Modeling the molecular basis of atovaquone resistance in parasites and pathogenic fungi

Atovaquone is a substituted hydroxynaphthoquinone that is used therapeutically for treating Plasmodium falciparum malaria, Pneumocystis jirovecii pneumonia and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting parasite and fungal respiration by binding to the cytochrome bc1 complex. The recent, growing failure of atovaquone treatment and increased mortality of patients with malaria or Pneumocystis pneumonia has been linked to the appearance of mutations in the cytochrome b gene. To better understand the molecular basis of drug resistance, we have developed the yeast and bovine bc1 complexes as surrogates to model the molecular interaction of atovaquone with human and resistant pathogen enzymes.     

Cytochrome b mutations that modify the ubiquinol-binding pocket of the cytochrome bc1 complex and confer anti-malarial drug resistance in Saccharomyces cerevisiae

Atovaquone is a new anti-malarial agent that specifically targets the cytochrome bc1 complex and inhibits parasite respiration. A growing number of failures of this drug in the treatment of malaria have been genetically linked to point mutations in the mitochondrial cytochrome b gene. To better understand the molecular basis of atovaquone resistance in malaria, we introduced five of these mutations, including the most prevalent variant found in Plasmodium falciparum (Y268S), into the cytochrome b gene of the budding yeast Saccharomyces cerevisiae and thus obtained cytochrome bc1 complexes resistant to inhibition by atovaquone. By modeling the variations in cytochrome b structure and atovaquone binding with the mutated bc1 complexes, we obtained the first quantitative explanation for the molecular basis of atovaquone resistance in malaria parasites.     

Uncovering the molecular mode of action of the antimalarial drug atovaquone using a bacterial system

Atovaquone is an antiparasitic drug that selectively inhibits electron transport through the parasite mitochondrial cytochrome bc1 complex and collapses the mitochondrial membrane potential at concentrations far lower than those at which the mammalian system is affected. Because this molecule represents a new class of antimicrobial agents, we seek a deeper understanding of its mode of action. To that end, we employed site-directed mutagenesis of a bacterial cytochrome b, combined with biophysical and biochemical measurements. A large scale domain movement involving the iron-sulfur protein subunit is required for electron transfer from cytochrome b-bound ubihydroquinone to cytochrome c1 of the cytochrome bc1 complex. Here, we show that atovaquone blocks this domain movement by locking the iron-sulfur subunit in its cytochrome b-binding conformation. Based on our malaria atovaquone resistance data, a series of cytochrome b mutants was produced that were predicted to have either enhanced or reduced sensitivity to atovaquone. Mutations altering the bacterial cytochrome b at its ef loop to more closely resemble Plasmodium cytochrome b increased the sensitivity of the cytochrome bc1 complex to atovaquone. A mutation within the ef loop that is associated with resistant malaria parasites rendered the complex resistant to atovaquone, thereby providing direct proof that the mutation causes atovaquone resistance. This mutation resulted in a 10-fold reduction in the in vitro activity of the cytochrome bc1 complex, suggesting that it may exert a cost on efficiency of the cytochrome bc1 complex.     

Resistance mutations reveal the atovaquone-binding domain of cytochrome b in malaria parasites

Atovaquone represents a class of antimicrobial agents with a broad-spectrum activity against various parasitic infections, including malaria, toxoplasmosis and Pneumocystis pneumonia. In malaria parasites, atovaquone inhibits mitochondrial electron transport at the level of the cytochrome bc1 complex and collapses mitochondrial membrane potential. In addition, this drug is unique in being selectively toxic to parasite mitochondria without affecting the host mitochondrial functions. A better understanding of the structural basis for the selective toxicity of atovaquone could help in designing drugs against infections caused by mitochondria-containing parasites. To that end, we derived nine independent atovaquone-resistant malaria parasite lines by suboptimal treatment of mice infected with Plasmodium yoelii; these mutants exhibited resistance to atovaquone-mediated collapse of mitochondrial membrane potential as well as inhibition of electron transport. The mutants were also resistant to the synergistic effects of atovaquone/ proguanil combination. Sequencing of the mitochondrially encoded cytochrome b gene placed these mutants into four categories, three with single amino acid changes and one with two adjacent amino acid changes. Of the 12 nucleotide changes seen in the nine independently derived mutants 11 replaced A:T basepairs with G:C basepairs, possibly because of reactive oxygen species resulting from atovaquone treatment. Visualization of the resistance-conferring amino acid positions on the recently solved crystal structure of the vertebrate cytochrome bc1 complex revealed a discrete cavity in which subtle variations in hydrophobicity and volume of the amino acid side-chains may determine atovaquone-binding affinity, and thereby selective toxicity. These structural insights may prove useful in designing agents that selectively affect cytochrome bc1 functions in a wide range of eukaryotic pathogens.     

