Understanding the Molecular Basis of the Multiple Mitochondrial Dysfunctions Syndrome 2: The Disease-Causing His96Arg Mutation of BOLA3

Multiple mitochondrial dysfunctions syndrome type 2 with hyperglycinemia (MMDS2) is a severe disorder of mitochondrial energy metabolism, associated with biallelic mutations in the gene encoding for BOLA3, a protein with a not yet completely understood role in iron-sulfur (Fe-S) cluster biogenesis, but essential for the maturation of mitochondrial [4Fe-4S] proteins. To better understand the role of BOLA3 in MMDS2, we have investigated the impact of the p.His96Arg (c.287A > G) point mutation, which involves a highly conserved residue, previously identified as a [2Fe-2S] cluster ligand in the BOLA3-[2Fe-2S]-GLRX5 heterocomplex, on the structural and functional properties of BOLA3 protein. The His96Arg mutation has been associated with a severe MMDS2 phenotype, characterized by defects in the activity of mitochondrial respiratory complexes and lipoic acid-dependent enzymes. Size exclusion chromatography, NMR, UV-visible, circular dichroism, and EPR spectroscopy characterization have shown that the His96Arg mutation does not impair the interaction of BOLA3 with its protein partner GLRX5, but leads to the formation of an aberrant BOLA3-[2Fe-2S]-GLRX5 heterocomplex, that is not functional anymore in the assembly of a [4Fe-4S] cluster on NFU1. These results allowed us to rationalize the severe phenotype observed in MMDS2 caused by His96Arg mutation.