What question did this study set out to answer?

The study aims to investigate the role of VLCAD in facilitating interactions between fatty acid oxidation and electron transport chain proteins in mitochondria.

January 22, 2026Open Access

Restoration of Interaction Between Fatty Acid Oxidation and Electron Transport Chain Proteins In Vitro by Addition of Recombinant VLCAD

Key Points

The study aims to investigate the role of VLCAD in facilitating interactions between fatty acid oxidation and electron transport chain proteins in mitochondria.
Conducted mitochondrial functional studies on mutant and VLCAD knockout mouse mitochondria.
Measured levels of FAO and ETC proteins in VLCAD-deficient heart mitochondria.
Assessed enzyme activities and FAO-ETC flux before and after VLCAD reconstitution.
VLCAD knockout mice showed disrupted mitochondria supercomplexes and reduced specific protein levels.
Oxidative phosphorylation enzyme activities declined in VLCAD-deficient mice, though total protein amounts remained stable.
Reconstitution with recombinant VLCAD improved protein levels and enzyme activities, restoring FAO-ETC flux and reducing mitochondrial ROS levels.

Abstract

Background/Objectives: We have previously demonstrated that fatty acid oxidation (FAO) enzymes physically and functionally interact with electron transfer chain supercomplexes (ETC-SC) at two contact points. The FAO trifunctional protein (TFP) and electron transfer flavoprotein dehydrogenase (ETFDH) interact with the NADH+-binding domain of ETC complex I (com I) and the core 2 subunit of complex III (com III), respectively. In addition, the FAO enzyme very-long-chain acyl-CoA dehydrogenase (VLCAD) interacts with TFP. These interactions define a functional FAO-ETC macromolecular complex (FAO-ETC MEC) in which FAO-generated NADH+ and FADH2 can safely transfer electron equivalents to ETC in order to generate ATP. Methods: In this study, we use multiple mitochondrial functional studies to demonstrate the effect of added VLCAD protein on mutant mitochondria. Results: We demonstrate that heart mitochondria from a VLCAD knockout (KO) mouse exhibit disrupted supercomplexes, with significantly reduced levels of TFPα and TFPβ subunits, electron transfer flavoprotein a-subunit (ETFα), and NDUFV2 subunit of com I in the FAO-ETC MEC. In addition, the activities of individual oxidative phosphorylation (OXPHOS) enzymes are decreased, as is the transfer of reducing equivalents from palmitoyl-CoA to ETC (FAO-ETC flux). However, the total amount of these proteins did not decrease in VLCAD KO animals. These results suggest that loss of VLCAD affects the interactions of FAO and ETC proteins in the FAO-ETC MEC. Reconstitution of VLCAD-deficient heart mitochondria with recombinant VLCAD improved the levels of FAO-ETC MEC proteins and enzyme activities, as well as restoring FAO-ETC flux. It also reduced mitochondrial ROS levels, previously demonstrated to be elevated in VLCAD-deficient mitochondria. In contrast, incubation of VLCAD KO mitochondria with two VLCADs with mutations in the C-terminal domain of the enzyme (A450P and L462P) did not restore FAO-ETC MECs. Conclusions: These results suggest that VLCAD is a necessary component of the FAO-ETC MEC and plays a major role in assembly of the macro-supercomplex. These studies provide evidence that both the level of enzyme and its structural confirmation are necessary to stabilize the FAO-ETC MEC.

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Wang et al. (Tue,) studied this question.

synapsesocial.com/papers/6971bea8642b1836717e35b2 https://doi.org/https://doi.org/10.3390/biomedicines14010222

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