Parameters determining the relative efficacy of hydroxy-naphthoquinone inhibitors of the cytochrome bc1 complex

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc(1) complex that bind to the ubiquinol oxidation site between cytochrome b and the iron-sulfur protein and presumably mimic a transition state in the ubiquinol oxidation reaction catalyzed by the enzyme. The parameters that affect efficacy of binding of these inhibitors to the bc(1) complex are not well understood. Atovaquone, a hydroxy-naphthoquinone, has been used therapeutically to treat Pneumocystis carinii and Plasmodium infections. As the pathogens have developed resistance to this drug, it is important to understand the molecular basis of the drug resistance and to develop new drugs that can circumvent the drug resistance. We previously developed the yeast and bovine bc(1) complexes as surrogates to model the interaction of atovaquone with the bc(1) complexes of the target pathogens and human host. As a first step to identify new cytochrome bc(1) complex inhibitors with therapeutic potential and to better understand the determinants of inhibitor binding, we have screened a library of 2-hydroxy-naphthoquinones with aromatic, cyclic, and non-cyclic alkyl side-chain substitutions at carbon-3 on the hydroxy-quinone ring. We found a group of compounds with alkyl side-chains that effectively inhibit the yeast bc(1) complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc(1) complex provides structural and quantitative explanations for their binding efficacy to the target enzyme. In addition we also identified a 2-hydroxy-naphthoquinone with a branched side-chain that has potential for development as an anti-fungal and anti-parasitic therapeutic.     

Plasmodium falciparum: the effects of atovaquone resistance on respiration

Atovaquone is an antimalarial agent that specifically inhibits the cytochrome bc(1) complex of the cytochrome pathway. High-level atovaquone resistance is associated with a point mutation in the cytochrome b gene. A pair of isogenic clinical isolates of Plasmodium falciparum derived from before and after the acquisition of atovaquone resistance was used to determine whether the change in the cytochrome b gene resulted in changes in respiration in response to atovaquone. Since P. falciparum appears to utilize a branched respiratory system comprising both the cytochrome and an alternative respiratory pathway, the proportion of each pathway utilized by the sensitive and resistant parasites was investigated. Atovaquone inhibited total parasite oxygen consumption by up to 66% in the sensitive isolate but only up to 28% in the resistant isolate. Both the atovaquone-sensitive and the atovaquone-resistant parasites were comparably sensitive to the alternative pathway inhibitor, salicylhydroxamic acid. Atovaquone appeared to partially inhibit the rate of oxygen consumed through the alternative pathway in only the atovaquone-sensitive isolate. Cross resistance was noted between atovaquone and a new antimalarial agent WR243251. However, the level of WR243251 resistance was very modest compared to the level of atovaquone resistance. WR243251 was shown to rapidly reduce the rate of parasite oxygen consumption by almost 80% in the atovaquone-sensitive isolate and by 57% in the atovaquone-resistant isolate. Drug interaction studies suggest that atovaquone and WR243251 may inhibit growth additively or with mild synergy. Together, these results suggest that while WR243251 may inhibit respiration, its target of action probably differs from that of atovaquone.     

Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ～40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.     

Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine

Background

Atovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone^®) and inhibits the cytochrome bc₁ complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance.

Methods

The prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed.

Results

Four/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found.

Conclusion

In the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare.     

Understanding the Mechanism of Atovaquone Drug Resistance in Plasmodium falciparum Cytochrome b Mutation Y268S Using Computational Methods

The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.     

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.     

