Human disease-related mutations in cytochrome b studied in yeast

Several mutations in the mitochondrially encoded cytochrome b have been reported in patients. To characterize their effect, we introduced six "human" mutations, namely G33S, S152P, G252D, Y279C, G291D, and Delta252-259 in the highly similar yeast cytochrome b. G252D showed wild type behavior in standard conditions. However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation. The mutations G33S, S152P, G291D, and Delta252-259 were clearly pathogenic. They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc(1) complex, as observed by immunodetection. Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc(1) complex. Y279C caused a significant decrease of the bc(1) function and perturbed the quinol binding. The EPR spectra showed an altered signal, indicative of a lower occupancy of the Q(o) site. The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Q(o) site properties. Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.     

Functional insensitivity of the cytochrome b6f complex to structure changes in the hinge region of the Rieske iron-sulfur protein

Structure analysis of the cytochrome bc1 complex in the presence and absence of Qp quinol analog inhibitors implied that a large amplitude motion of the Rieske iron-sulfur protein (ISP) is required to mediate electron transfer from ubiquinol to cytochrome c1. Studies of the functional consequences of mutagenesis of an 8-residue ISP "hinge" region in the bc1 complex showed it to be sensitive to structure perturbation, implying that optimum flexibility and length are required for the large amplitude motion. Mutagenesis-function analysis carried out on the ISP hinge region of the cytochrome b6 f complex using the cyanobacterium Synechococcus sp. PCC 7002 showed the following. (i) Of three petC genes, only that in the petCA operon codes for functional ISP. (ii) The function of the complex was insensitive to changes in the hinge region that increased flexibility, decreased flexibility by substitutions of 4-6 Pro residues, shortened the hinge by a 1-residue deletion, or elongated it by insertion of 4 residues. The latter change increased sensitivity to Qp inhibitors, whereas deletion of 2 residues resulted in a loss of inhibitor sensitivity and a decrease in activity, indicating a minimum hinge length of 7 residues required for optimum binding of ISP at the Qp site. Thus, in contrast to the bc1 complex, the function of the b6 f complex was insensitive to sequence changes in the ISP hinge that altered its length or flexibility. This implies that either the barriers to motion or the amplitude of ISP motion required for function is smaller than in the bc1 complex.     

Substituting leucine for alanine-86 in the tether region of the iron-sulfur protein of the cytochrome bc1 complex affects the mobility of the [2Fe2S] domain

Mutating three conserved alanine residues in the tether region of the iron-sulfur protein of the yeast cytochrome bc(1) complex resulted in 22-56% decreases in enzymatic activity [Obungu et al. (2000) Biochim. Biophys. Acta 1457, 36-44]. The activity of the cytochrome bc(1) complex isolated from A86L was decreased 60% compared to the wild-type without loss of heme or protein and without changes in the 2Fe2S cluster or proton-pumping ability. The activity of the bc(1) complex from mutant A92R was identical to the wild-type, while loss of both heme and activity was observed in the bc(1) complex isolated from mutant A90I. Computer simulations indicated that neither mutation A86L nor mutation A92R affects the alpha-helical backbone in the tether region; however, the side chain of the leucine substituted for Ala-86 interacts with the side chain of Leu-89. The Arrhenius plot for mutant A86L was apparently biphasic with a transition observed at 17-19 degrees C and an activation energy of 279.9 kJ/mol below 17 degrees C and 125.1 kJ/mol above 17 degrees C. The initial rate of cytochrome c(1) reduction was lowered 33% in mutant A86L; however, the initial rate of cytochrome b reduction was unaffected, suggesting that movement of the tether region of the iron-sulfur protein is necessary for maximum rates of enzymatic activity. Substituting a leucine for Ala-86 impedes the unwinding of the alpha-helix and hence movement of the tether.     

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.     

The iron-sulfur protein of cytochrome bc1 complex. Its occurrence in the mitochondrial inner membrane in excess of the amount constituting the complex

Radioimmunoassay and quantitative immunoblot analysis have been developed for quantitation of the iron-sulfur protein of cytochrome bc1 complex in order to compare its content in isolated cytochrome bc1 complex with that in electron transport particles. The result by radioimmunoassay indicated that the content of the iron-sulfur protein/mol of cytochrome b is higher by approximately 30%, on the average, in electron transport particles than in cytochrome bc1 complex. This observation was supported by the data of immunoblot analysis. Since approximately 1/3 of cytochrome b in electron transport particles is not attributed to cytochrome bc1 complex, but to succinate-ubiquinone oxidoreductase complex (Davis, K.A., Hatefi, Y., Poff, K. L., and Butler, W. L. (1973) Biochim. Biophys. Acta 325, 341-356), the ratio of the iron-sulfur protein detectable by radioimmunoassay in electron transport particles to that in cytochrome bc1 complex is calculated to be approximately 2 on the basis of the content of 2 mol of b-type heme/mol of the complex. Therefore, it appears that the mitochondrial inner membrane contains approximately two times as much of the immunoreactive iron-sulfur protein as what is expected from the stoichiometry of one iron-sulfur center and two b-type hemes for cytochrome bc1 complex. This finding affords an interesting aspect in the study of biogenesis of cytochrome bc1 complex.     

A compensatory double mutation of the alanine-86 to leucine mutant located in the hinge region of the iron-sulfur protein of the yeast cytochrome bc1 complex

Mutations in the hinge region connecting the membrane anchor to the extra-membranous head-group of the iron-sulfur protein can impede proper assembly and function of the cytochrome bc(1) complex. Mutating the conserved alanines, residues 86, 90, and 92, located in the hinge region resulted in a 30-50% decrease in enzymatic activity without loss of the iron-sulfur protein [J. Bioenerg. Biomembr. 31 (1999) 215]. The lowered enzymatic activity in the A86L mutant was shown to result from steric interference between the side chains of Leu-86 and Leu-89 [Biochemistry 40 (2001) 327]. The compensatory double mutant A86L/L89A restored activity to wild type levels and relieved the steric hindrance; however, the L89A mutant did not assemble properly into the bc(1) complex. Molecular modeling studies of these mutants compared to the wild type have suggested that the hydrophobic residues located in the hinge region are critical to the motion of the head group of the iron-sulfur protein during catalysis.     

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.     

Movement of the iron-sulfur subunit beyond the ef loop of cytochrome b is required for multiple turnovers of the bc1 complex but not for single turnover Qo site catalysis.

Recent kinetics experiments using mutants of the bc(1) complex (ubihydroquinone-cytochrome c oxidoreductase) iron-sulfur subunit with modified hinge regions have revealed the crucial role played by the large scale movement of its [2Fe-2S] cluster domain during the activity of this enzyme. In particular, one of these mutants (+1Ala) with an insertion of one alanine residue in the hinge region is partially deficient in performing this movement. We found that this defect can be overcome by the appearance of a second mutation substituting the leucine at position 286 in the ef loop of cytochrome b with a phenylalanine. Detailed studies of these mutants and their derivatives revealed that the ef loop acts as a barrier that needs to be crossed for multiple turnovers of the enzyme but not for a single turnover ubihydroquinone oxidation site catalysis. These findings indicate that the movement of the iron-sulfur subunit is composed of two discrete parts: a "micro-movement" at the cytochrome b interface, during which the [2Fe-2S] cluster interacts with ubihydroquinone oxidation site occupants and catalyzes ubihydroquinone oxidation, and a "macro-movement," during which the cluster domain swings away from cytochrome b interface, crosses the ef loop, and reaches a position close to cytochrome c(1) heme, to which it ultimately transfers an electron.     

The chloroplast Rieske iron-sulfur protein. At the crossroad of electron transport and signal transduction

We have addressed the functional and structural roles of three domains of the chloroplast Rieske iron-sulfur protein; that is, the flexible hinge that connects the transmembrane helix to the soluble cluster-bearing domain, the N-terminal stromal protruding domain, and the transmembrane helix. To this aim mutants were generated in the green alga Chlamydomonas reinhardtii. Their capacities to assemble the cytochrome b6f complex, perform plastoquinol oxidation, and signal redox-induced activation of the light-harvesting complex II kinase during state transition were tested in vivo. Deletion of one residue and extensions of up to five residues in the flexible hinge had no significant effect on complex accumulation or electron transfer efficiency. Deletion of three residues (Delta3G) dramatically decreased reaction rates by a factor of approximately 10. These data indicate that the chloroplast iron-sulfur protein-linking domain is much more flexible than that of its counterpart in mitochondria. Despite greatly slowed catalysis in the Delta3G mutant, there was no apparent delay in light-harvesting complex II kinase activation or state transitions. This indicates that conformational changes occurring in the Rieske protein did not represent a limiting step for kinase activation within the time scale tested. No phenotype could be associated with mutations in the N-terminal stromal-exposed domain. In contrast, the N17V mutation in the Rieske protein transmembrane helix resulted in a large decrease in the cytochrome f synthesis rate. This reveals that the Rieske protein transmembrane helix plays an active role in assembly-mediated control of cytochrome f synthesis. We propose a structural model to interpret this phenomenon based on the C. reinhardtii cytochrome b6f structure.     

QO site deficiency can be compensated by extragenic mutations in the hinge region of the iron-sulfur protein in the bc1 complex of Saccharomyces cerevisiae

The mitochondrial bc(1) complex catalyzes the oxidation of ubiquinol and the reduction of cytochrome (cyt) c. The cyt b mutation A144F has been introduced in yeast by the biolistic method. This residue is located in the cyt b cd(1) amphipathic helix in the quinol-oxidizing (Q(O)) site. The resulting mutant was respiration-deficient and was affected in the quinol binding and electron transfer rates at the Q(O) site. An intragenic suppressor mutation was selected (A144F+F179L) that partially alleviated the defect of quinol oxidation of the original mutant A144F. The suppressor mutation F179L, located at less than 4 A from A144F, is likely to compensate directly the steric hindrance caused by phenylalanine at position 144. A second set of suppressor mutations was obtained, which also partially restored the quinol oxidation activity of the bc(1) complex. They were located about 20 A from A144F in the hinge region of the iron-sulfur protein (ISP) between residues 85 and 92. This flexible region is crucial for the movement of the ISP between cyt b and cyt c(1) during enzyme turnover. Our results suggested that the compensatory effect of the mutations in ISP was due to the repositioning of this subunit on cyt b during quinol oxidation. This genetic and biochemical study thus revealed the close interaction between the cyt b cd(1) helix in the quinol-oxidizing Q(O) site and the ISP via the flexible hinge region and that fine-tuning of the Q(O) site catalysis can be achieved by subtle changes in the linker domain of the ISP.     

Assembly of the iron-sulfur protein into the cytochrome b-c1, complex of yeast mitochondria.

The assembly of the iron-sulfur protein into the cytochrome bc1 complex after import and processing of the precursor form into mitochondria in vitro was investigated by immunoprecipitation of the radiolabeled iron-sulfur protein from detergent-solubilized mitochondria with specific antisera. After import in vitro, the labeled mature form of the iron-sulfur protein was immunoprecipitated by antisera against both the iron-sulfur protein and the entire bc1 complex from mitochondria solubilized with either Triton X-100 or dodecyl maltoside. After sodium dodecyl sulfate solubilization of mitochondria, however, the antiserum against the iron-sulfur protein, but not that against the bc1 complex, immunoprecipitated the radiolabeled iron-sulfur protein. These results suggest that in mitochondria the mature form of the iron-sulfur protein is assembled with other subunits of the bc1 complex that are recognized by the antiserum against the bc1 complex. By contrast, the intermediate and precursor forms of the iron-sulfur protein that accumulated in the matrix when proteolytic processing was blocked with EDTA and o-phenanthroline were not efficiently assembled into the bc1 complex. The import and processing of the iron-sulfur protein also occurred in mitochondria lacking either cytochrome b (W-267) or the iron-sulfur protein (JPJ1). The mature form of the iron-sulfur protein was immunoprecipitated by antisera against the bc1 complex or core protein I after import in vitro into these mitochondria, suggesting that the mature form is associated with other subunits of the bc1 complex in these strains.     

Mutations in the tether region of the iron-sulfur protein affect the activity and assembly of the cytochrome bc(1) complex of yeast mitochondria

Resolution of the crystal structure of the mitochondrial cytochrome bc(1) complex has indicated that the extra-membranous extrinsic domain of the iron-sulfur protein containing the 2Fe2S cluster is connected by a tether to the transmembrane helix that anchors the iron-sulfur protein to the complex. To investigate the role of this tether in the cytochrome bc(1) complex, we have mutated the conserved amino acid residues Ala-86, Ala-90, Ala-92, Lys-93 and Glu-95 and constructed deletion mutants DeltaVLA(88-90) and DeltaAMA(90-92) and an insertion mutant I87AAA88 in the iron-sulfur protein of the yeast, Saccharomyces cerevisiae. In cells grown at 30 degrees C, enzymatic activities of the bc(1) complex were reduced 22-56% in mutants A86L, A90I, A92C, A92R and E95R, and the deletion mutants, DeltaVLA(88-90) and DeltaAMA(90-92), while activity of the insertion mutant was reduced 90%. No loss of cytochromes b or c-c(1), detected spectrally, or the iron-sulfur protein, determined by quantitative immunoblotting, was observed in these mutants with the exception of the mutants of Ala-92 in which the loss of activity paralleled a loss in the amount of the iron-sulfur protein. EPR spectroscopy revealed no changes in the iron-sulfur cluster of mutants A86L, A90I, A92R or the deletion mutant DeltaVLA(88-90). Greater losses of both protein and activity were observed in all of the mutants of Ala-92 as well as in A90F grown at 37 degrees C. suggesting that these conserved alanine residues may be involved in maintaining the stability of the iron-sulfur protein and its assembly into the bc(1) complex. By contrast, no significant loss of iron-sulfur protein was observed in the mutants of Ala-86 in cells grown at either 30 degrees C or 37 degrees C despite the 50-70% loss of enzymatic activity suggesting that Ala-86 may play a critical role in catalysis in the bc(1) complex.     

Evidence for a concerted mechanism of ubiquinol oxidation by the cytochrome bc1 complex

To better understand the mechanism of divergent electron transfer from ubiquinol to the iron-sulfur protein and cytochrome b(L) within the cytochrome bc(1) complex, we have examined the effects of antimycin on the presteady state reduction kinetics of the bc(1) complex in the presence or absence of endogenous ubiquinone. When ubiquinone is present, antimycin slows the rate of cytochrome c(1) reduction by approximately 10-fold but had no effect upon the rate of cytochrome c(1) reduction in bc(1) complex lacking endogenous ubiquinone. In the absence of endogenous ubiquinone cytochrome c(1), reduction was slower than when ubiquinone was present and was similar to that in the presence of ubiquinone plus antimycin. These results indicate that the low potential redox components, cytochrome b(H) and b(L), exert negative control on the rate of reduction of cytochrome c(1) and the Rieske iron-sulfur protein at center P. If electrons cannot equilibrate from cytochrome b(H) and b(L) to ubiquinone, partial reduction of the low potential components slows reduction of the high potential components. We also examined the effects of decreasing the midpoint potential of the iron-sulfur protein on the rates of cytochrome b reduction. As the midpoint potential decreased, there was a parallel decrease in the rate of b reduction, demonstrating that the rate of b reduction is dependent upon the rate of ubiquinol oxidation by the iron-sulfur protein. Together these results indicate that ubiquinol oxidation is a concerted reaction in which both the low potential and high potential redox components control ubiquinol oxidation at center P, consistent with the protonmotive Q cycle mechanism.     

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.     

Aspartate-186 in the head group of the yeast iron-sulfur protein of the cytochrome bc1 complex contributes to the protein conformation required for efficient electron transfer

Two conserved charged amino acids, aspartate-186 and arginine-190, localized in the aqueous head region of the iron-sulfur protein of the cytochrome bc(1) complex of yeast mitochondria, were mutated to alanine, glutamate, or asparagine and isoleucine, respectively. The R190I mutation resulted in the complete loss of antimycin- and myxothiazol-sensitive cytochrome c reductase activity due to loss of more than 60% of the iron-sulfur protein in the complex. Mitochondria isolated from the D186A mutant had a 50% decrease in cytochrome c reductase activity but no loss of the iron-sulfur protein or the [2Fe-2S] cluster. The midpoint potential of the [2Fe-2S] cluster of the D186A mutant was decreased from 281 to 178 mV. The D186E and D186N mutations did not result in a loss of cytochrome c reductase activity or content of iron-sulfur protein; however, the redox potential of the [2Fe-2S] cluster of D186N was decreased from 281 to 241 mV. Molecular modeling/dynamics studies predicted that substituting an alanine for Asp-186 causes global structural changes in the head group of the iron-sulfur protein resulting in changes in the orientation of the [2Fe-2S] cluster and consequently a lowered redox potential. The rate of electrogenic proton pumping in the bc(1) complex isolated from mutant D186A reconstituted into proteoliposomes decreased 64%; however, the H(+)/2e(-) ratio of 1.9 was identical in the mutant and the wild-type complexes. The carboxyl binding reagent, N-(ethoxycarbonyl)-2-ethoxyl-1,2-dihydroquinoline (EEDQ) blocked electrogenic proton pumping in the bc(1) complex reconstituted into proteoliposomes without affecting electron transfer resulting in a decrease in the H(+)/2e(-) ratio to 1.2 and 1.1, respectively. EEDQ was bound to the iron-sulfur protein and core protein II in both the wild type and the D186A mutant, indicating that Asp-186 of the iron-sulfur protein is not required for proton translocation in the bc(1) complex.     

Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b.

Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Q(o) inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases. Specific residues in the Q(o) site of yeast cytochrome b were modified to obtain four new forms mimicking the Q(o) binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma. These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc(1) complex activity. In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied. The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin. The pattern of resistance conferred by F129L and L275F was different. Interestingly G143A had a slightly deleterious effect on the bc(1) function in V. inaequalis, S. fuliginea and P. megasperma Q(o) site mimics but not in that for E. graminis. Thus small variations in the Q(o) site seem to affect the impact of the G143A mutation on bc(1) activity. Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially. If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Confirmation of the involvement of protein domain movement during the catalytic cycle of the cytochrome bc1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein

To study the essentiality of head domain movement of the Rieske iron-sulfur protein (ISP) during bc(1) catalysis, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with three pairs of cysteines engineered (one cysteine each) on the interface between cytochrome b and ISP, A185C(cytb)/K70C(ISP), I326C(cytb)/G165C(ISP), and T386C(cytb)/K164C(ISP), were generated and characterized. Formation of an intersubunit disulfide bond between cytochrome b and ISP is detected in membrane (intracytoplasmic membrane and air-aged chromatophore), and purified bc(1) complex was prepared from the A185C(cytb)/K70C(ISP) mutant cells. Formation of the intersubunit disulfide bond in this cysteine pair mutant complex is concurrent with the loss of its bc(1) activity. Reduction of this disulfide bond by beta-mercaptoethanol restores activity, indicating that mobility of the head domain of ISP is functionally important in the cytochrome bc(1) complex. The rate of intramolecular electron transfer, between 2Fe2S and heme c(1), in the A185C(cytb)/K70C(ISP) mutant complex is much lower than that in the wild type or in their respective single cysteine mutant complexes, indicating that formation of an intersubunit disulfide bond between cytochrome b and ISP arrests the head domain of ISP in the "fixed state" position, which is too far for electron transfer to heme c(1).     

Function of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions of the mitochondrial respiratory chain

Resolution and reconstitution has been used to examine the involvement of the iron-sulfur protein of the cytochrome b-c1 segment in electron transfer reactions in this region of the mitochondrial respiratory chain. The iron-sulfur protein is required for electron transfer from succinate and from ubiquinol to cytochrome c1. It is not required for reduction of cytochrome b under these conditions, but it is required for oxidation of cytochrome b by cytochrome c plus cytochrome c oxidase. Removal of the iron-sulfur protein from the b-c1 complex prevents reduction of both cytochromes b and c1 by succinate or ubiquinol if antimycin is added to the depleted complex. As increasing amounts of iron-sulfur protein are reconstituted to the depleted complex, the amounts of cytochromes b and c1 reduced by succinate in the presence of antimycin increase and closely parallel the amounts of ubiquinol-cytochrome c reductase activity restored to the reconstituted complex, measured before addition of antimycin. The function of the iron-sulfur protein in these oxidation-reduction reactions is consistent with a cyclic pathway of electron transfer through the cytochrome b-c1 complex, in which the iron-sulfur protein functions as a ubiquinol-cytochrome c1/ubisemiquinone-cytochrome b oxidoreductase.