Unraveling the molecular determinants of a rare human mitochondrial disorder caused by the <scp>P144L</scp> mutation of <scp>FDX2</scp>

Episodic mitochondrial myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL) is a rare, orphan autosomal recessive disorder caused by mutations in ferredoxin-2 (FDX2), which is a [2Fe-2S] cluster-binding protein participating in the formation of iron-sulfur clusters in mitochondria. In this biosynthetic pathway, FDX2 works as electron donor to promote the assembly of both [2Fe-2S] and [4Fe-4S] clusters. A recently identified missense mutation of MEOAL is the homozygous mutation c.431C>T (p.P144L) described in six patients from two unrelated families. This mutation alters a highly conserved proline residue located in a loop of FDX2 that is distant from the [2Fe-2S] cluster. How this Pro to Leu substitution damages iron-sulfur cluster biosynthesis is unknown. In this work, we have first compared the structural, dynamic, cluster binding and redox properties of WT and P144L [2Fe-2S] FDX2 to have clues on how the pathogenic P144L mutation can perturb the FDX2 function. Then, we have investigated the interaction of both WT and P144L [2Fe-2S] FDX2 with its physiological electron donor, ferredoxin reductase FDXR, comparing their electron transfer efficiency and protein-protein recognition patterns. Overall, the data indicate that the pathogenic P144L mutation negatively affects the FDXR-dependent electron transfer pathway from NADPH to FDX2, thereby reducing the capacity of FDX2 in assembling both [2Fe-2S] and [4Fe-4S] clusters. Our study also provided solid molecular evidences on the functional role of the C-terminal tail of FDX2 in the electron transfer between FDX2 and FDXR.