Antimalarial quinolones: synthesis, potency, and mechanistic studies

In the present article we examine the antiplasmodial activities of novel quinolone derivatives bearing extended alkyl or alkoxy side chains terminated by a trifluoromethyl group. In the series under investigation, the IC50 values ranged from 1.2 to approximately 30 nM against chloroquine-sensitive and multidrug-resistant Plasmodium falciparum strains. Modest to significant cross-resistance was noted in evaluation of these haloalkyl- and haloalkoxyquinolones for activity against the atovaquone-resistant clinical isolate Tm90-C2B, indicating that a primary target for some of these compounds is the parasite cytochrome bc1 complex. Additional evidence to support this biochemical mechanism includes the use of oxygen biosensor plate technology to show that the quinolone derivatives block oxygen consumption by parasitized red blood cells in a fashion similar to atovaquone in side-by-side experiments. Atovaquone is extremely potent and is the only drug in clinical use that targets the Plasmodium bc1 complex, but rapid emergence of resistance to it in both mono- and combination therapy is evident and therefore additional drugs are needed to target the cytochrome bc1 complex which are active against atovaquone-resistant parasites. Our study of a number of halogenated alkyl and alkoxy 4(1H)-quinolones highlights the potential for development of "endochin-like quinolones" (ELQ), bearing an extended trifluoroalkyl moiety at the 3-position, that exhibit selective antiplasmodial effects in the low nanomolar range and inhibitory activity against chloroquine and atovaquone-resistant parasites. Further studies of halogenated alkyl- and alkoxy-quinolones may lead to the development of safe and effective therapeutics for use in treatment or prevention of malaria and other parasitic diseases.     

Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor

The cytochrome bc1 complex resides in the inner membrane of mitochondria and transfers electrons from ubiquinol to cytochrome c. This electron transfer is coupled to the translocation of protons across the membrane by the protonmotive Q cycle mechanism. This mechanism topographically separates reduction of quinone and reoxidation of quinol at sites on opposite sites of the membrane, referred to as center N (Qn site) and center P (Qp site), respectively. Both are located on cytochrome b, a transmembrane protein of the bc1 complex that is encoded on the mitochondrial genome. To better understand the parameters that affect ligand binding at the Qn site, we applied the Qn site inhibitor ilicicolin H to select for mutations conferring resistance in Saccharomyces cerevisiae. The screen resulted in seven different single amino acid substitutions in cytochrome b rendering the yeast resistant to the inhibitor. Six of the seven mutations have not been previously linked to inhibitor resistance. Ubiquinol-cytochrome c reductase activities of mitochondrial membranes isolated from the mutants confirmed that the differences in sensitivity toward ilicicolin H originated in the cytochrome bc1 complex. Comparative in vivo studies using the known Qn site inhibitors antimycin and funiculosin showed little cross-resistance, indicating different modes of binding of these inhibitors at center N of the bc1 complex.     

Using an antimalarial in mosquitoes overcomes Anopheles and Plasmodium resistance to malaria control strategies

The spread of insecticide resistance in Anopheles mosquitoes and drug resistance in Plasmodium parasites is contributing to a global resurgence of malaria, making the generation of control tools that can overcome these roadblocks an urgent public health priority. We recently showed that the transmission of Plasmodium falciparum parasites can be efficiently blocked when exposing Anopheles gambiae females to antimalarials deposited on a treated surface, with no negative consequences on major components of mosquito fitness. Here, we demonstrate this approach can overcome the hurdles of insecticide resistance in mosquitoes and drug resistant in parasites. We show that the transmission-blocking efficacy of mosquito-targeted antimalarials is maintained when field-derived, insecticide resistant Anopheles are exposed to the potent cytochrome b inhibitor atovaquone, demonstrating that this drug escapes insecticide resistance mechanisms that could potentially interfere with its function. Moreover, this approach prevents transmission of field-derived, artemisinin resistant P. falciparum parasites (Kelch13 C580Y mutant), proving that this strategy could be used to prevent the spread of parasite mutations that induce resistance to front-line antimalarials. Atovaquone is also highly effective at limiting parasite development when ingested by mosquitoes in sugar solutions, including in ongoing infections. These data support the use of mosquito-targeted antimalarials as a promising tool to complement and extend the efficacy of current malaria control interventions.     

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.     

