Abstract
The crystal structure of the bovine Rieske iron-sulfur protein indicates a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (Sgamma) of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, respectively. We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske iron-sulfur protein by site-directed mutagenesis of the iron-sulfur protein gene to examine how these hydrogen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzyme. Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to Sgamma of Cys-159 lowers the midpoint potential by 65 mV. Eliminating both hydrogen bonds has an approximately additive effect, lowering the midpoint potential by 180 mV. Thus, these hydrogen bonds contribute significantly to the positive midpoint potential of the cluster but are not essential for its assembly. The activity of the bc1 complex decreases with the decrease in midpoint potential, confirming that oxidation of ubiquinol by the iron-sulfur protein is the rate-limiting partial reaction in the bc1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.
Highlights
Eliminating the hydrogen bond from the hydroxyl group of Ser-183 to S-1 of the cluster lowers the midpoint potential of the cluster by 130 mV, and eliminating the hydrogen bond from the hydroxyl group of Tyr-185 to S␥ of Cys-159 lowers the midpoint potential by 65 mV
We show that conservative substitutions that eliminate these two hydrogen bonds lower the midpoint potential, without affecting the stability of the protein, and that these changes are accompanied by decreases in turnover numbers of the bc1 complexes containing the altered forms of the Rieske protein
In the bovine crystal structure, this residue is on the surface of the protein, in the loop that extends from the right of the protein in the orientation shown in Fig. 1, and distal from the cluster
Summary
Materials—SDS, acrylamide, and bisacrylamide were from Bio-Rad. Urea and agarose (Ultra Pure) were from Life Technologies, Inc. Purification of Cytochrome bc Complexes—For all of the yeast mutants in which the iron-sulfur protein was detectable by Western blotting of the mitochondrial membranes, the cytochrome bc complexes were purified in two different laboratories and by two different methods. Turnover numbers of the bc complexes in situ and of the purified enzymes were calculated on the basis of the concentration of cytochrome b, which was determined from optical spectra of the dithionite reduced minus ferricyanide oxidized samples [20]. This program does not calculate true energy minimization’s for substituted amino acids, it does calculate a “score” for each rotamer position of a substituted amino acid according to the empirical formula S ϭ 4X ϩ 3Y ϩ 2Z Ϫ nH, where X is the number of clashes with backbone nitrogen or carbon atoms, including the ␣ carbon, Y is the number of clashes with backbone oxygen atoms, Z is the number of clashes with side-chain atoms, and H is the number of potential hydrogen bonds
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