Investigating the Impact of Natural SNPs on hETF Conformational Dynamics and FAD Cofactor Alteration

Human electron transfer flavoprotein (hETF) is a critical mitochondrial enzyme essential for fatty acid β-oxidation. Genetic variations, mainly single nucleotide polymorphisms (SNPs) in the electron transfer flavoprotein dehydrogenase (ETFDH) gene, can lead to multiple acyl-CoA dehydrogenase deficiency (MADD) and severe metabolic dysfunction. Many disease-associated missense mutations cluster at the α/β-subunit interface, affecting protein structure and stability at the molecular level. Recent studies demonstrated that the flavin adenine dinucleotide (FAD) cofactor within hETF is susceptible to oxidation into 8-formyl-FAD (8f-FAD), potentially altering enzyme kinetics. This study employs in silico methodologies to explore the impact of three naturally occurring SNPs at the α/β-subunit interface—hETF-βE165K, hETF-αN259S, and hETF-αN259D—on hETF structure, stability, and the potential for FAD modification to 8-fromyl-FAD (8f-FAD). Our findings suggest that these SNPs likely favor an open hETF conformation due to disrupted inter-subunit interactions, potentially increasing the susceptibility of the FAD cofactor oxidation to 8f-FAD. This hypothesis is supported by comparative analyses with previously studied experimental variants (hETF-αN259A and hETF-βE165A).This in silico investigation provides novel insights into the structural and energetic consequences of these natural hETF variants at the molecular level, highlighting the role of molecular interactions in protein function and dysfunction. These findings lay the groundwork for future experimental studies aimed at elucidating their role in metabolic disorders and informing the development of targeted diagnostic and therapeutic strategies at the molecular level.