Natural resistance to inhibitors of the ubiquinol cytochrome c oxidoreductase of Rubrivivax gelatinosus: sequence and functional analysis of the cytochrome bc(1) complex

Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc(1) complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q(0) and Q(i) sites. Because the resistance to cytochrome bc(1) inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc(1) complex of Rubrivivax gelatinosus was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc(1) inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b(H) and b(L) hemes, respectively. These characteristics imply different functional properties of the cytochrome bc(1) complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of R. gelatinosus cytochrome bc(1) are discussed with reference to the structure and function of the cytochrome bc(1) complexes from other organisms.     

Mutations conferring resistance to quinol oxidation (Qz) inhibitors of the cyt bc1 complex of Rhodobacter capsulatus.

Several spontaneous mutants of the photosynthetic bacterium Rhodobacter capsulatus resistant to myxothiazol, stigmatellin and mucidin--inhibitors of the ubiquinol: cytochrome c oxidoreductase (cyt bc1 complex)--were isolated. They were grouped into eight different classes based on their genetic location, growth properties and inhibitor cross-resistance. The petABC (fbcFBC) cluster that encodes the structural genes for the Rieske FeS protein, cyt b and cyt c1 subunits of the cyt bc1 complex was cloned out of the representative isolates and the molecular basis of inhibitor-resistance was determined by DNA sequencing. These data indicated that while one group of mutations was located outside the petABC(fbcFBC) cluster, the remainder were single base pair changes in codons corresponding to phylogenetically conserved amino acid residues of cyt b. Of these substitutions, F144S conferred resistance to myxothiazol, T163A and V333A to stigmatellin, L106P and G152S to myxothiazol + mucidin and M140I and F144L to myxothiazol + stigmatellin. In addition, a mutation (aer126) which specifically impairs the quinol oxidase (Qz) activity of the cyt bc1 complex of a non-photosynthetic mutant (R126) was identified to be a glycine to an aspartic acid replacement at position 158 of cyt b. Six of these mutations were found between amino acid residues 140 and 163, in a region linking the putative third and fourth transmembrane helices of cyt b. The non-random clustering of several inhibitor-resistance mutations around the non-functional aer126 mutation suggests that this region may be involved in the formation of the Qz inhibitor binding/quinol oxidation domain(s) of the cyt bc1 complex. Of the two remaining mutations, the V333A replacement conferred resistance to stigmatellin exclusively and was located in another region toward the C terminus of cyt b. The L106P substitution, on the other hand, was situated in the transmembrane helix II that carries two conserved histidine residues (positions 97 and 111 in R. capsulatus) considered to be the axial ligands for the heme groups of cyt b. The structural and functional roles of the amino acid residues involved in the acquisition of Qz inhibitor resistance are discussed in terms of the primary structure of cyt b and in relation to the natural inhibitor-resistance of various phylogenetically related cyt bc/bf complexes.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Probing binding determinants in center P of the cytochrome bc1 complex using novel hydroxy-naphthoquinones

Atovaquone is a substituted 2-hydroxy-naphthoquinone used therapeutically against Plasmodium falciparum (malaria) and Pneumocystis pathogens. It acts by inhibiting the cytochrome bc₁ complex via interactions with the Rieske iron-sulfur protein and cytochrome b in the ubiquinol oxidation pocket. As the targeted pathogens have developed resistance to this drug there is an urgent need for new alternatives. To better understand the determinants of inhibitor binding in the ubiquinol oxidation pocket of the bc₁ complex we synthesized a series of hydroxy-naphthoquinones bearing a methyl group on the benzene ring that is predicted to interact with the nuclear encoded Rieske iron-sulfur protein. We have also attempted to overcome the metabolic instability of a potent cytochrome bc₁ complex inhibitor, a 2-hydroxy-naphthoquinone with a branched side chain, by fluorinating the terminal methyl group. We have tested these new 2-hydroxy-naphthoquinones against yeast and bovine cytochrome bc₁ complexes to model the interaction with pathogen and human enzymes and determine parameters that affect efficacy of binding of these inhibitors. We identified a hydroxy-naphthoquinone with a trifluoromethyl function that has potential for development as an anti-fungal and anti-parasitic therapeutic.