Abstract
Mutation of a serine that forms a hydrogen bond to the iron-sulfur cluster of the Rieske iron-sulfur protein to a cysteine results in a respiratory-deficient yeast strain due to formation of iron-sulfur protein lacking the iron-sulfur cluster. The Rieske apoprotein lacking the iron-sulfur cluster is inserted into both monomers of the dimeric cytochrome bc(1) complex and processed to mature size, but the protein lacking iron-sulfur cluster is more susceptible to proteolysis. In addition, the protein environment of center P in one half of the dimer is affected by failure to insert the iron-sulfur cluster as indicated by the fact that only one molecule of myxothiazol can be bound to the cytochrome bc(1) dimer. Although the bc(1) complex lacking the Rieske iron-sulfur cluster cannot oxidize ubiquinol through center P, rates of reduction of cytochrome b by menaquinol through center N are normal. However, less cytochrome b is reduced through center N, and only one molecule of antimycin can be bound at center N in the bc(1) dimer lacking iron-sulfur cluster. These results indicate that failure to insert the [2Fe-2S] cluster impairs assembly of the Rieske protein into the bc(1) complex and that this interferes with proper assembly of both center P and center N in one half of the dimeric enzyme.
Highlights
The Rieske iron-suflur protein is an essential subunit of mitochondrial cytochrome bc1 complexes that is encoded by a nuclear gene, synthesized on cytoplasmic ribosomes, and imported into the mitochondria and assembled into the cytochrome bc1 complex in the inner mitochondrial membrane [1]
We showed that the apoprotein can be assembled into the bc1 complex if the cluster is not inserted [5], which suggests that the [2Fe-2S] cluster is added after the apoprotein is assembled into the bc1 complex
The striking finding from our results is that failure to insert the [2Fe-2S] cluster into the Rieske iron-sulfur affects the structure of center N, which is on the opposite side of the membrane from the extrinsic domain where the cluster is located
Summary
The Rieske iron-suflur protein is an essential subunit of mitochondrial cytochrome bc1 complexes that is encoded by a nuclear gene, synthesized on cytoplasmic ribosomes, and imported into the mitochondria and assembled into the cytochrome bc1 complex in the inner mitochondrial membrane [1]. It was not necessary to include stigmatellin with the bc1 complex from the CHS14 mutant because the absence of iron-sulfur cluster prevents reduction of cytochrome b through center P.
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