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 (S ␥ ) 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 ironsulfur protein by site-directed mutagenesis of the ironsulfur 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 S ␥ 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 bc 1 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 bc 1 complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the iron-sulfur cluster.The Rieske iron-sulfur protein is a ubiquitous component of cytochrome bc 1 complexes (1-4) and has been shown to be essential for electron transfer and energy transduction by purification of the protein in a reconstitutively active form and reconstitution to iron-sulfur protein depleted bc 1 complex (5, 6). The electronic environment of the [2Fe-2S] cluster in the Rieske iron-sulfur protein differs from that in plant type [2Fe-2S] ferredoxins as evidenced by a distinct EPR spectrum (1) and a redox midpoint potential of the Rieske protein (e.g. ϩ280 mV) that is much more positive than the midpoint potentials typical of the ferredoxins (e.g. Ϫ420 mV; Ref. 2). The high midpoint potential of the iron-sulfur cluster is essential for the function of the Rieske protein in the Q cycle mechanism of the bc 1 complex (7,8), in which the Rieske protein is the primary electron acceptor and drives the electron transfer reaction by oxidizing ubiquinol and divergently transferring one electron to cytochrome c 1 , while the ubisemiquinone that is formed from ubiquinol reduces the low potential b heme.Recently, the crystal structure of the water-soluble part of the Rieske iron-sulfur protein of bovine heart mitochondrial bc 1 complex has been elucidated at 1.5 Å (9, 10). Ten ␤ strands form three layers of anti-parallel ␤ sheets in a flat spherical molecule as shown in Fig. 1A. The cluster binding fold is a small domain-like structure comprising approximately 46 residues; it consists of a distorted four-stranded antiparallel ␤-sheet and three loops. The loops between the strands ␤4...

QSR1 is an essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein required for joining of 40S and 60S subunits. Truncations of QSR1 predicted to encode C-terminally truncated forms of Qsr1p do not substitute for QSR1 but do act as dominant negative mutations, inhibiting the growth of yeast when expressed from an inducible promoter. The dominant negative mutants exhibit a polysome profile characterized by 'half-mer' polysomes, indicative of a subunit joining defect like that seen in other qsr1 mutants (D. P. Eisinger, F. A. Dick, and B. L. Trumpower, Mol. Cell. Biol. 17:5136-5145, 1997.) By screening a high-copy yeast genomic library, we isolated several clones containing overlapping inserts of a novel gene that rescues the slow-growth phenotype of the dominant negative qsr1 truncations. The suppressor of qsr1 truncation mutants, SQT1, is an essential gene, which encodes a 47.1-kDa protein containing multiple WD repeats and which interacts strongly with Qsr1p in a yeast two-hybrid system. SQT1 restores growth and the "half-mer" polysome profile of the dominant negative qsr1 mutants to normal, but it does not rescue temperature-sensitive qsr1 mutants or the original qsr1-1 missense allele. In yeast cell lysates, Sqt1p fractionates as part of an oligomeric protein complex that is loosely associated with ribosomes but is distinct from known eukaryotic initiation factor complexes. Loss of SQT1 function by down regulation from an inducible promoter results in formation of half-mer polyribosomes and decreased Qsr1p levels on free 60S subunits. Sqt1p thus appears to be involved in a late step of 60S subunit assembly or modification in the cytoplasm.The functions of individual eukaryotic ribosomal proteins are not well understood. We have undertaken a biochemical and genetic approach to elucidate the function of the recently discovered ribosomal protein, Qsr1p, in Saccharomyces cerevisiae (13). Qsr1p is a eukaryotic 60S ribosomal protein encoded by the essential single-copy S. cerevisiae gene QSR1 (13,54) and is the mammalian homolog of rat ribosomal protein L10 (9).Characterization of Qsr1p and of temperature-sensitive qsr1 mutants has revealed that Qsr1p is required for joining of 40S and 60S subunits into either active 80S ribosomes or inactive 80S couples (14). Mutant alleles that are either temperature sensitive or conditional for the synthesis of Qsr1p have 60S subunits that are devoid of Qsr1p and are unable to join with 40S subunits. Whereas most conditional mutants of 60S ribosomal protein genes are defective in the biogenesis of 60S subunits (11,12,35,38,39,45,47,48), which precludes insights into any postassembly functions, the biogenesis of 60S subunits is only slightly impaired in qsr1 mutants (14). Further investigation of Qsr1p is thus likely to lead to a better understanding of the late stages of translation initiation or ribosomal subunit joining, of which little is currently known.To learn more about the function of Qsr1p, we constructed dominant-negative qsr1 mutants and...

The iron-sulfur protein of the cytochrome bc1 complex is one of a small number of proteins that are processed in two sequential steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP) during import into Saccharomyces cerevisiae mitochondria. To test whether two-step processing is necessary for import and assembly of the iron-sulfur protein into the cytochrome bc1 complex, we mutagenized the presequence of the iron-sulfur protein to eliminate the original MPP site and replace the MIP site with a new MPP site. The mutated presequence is cleaved and forms mature-sized protein in a single step, and the mature-sized iron-sulfur protein is correctly targeted to the outer side of the inner mitochondrial membrane in vitro. Mutant iron-sulfur protein which is processed to mature size in one step complements the respiratory deficient phenotype of a yeast strain in which the endogenous gene for the iron-sulfur protein is deleted. These results establish that mature-sized iron-sulfur protein can be formed by single-step processing and assembled into a functionally active form in the cytochrome bc1 complex in S. cerevisiae.

We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron-sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron-sulfur protein of the yeast cytochrome bc1 complex. The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207. Greater than 70% of the catalytic activity was retained for all of the mutated iron-sulfur proteins, except for those containing a W152L and a W176L-F177L double mutation, for which the activity was approximately 45%. The crystal structures of the bc1 complex indicate that F177 and H184 are at the surface of the iron-sulfur protein near the surface of cytochrome c1, but not directly in a linear pathway between the iron-sulfur cluster and the c1 heme. The pre-steady-state rates of reduction of cytochromes b and c1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70-85% of the wild-type rates. There was a large decrease in iron-sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L-F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins. These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability.