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The iron-sulfur protein, a subunit of the bc1 complex, is encoded in the nucleus and subsequently imported into the mitochondria where it is assembled with the rest of the subunits into a functional cytochrome bc1 complex. The assembly of deletion mutants of the iron-sulfur protein into the bc1 complex has been investigated by immunoprecipitation by specific antisera against either the iron-sulfur protein or the intact bc1 complex. These studies have suggested that the iron-sulfur protein may be integrated into the bc1 complex of mitochondria through multiple interactions of different regions of the protein with other subunits of the bc1 complex by a process involving hydrophobic and hydrophilic interactions. Furthermore this investigation suggested that amino acid residues 138–153 located in the α1-β4 loop of the extrinsic globular domain of the protein might be involved in the assembly process. To test this hypothesis, charged amino acids located in the α1-β4 loop of the iron-sulfur protein were mutated to uncharged residues and tryptophan-152 to phenylalanine. The mutant genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur protein gene. The effects of these mutations on growth, enzyme activity and protein expression have been determined. The results of this study suggest that several charged amino acids and Trp-152 located in the α1-β4 loop of the iron-sulfur protein of the bc1 complex are required to maintain the stability of the protein in vivo . These investigations were extended to mutations of charged and uncharged amino acids located in the α-1 helix of the iron-sulfur protein. The results of these investigations suggest that several amino acids located in the α-1 helix of the protein are important in maintaining the stability and proper assembly of the iron-sulfur protein into the complex. Recent crystallographic studies have proposed that the iron-sulfur protein undergoes some movement to facilitate electron transfer. To establish the structural requirements of the proposed movement, the effects of mutating alanines 86, 90 and 92 in the putative flexible loop have been investigated. The results of this investigation suggest that if movement does occur then rigid structural requirements in this region of the protein are not critical.