Intermediate length Rieske iron-sulfur protein is present and functionally active in the cytochrome bc1 complex of Saccharomyces cerevisiae

To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP. When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP. These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex.     

Cloning and sequence analysis of a cDNA encoding the Rieske iron-sulfur protein of rat mitochondrial cytochrome bc1 complex

We have isolated a cDNA clone for the Rieske iron-sulfur protein of rat cytochrome bc1 complex, by screening a rat liver cDNA expression library using antiserum directed against the corresponding protein of bovine. The amino acid sequence deduced from the nucleotide sequence of the cDNA indicated that the mature polypeptide of the rat protein consists of 196 amino acid residues with a molecular weight of 21,465, and that it is formed as a precursor with an amino-terminal extension. Northern blot analysis indicated that rat liver possibly contains different sizes of mRNAs for the Rieske iron-sulfur protein, and Southern blot analysis demonstrated that rats and mice possess a single gene for this protein.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. II. Biochemical characterization of temperature-sensitive RIP1- mutations

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.     

Failure to insert the iron-sulfur cluster into the Rieske iron-sulfur protein impairs both center N and center P of the cytochrome bc1 complex

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.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex

The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.     

Structure and function of the mitochondrial bc1 complex. A mutational analysis of the yeast Rieske iron-sulfur protein

Respiratory-defective mutants of Saccharomyces cerevisiae assigned to a single complementation group (G12) have been determined to have lesions in the iron-sulfur protein (Rieske protein) of ubiquinol: cytochrome c reductase. Mutants capable of expressing the protein were chosen for further studies. The genes from 13 independent isolates were cloned and their mutations sequenced. Twelve mutations were ascertained to cause single amino acid substitutions in the carboxyl-terminal regions of the protein between residues 127 and 173. This region is proposed to be part of the catalytic domain with the ligands responsible for co-ordinating the two irons of the 2Fe-2S cluster. Based on the catalytic properties of the ubiquinol: cytochrome c reductase complex and the electron paramagnetic resonance (e.p.r.) signals of the iron-sulfur protein, the mutants describe two different phenotypes. A subset of mutants have no detectable iron-sulfur cluster and are completely deficient in ubiquinol: cytochrome c reductase activity. These strains identify mutations in residues considered to be essential for binding of the iron or for maintaining a proper tertiary structure of the catalytic domain. A second group of mutants have reduced levels of enzymatic activity and exhibit e.p.r. spectra characteristic of the Rieske iron-sulfur cluster. The mutations in the latter strains have been ascribed to residues that influence the redox properties of the cluster by distorting the iron-binding pocket. A secondary and tertiary structure model is presented of the carboxyl-terminal 65 residues constituting the catalytic domain of the iron-sulfur protein. It is postulated that the two irons of the cluster are co-ordinated by three cysteine and a single histidine residue located in a loop structure. The catalytic domain also contains two short alpha-helices and three beta-strands that form a partial beta-barrel. Most of the hydrophilic amino acids are present in turns that map to one pole of the domain. When viewed in the context of the model, mutations that abolish the iron-sulfur cluster are mostly in residues defining the boundaries of the alpha-helices and beta-strands. The notable exception is a cysteine residue that has been assigned to the loop with the iron ligands. This cysteine residue is proposed to co-ordinate one iron of the cluster. Mutations that reduce ubiquinol: cytochrome c reductase activity and alter the redox potential of the cluster occur in residues located in the loop that contains the ligands of the cluster.     

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. I. Construction of a RIP1 deletion strain and isolation of temperature-sensitive mutants

A protocol has been devised to permit mutational analysis of the Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex of Saccharomyces cerevisiae. The gene for this iron-sulfur protein (RIP1) has recently been cloned and sequenced (Beckmann, J. D., Ljungdahl, P. O., Lopez, J. L., and Trumpower, B. L. (1987) J. Biol. Chem. 262, 8901-8909). We have constructed a stable yeast deletion strain, JPJ1, in which the chromosomal copy of RIP1 was displaced by the yeast LEU2 gene by homologous recombination. A linear DNA fragment containing the LEU2 gene was inserted at the breakpoints of an 800-base pair deletion of the iron-sulfur protein gene and used to transform a leu- yeast strain. Leu+ transformants were obtained which were unable to grow on nonfermentable carbon sources. Southern analysis of the transformant, JPJ1, confirmed that the chromosomal copy of the RIP1 gene was deleted and replaced by the LEU2 gene. The genotype of JPJ1 was confirmed by genetic crosses. JPJ1 cannot grow on nonfermentable carbon sources but can be complemented to respiratory competence and transformed by yeast vectors containing the wild type RIP1 gene. The ability to complement strain JPJ1 with episomally encoded iron-sulfur protein provided the basis of a selection protocol by which mutagenized plasmids containing the RIP1 gene were assayed for mutations affecting respiratory growth. Five mutants of RIP1 were identified by their ability to complement JPJ1 to temperature-sensitive respiratory growth. DNA sequence analysis demonstrated that temperature-sensitive respiratory growth resulted from single point mutations within the protein coding region of RIP1. These mutations altered a single amino acid residue in each case. Mutations were dispersed throughout the terminal two-thirds of the protein. Each mutation was recessive and did not affect fermentative growth on dextrose. However, each mutation exerted unique temperature-sensitive growth characteristics on media containing the nonfermentable carbon source glycerol.     

