Iron-sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron-sulfur or Fe-Sproteins) are present inmitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe- S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the L-cysteine desulfurase activityofNFS1was barely presentwhenco-expressed with mutant ISD11.Ourfindings are consistent with a defect in the early step of ISCassemblyaffecting abroad variety of Fe-Sproteins.Thedifferences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.