Date of Graduation
1998
Document Type
Thesis
Degree Type
MS
College
School of Medicine
Department
Biochemistry
Committee Chair
Diana S. Beattie
Committee Member
James E. Mahaney
Committee Member
Andrew K. Shiemke
Committee Member
Stephen G. Graber
Abstract
The Rieske iron sulfur protein is a subunit of the cytochrome bc1 complex of the mitochondrial electron transport chain. It is hypothesized that electron transfer by the iron sulfur protein requires movement of a flexible "tether" region that connects the transmembrane portion of the protein to its extramembranous domain (Zhang et al., 1998). To determine which amino acid residues are important for catalysis and stability of the Saccharomyces cerevisiae iron sulfur protein, site-directed mutagenesis was performed on two charged residues adjacent to the proposed flexible region (lysine 93 to leucine and glutamate 95 to arginine). In addition, a hydrophobic residue in the membrane-spanning domain, leucine 65, was mutated to arginine. The mutations K93L and E95R did not affect the growth of yeast in nonfermentable media at the permissive temperature of 30o C, the enzyme activity of the cytochrome bc1 complex, or the levels of cytochromes b and c1 and the iron sulfur protein. When these mutant yeast cells were grown at the nonpermissive temperature of 37o C, the growth of the both the K93L and E95R cells were decreased, as was the enzymatic activity. No reduction of cytochromes b or c1 was observed in these cells; however, the level of iron sulfur protein was decreased, suggesting that these charged residues are not required for proper function of the iron sulfur protein at 30o C, but may be
important for the stability of the protein when the yeast are grown at nonpermissive temperatures. The mutation made in the membrane-spanning region, L65R, caused yeast cells to grow more slowly than the wild type at 30o C with concomitant decreases in enzymatic activity and levels of cytochrome b and the iron sulfur protein. In cells grown at 37o C, the L65R mutant cells grew more slowly than the wild type and had decreased enzymatic activity and cytochrome b content. Almost no iron sulfur protein could be detected, suggesting that the hydrophobic leucine at position 65 may be important for stabilizing the protein in the cytochrome bc1 complex, perhaps through interactions with membrane phospholipids or with other embedded subunits.
Recommended Citation
Amyot, Suzelle Madeleine, "The role of amino acids in the transmembrane and extra-membranous domains of the S. cerevisiae iron sulfur protein in its activity and stability in the cytochrome bc1 complex." (1998). Graduate Theses, Dissertations, and Problem Reports. 10367.
https://researchrepository.wvu.edu/etd/10367