Mitochondrial complex III Rieske Fe-S protein processing and assembly

Regulation of the mitochondrial respiratory chain biogenesis is a matter of great interest because of its implications for mitochondrial disease. One of the mitochondrial disease genes recently discovered associated to encephalopathy and mitochondrial complex III (cIII) deficiency is TTC19. Our study of TTC19-deficient human and mouse models, has led us to propose a post-assembly quality control role or ‘husbandry’ function for this factor that is linked to Rieske Fe-S protein (UQCRFS1). UQCRFS1 is the last incorporated cIII subunit, and its presence is essential for enzymatic activity. During UQCRFS1 assembly, the precursor is cleaved and its N-terminal part remains bound to the complex, between the two core subunits (UQCRC1 and UQCRC2). In the absence of TTC19 there is a prominent accumulation of these UQCRFS1-derived N-terminal fragments that proved to be detrimental for cIII function. In this article we will discuss some ideas around the UQCRFS1 processing and assembly and its importance for the regulation of cIII activity and biogenesis.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

Elimination of the disulfide bridge in the Rieske iron-sulfur protein allows assembly of the [2Fe-2S] cluster into the Rieske protein but damages the ubiquinol oxidation site in the cytochrome bc1 complex

The [2Fe-2S] cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster. EPR spectra of the Rieske proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the [2Fe-2S] iron-sulfur center. In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske protein from wild-type yeast to 150 mV in the protein from the C164Y/C180L mutant and to 160 mV in the protein from the C164Y/C180V mutant. Ubiquinol-cytochrome c reductase activities of the bc(1) complexes with Rieske proteins lacking the disulfide bridge are less than 1% of the activity of the bc(1) complex from wild-type yeast, even though normal amounts of the iron-sulfur protein are present as judged by Western blot analysis. These activities are lower than the 105-115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for. Pre-steady-state reduction of the bc(1) complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N. In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc(1) complexes with Rieske proteins lacking the disulfide bridge. Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc(1) complexes with Rieske proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske protein.     

Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p

Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.     

Aromatic amino acids in the Rieske iron-sulfur protein do not form an obligatory conduit for electron transfer from the iron-sulfur cluster to the heme of cytochrome c1 in the cytochrome bc1 complex

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.     

Significance of the "Rieske" iron-sulfur protein for formation and function of the ubiquinol-oxidation pocket of mitochondrial cytochrome c reductase (bc1 complex).

The binding of specific inhibitors to the ubiquinol oxidation pocket ("QP center") of cytochrome c reductase was analyzed before and after removal of bound phospholipid and the "Rieske" iron-sulfur protein using optical spectroscopy and fluorescence quench binding assays. The enzyme lacking iron-sulfur protein showed almost unchanged, tight binding of the E-beta-methoxyacrylate inhibitors oudemansin A and MOA-stilbene, whereas binding of the chromone inhibitor stigmatellin was almost completely abolished. The affinity of the weak inhibitor 3-undecyl-2-hydroxy-naphthoquinone was decreased. Oudemansin A binding to the defective pocket of the iron-sulfur protein-depleted enzyme was lowered by added phospholipid. It was deduced from these results that the QP center is a spacious pocket formed by domains of cytochrome b, bearing the E-beta-methoxcyacrylate binding site, and the iron-sulfur protein, bearing the stigmatellin binding site. Moreover, removal of the iron-sulfur protein leaves this pocket defective but essentially unchanged in its remaining binding capability. The affinity of three preparations of cytochrome c reductase, the complete, the delipidated, and the iron-sulfur depleted enzyme for E-beta-methoxyacrylate-stilbene, was analyzed for different redox states of the catalytic centers of cytochrome c reductase. The apparent Kd values for the different redox states were interpreted in terms of two conformational states. It is suggested that these changes reflect the two states of the "catalytic switch" proposed recently for the QP pocket of cytochrome c reductase (Brandt, U., and von Jagow, G. (1991) Eur. J. Biochem. 195, 163-170). According to the refined model presented in this work, changeover to the "b" state is triggered by reduction of the iron-sulfur cluster, and changeover back to the "FeS" state is triggered by electron transfer from the low potential onto the high potential heme b center. Our interpretation implies that the stability of the two states is affected by the redox states of the enzyme, but that additionally changing the redox states of the two centers is required for "switching" on a catalytic time scale.     

