Although the function of the Rieske iron-sulfur protein is generally understood, little is known of how the structure of this protein supports its mechanistic role in electron transfer in the cytochrome bc1 complex. To better understand the structural basis of iron-sulfur protein function, we have undertaken a mutational analysis of the gene encoding this protein and initially isolated five temperature-sensitive iron-sulfur protein mutants (Beckmann, J. D., Ljungdahl, P. O., and Trumpower, B. L. (1989) J. Biol. Chem. 264, 3713-3722). Each of the five ts-rip1- mutants exhibited pleiotropic effects. Although the mutant iron-sulfur proteins manifest several in vitro phenotypes in common, each exhibited unique characteristics. All of the ts-rip1- mutations resulted in membranes with decreased ubiquinol-cytochrome c oxidoreductase activities and decreased thermostability compared to membranes containing wild type iron-sulfur protein. All of the mutations conferred slight but significant resistance to the respiratory inhibitor myxothiazol, and one mutant was hypersensitive to inhibition by UHDBT, a structural analog of ubiquinone. In addition, one of the mutations completely blocks post-translational processing of the iron-sulfur protein, leading to accumulation of pre-iron-sulfur protein in mitochondrial membranes at nonpermissive temperatures. Finally, a mutation 12-amino acid residues away from the carboxyl terminus (203S) results in an extremely unstable protein. This region of the protein may be essential in blocking degradation of pre-iron-sulfur protein by cytoplasmic proteases as the protein is imported into the mitochondria, or may be a "degradation signal," which tags the iron-sulfur protein for turnover.