THE ROLE OF CHARGED AMINO-ACIDS IN THE ALPHA-1-BETA-4 LOOP OF THE IRON-SULFUR PROTEIN OF THE CYTOCHROME BC(1) COMPLEX OF YEAST MITOCHONDRIA

Previous experiments using deletion mutants of the iron-sulfur protein had indicated that amino acid residues 138-153 might be involved in t he assembly of this protein into the cytochrome bc(1) complex. To dete rmine which specific residues might be involved in the assembly proces s, charged amino acids located in the alpha 1-beta 4 loop of the iron- sulfur protein were mutated to uncharged residues and tryptophan 152 t o phenylalanine. The mutant genes were used to transform yeast cells ( JPJ1) lacking the iron-sulfur protein gene. Mutants R146I and W152F ha d almost undetectable growth in medium containing glycerol/ethanol, wh ereas mutants D143A, K148I, and D149A grew more slowly than the wild t ype. Activity of the cytochrome bc(1) complex was decreased 50, 90, 67 , 89, and 90% in mutants D143A, R146I, K148I, D149A, and W152F, respec tively, but unchanged in mutants D139A, Q141I, D145L, and V147S, In al l of these mutants except W152F, the cytochrome c(1) content, determin ed by immunoblotting, was comparable with that of wild-type cells. How ever, immunoblotting revealed that the content of the iron-sulfur prot ein was decreased proportionately in the five mutants with lowered enz ymatic activity and growth suggesting that these amino acids are criti cal for maintaining the stability of the iron-sulfur protein. The effi ciency of assembly in vitro compared with the wild type determined by selective immunoprecipitation was unchanged in the mutants with the ex ception of R146I, D149A, and W152F where decreases of 80, 60, and 60%, respectively, were observed suggesting that these amino acids are cri tical for the proper assembly of the iron-sulfur protein into the bc(1 ) complex.