α,ω-Hexadecanedioic acid induces proton-translocating decoupling at complex III via Q-cycle disruption: evidence from...

This study investigates the interaction between α,ω-hexadecanedioic acid (HDA (D)) and сomplex III (E_III) electron transport chain in liver mitochondria, focusing on DE_III complex formation during succinate and glutamate/malate oxidation. A key emphasis is placed on the "idling" state of DE_III, where electron transfer occurs without proton translocation. The decoupling effect of HDA was quantified using three parameters: 1) K_d^ap and K_d^∗: apparent dissociation constants of the DE_III complex, calculated based on the total HDA concentration and the HDA quantity within the mitochondrial effective volume, respectively; 2) J_Dmax: the maximal mitochondrial respiration rate under saturating HDA concentrations (as [HDA] approaches infinity); 3) K_0.5: the HDA concentration at which the decoupling effect (J_D-J₄) equals half of its maximal value (J_Dmax-J₄), where J₄ represents the mitochondrial respiration rate in State 4. Methodologies for parameter determination were established through HDA concentration-dependent respiration profiles. Key findings reveal that K_d^ap remains substrate-independent but in contrast to K_d^∗ varies with mitochondrial protein concentration. In contrast, K_0.5 and J_Dmax/J₄ were significantly higher during succinate oxidation compared to glutamate/malate. Classical protonophores 3,5-di(tret-butyl)-4-hydroxybenzylidenemalononitrile (SF6847) and 2,4-dinitrophenol (DNP), as well as chenodeoxycholic acid (CDCA) at low concentrations, increased K_d^ap without affecting J_Dmax/J₄, suggesting reduced HDA efficacy. Molecular docking identified potential HDA binding sites on сomplex III. Based on these findings, we discuss a possible mechanism underlying the decoupling action (intrinsic uncoupling of complex III) of HDA by shuttling its protonated and anionic forms.