On the interaction of mitochondrial complex III with the Rieske iron-sulfur protein (subunit V).

Limited proteolysis of solubilized beef heart mitochondrial complex III with trypsin yields a product previously identified as fragment V" (González-Halphen, D., Lindorfer, M. A., and Capaldi, R. A. (1988) Biochemistry 27, 7021-7031). In this work, fragment V" was generated by trypsin treatment of both the intact complex III and the purified Rieske iron-sulfur protein. Thus, in its bound or isolated form, the same sites of subunit V are sensitive to protease action. Fragment V" was a soluble protein that retained its iron-sulfur moiety. It was purified by exclusion from a hydrophobic phenyl-Sepharose CL-4B column followed by gel filtration. In contrast to the pure, intact subunit V, fragment V" did not reconstitute oxidoreductase activity when combined with complex III devoid of subunit V. However, a 20-amino acid synthetic peptide carrying the sequence between amino acids Lys33 and Lys52 of the Rieske iron-sulfur protein competed with intact subunit V in reconstitution assays. The results obtained suggest that the iron-sulfur protein binds to complex III by hydrophobic protein-protein interactions, and that a nontransmembrane 18-amino acid amphipathic stretch accounts for the association of this subunit to the rest of the complex.     

Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc1 complex

Long-range movement of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in electron transfer within the cyt bc1 complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc1 were prepared to examine the role of this loop in controlling the capture and release of the ISP from cyt b. Electron transfer in the cyt bc1 complex was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 mus and initiate the reaction. The rate constant for electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c1 was k1 = 60 000 s-1. Famoxadone binding to the Qo site decreases k1 to 5400 s-1, indicating that a conformational change on the surface of cyt b decreases the rate of release of the ISP from cyt b. The mutation I292A on the surface of the ISP-binding crater decreased k1 to 4400 s-1, while the addition of famoxadone further decreased it to 3000 s-1. The mutation L286A at the tip of the ef loop decreased k1 to 33 000 s-1, but famoxadone binding caused no further decrease, suggesting that this mutation blocked the conformational change induced by famoxadone. Studies of all of the mutants provide further evidence that the ef loop plays an important role in regulating the domain movement of the ISP to facilitate productive electron transfer and prevent short-circuit reactions.     

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Stigmatellin induces reduction of iron-sulfur protein in the oxidized cytochrome bc1 complex

Stigmatellin, a Q(P) site inhibitor, inhibits electron transfer from iron-sulfur protein (ISP) to cytochrome c1 in the bc1 complex. Stigmatellin raises the midpoint potential of ISP from 290 mV to 540 mV. The binding of stigmatellin to the fully oxidized complex, oxidized completely by catalytic amounts of cytochrome c oxidase and cytochrome c, results in ISP reduction. The extent of ISP reduction is proportional to the amount of inhibitor used and reaches a maximum when the ratio of inhibitor to enzyme complex reaches unity. A g = 2.005 EPR peak, characteristic of an organic free radical, is also observed when stigmatellin is added to the oxidized complex, and its signal intensity depends on the amount of stigmatellin. Addition of ferricyanide, a strong oxidant, to the oxidized complex also generates a g = 2.005 EPR peak that is oxidant concentration-dependent. Oxygen radicals are generated when stigmatellin is added to the oxidized complex in the absence of the exogenous substrate, ubiquinol. The amount of oxygen radical formed is proportional to the amount of stigmatellin added. Oxygen radicals are not generated when stigmatellin is added to a mutant bc1 complex lacking the Rieske iron-sulfur cluster. Based on these results, it is proposed that ISP becomes a strong oxidant upon stigmatellin binding, extracting electrons from an organic compound, likely an amino acid residue. This results in the reduction of ISP and generation of organic radicals.