Objective Participation of the Fe‐S‐IBG subsystem in the assembly of the [2Fe‐2S] cluster into the bc1 complex. Methods Mutants of S. cerevisiae: Δiba57, Δisa1, Δgrx5, and Δrip1 were grown in YPD medium at 30°C. Mitochondria were isolated by differential centrifugation according to Auchére et al. (2008). Respiratory complex and aconitase activities were evaluated according to Gomez et al. (2014). Mitochondria were treated with digitonin to evaluate the formation of respiratory supercomplexes by electrophoresis in Polyacrylamide Blue Native Gels (BN‐PAGE), and after 2‐dimensional SDS‐PAGE analysis according to Schagger et al. (2000). Detection of the Rip1p subunit was performed by Western blot using the anti‐Rip1p monoclonal antibody at 1:20,000 dilution and developed with anti‐mouse IgG HRP‐conjugate. Abstract The iron‐sulfur clusters [Fe‐S] are inorganic cofactors contained in enzymes involved in multiple cellular processes. In eukaryotes, [Fe‐S] synthesis mainly occurs in the mitochondrial matrix by the biogenesis of Iron Sulfur Cluster system (ISC). This system involves two steps: the novo assembly of the [Fe–S] clusters on scaffold proteins, and the transference of the [Fe–S] cluster to target apo‐proteins. These steps involve the participation of several proteins, which perform specific reactions. The ISC subsystem Fe‐S‐IBG conformed by the Grx5p, Isa1p, Isa2p, and Iba57p proteins is essential for the synthesis of the [4Fe‐4S] clusters. Yeast mutants in the Fe‐S‐IBG subsystem showed deficiency in respiration and excessive ROS generation was found, similarly to the mutant Δrip1, which gene encodes for the Rieske subunit of cytochrome bc1. In this work, the activity of the cytochrome bc1 in mitochondria from the Δiba57,Δisa1, Δgrx5, and Δrip1 mutants strains were determined. Results showed that the cytochrome bc1 complex in mitochondria from the Δiba57,Δisa1, and Δrip1 mutants was dysfunctional; whereas in the Δgrx5, the cytochrome bc1 activity was partially decreased in comparison with the WT strain. BN‐PAGE analysis shows that the respiratory complex lost the capacity to form supercomplexes in mitochondria from the Δiba57 and Δisa1 mutants, Immunodetection assays showed that Rip1p was absent in the iba57Δ and Δisa1 mutants; whereas the Δgrx5 mutant shows an unaltered pattern profile of the supercomplexes bands in BN‐PAGE, without the loss of Rieske protein. In conclusion, results indicate that the Isa1p and Iba57p proteins are involved in the insertion of the [2Fe‐2S] cluster on the Rip1p apoprotein, suggesting that the Fe‐S‐IBG subsystem is not exclusive for the maturation/assembly of proteins with [4Fe‐4S] clusters, but also is involved in [2Fe‐2S] clusters assembly such as occurred in the Rieske subunit.