Investigation of the Physiochemical Properties of the Phospholipid Cardiolipin: Implications for Oxphos Regulation and Barth Syndrome

Abstract

Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane, which houses respiratory complexes that generate electrochemical ion gradients to drive processes such as ATP synthesis by oxidative phosphorylation. Nascent CL, which is enriched in saturated acyl chains of variable lengths, is subject to a two-stage remodeling process: a deacylase first removes an acyl chain to generate the monolysocardiolpin (MLCL) intermediate, and the transacylase tafazzin regenerates the four-chain lipid with highly unsaturated acyl chains with a high degree of symmetry. Mutations in the TAZ gene that encodes tafazzin underpin Barth syndrome, a multi-system mitochondrial disorder characterized by gross abnormalities in lipid profiles, cardiac and skeletal myopathy, neutropenia, growth retardation and mortality in early childhood. First, we analyze the role of CL in activating OXPHOS complexes using nanoscale lipid bilayers containing headgroup and acyl chain variants of CL. To this end, purified cytochrome c oxidase was reconstituted into nanodiscs of defined lipid composition and assayed for CL-dependent redox activity. Second, we have analyzed the binding characteristics of cytochrome c to bilayers containing CL variants. This work revealed that MLCL, which lacks a lipid tail, does not allow for the hydrophobic interactions and unfolding of cytochrome c, a requisite step in promoting peroxidase activity and of the apoptotic program. Finally, we use a combination of electrokinetic measurements and fluorescence-based approaches to address the ionization properties of the phosphate esters among CL variants. In contrast to the dominant paradigm in which a resonance-stabilized bicyclic headgroup structure promotes two disparate pKa values, we find the CL head group behaves as a strong dibasic acid, and exists as a dianion at physiological pH. Moreover, the ionization properties of the CL head group are similar for all variants examined.