin have been studied extensively but despite all these efforts there is still lingering controversy regarding the ionization of the two phosphate groups of cardiolipin. Results obtained in the 1990s and early 2000s suggested that cardiolipin has two disparate pKa values where one of the protons was proposed to be stabilized by an intramolecular hydrogen bond. This has led to extensive speculations on the roles of these two putative ionization states of cardiolipin in mitochondria. More recently the notion of two pKa values has been challenged and rejected by several groups. These studies relied on external measurements of proton adsorption or electrophoretic mobility of membranes but did not take into account the low pH phase behavior and chemical stability of cardiolipin. Here we used 31P NMR to show that in the physiologically relevant membrane phospholipid environment, cardiolipin carries two negative charges at physiological pH. We additionally demonstrate the pH dependent phase behavior and chemical stability of cardiolipin containing membranes.

Known unknowns of cardiolipin signaling: The best is yet to come

“…While the pKa of the phosphates of CL had originally been described as strong acids and were doubly negatively charged [53], a pH titration study comparing a native and 2'-deoxy-glycerol backbone suggested that there was both a low and high pKa[54]. Subsequently membrane measurements of CL using external proton adsorption, electrophoretic mobility of membranes and magic angle spinning P 31 NMR have clearly shown that the pKa of CL as well as lyso-CL is ~2-3 indicating that both phosphates are ionized at physiological pH in lipid bilayers [55-57]. Thus it seems logical or almost certain that at physiological pH, CLs in lipid bilayers adopt the states with two fully ionized phosphate groups.…”

Molecular Dynamics Analysis of Cardiolipin and Monolysocardiolipin on Bilayer Properties

“…The ionization state of the two phosphate moieties of CL has been a debated topic. Recent studies suggest that CL is doubly deprotonated at physiological pH and carries a charge of À2 (9)(10)(11)(12), though other literature indicates circumstances in which CL can carry a À1 charge (13)(14)(15)(16). The tuning of CL's charge state will affect the headgroup charge repulsion and modulate molecular geometry.…”

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons

“…15,16 Despite concurrent work which supported protonation of a CL phosphate in this pH regime, 25 recent studies question whether a CL phosphate ionizes near physiological pH. 26,27 Furthermore, the reverse micelle study, which identified site N, did not detect evidence for a CL binding site near Asn52. 14 Discrepancies in the magnitudes of binding constants for sites A and C also exist in the literature.…”