Molecular basis of resistance to cytochrome bc1 inhibitors

Inhibitors of the mitochondrial respiratory chain enzyme cytochrome bc ₁ (respiratory complex III) have been developed as antimicrobial agents. They are used in agriculture to control plant pathogenic fungi and in medicine against human pathogens, such as the malaria parasite Plasmodium falciparum , or Pneumocystis jiroveci (an opportunistic pathogenic fungus life-threatening in immuno-compromised patients). These respiratory inhibitors are thus effective against a broad range of important pathogens. Unfortunately, the problem of acquired resistance has rapidly emerged. A growing number of pathogen isolates resistant to inhibitor treatment have been reported, and this resistance is often linked to mutation within cytochrome b , one of the essential catalytic subunits of the complex. Saccharomyces cerevisiae is an invaluable model in order to assess the impact of the mutations on the sensitivity to the drugs, on the respiratory capacity and the fitness of cells. In this minireview, the inhibitors, their mode of action, and the mutations implicated in resistance and studied in yeast are briefly reviewed. Four mutations that are of particular importance in medicine and in agriculture are briefly reviewed and described in more detail and the molecular basis of resistance and of evolution of the mutations is discussed succinctly.     

Deletion of the gene encoding cytochrome b562 from Rhodobacter sphaeroides

Abstract Cryptococcus humicola strains secrete killer toxins inhibitory (at pH values ranging from 3 to 5.5) to many ascomycetous and basidiomycetous yeast-like fungi. RNA or DNA plasmids were not detected in the killers. The amino acid-containing toxins were of low M _r, soluble in methanol, resistant to proteolysis, thermostable, cellophane-diffusible and were specified as microcins. These findings show that the killer phenomenon in yeasts such as bacteriocinogeny may be due to excretion of two types of killer toxins: mycocins and microcins.     

The cytochrome bc1 complex of Rhodobacter sphaeroides can restore cytochrome c2-independent photosynthetic growth to a Rhodobacter capsulatus mutant lacking cytochrome bc1.

Plasmids encoding the structural genes for the Rhodobacter capsulatus and Rhodobacter sphaeroides cytochrome (cyt) bc1 complexes were introduced into strains of R. capsulatus lacking the cyt bc1 complex, with and without cyt c2. The R. capsulatus merodiploids contained higher than wild-type levels of cyt bc1 complex, as evidenced by immunological and spectroscopic analyses. On the other hand, the R. sphaeroides-R. capsulatus hybrid merodiploids produced only barely detectable amounts of R. sphaeroides cyt bc1 complex in R. capsulatus. Nonetheless, when they contained cyt c2, they were capable of photosynthetic growth, as judged by the sensitivity of this growth to specific inhibitors of the photochemical reaction center and the cyt bc1 complex, such as atrazine, myxothiazol, and stigmatellin. Interestingly, in the absence of cyt c2, although the R. sphaeroides cyt bc1 complex was able to support the photosynthetic growth of a cyt bc1-less mutant of R. capsulatus in rich medium, it was unable to do so when C4 dicarboxylic acids, such as malate and succinate, were used as the sole carbon source. Even this conditional ability of R. sphaeroides cyt bc1 complex to replace that of R. capsulatus for photosynthetic growth suggests that in the latter species the cyt c2-independent rereduction of the reaction center is not due to a structural property unique to the R. capsulatus cyt bc1 complex. Similarly, the inability of R. sphaeroides to exhibit a similar pathway is not due to some inherent property of its cyt bc1 complex.     

Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cytb providing resistance to Qo-inhibitors of the bc1 complex from Rhodobacter capsulatus

The substitutions M140I, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol-cytochrome c oxidoreductase (bc₁ complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Q_o inhibitors myxothiazol, mucidin and stigmatellin. Site-directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc₁, complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady-state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Q_o site. In particular, the presence of a methyl group on the β-carbon (He and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis-deficient mutants with assembled but impaired bc₁ complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of     

Behaviour of Nif− mutants of Rhodobacter capsulatus in photosynthetic, ammonia-limited chemostat cultures

Abstract Nif⁻ mutants of Rhodobacter capsulatus carrying mutations either in the nifR4 regulatory gene or in the nifH structural gene both outgrew the wild-type strain B10 in mixed chemostat cultures under conditions favouring nitrogenase-mediated H₂ production by the wild-type (ammonia as limiting nutrient, inert argon atmosphere, light as energy source), whereas under aerobic conditions in the dark, or in batch culture, the growth of Nif⁻ mutants was not favoured. Nitrogenase-mediated H₂ production therefore appears to be detrimental to the growth of R. capsulatus in nitrogen-limited continuous culture, as may also be the case for other nitrogen fixers.     

A novel membrane-associated c-type cytochrome, cyt cy, can mediate the photosynthetic growth of Rhodobacter capsulatus and Rhodobacter sphaeroides.

Mutants of Rhodobacter capsulatus lacking the soluble electron carrier cytochrome c2 are able to grow photosynthetically (Ps+), whereas Rhodobacter sphaeroides is unable to do so. To understand this unusual electron transfer pathway the gene required for cyt c2-independent growth of R.capsulatus was sought using chromosomal libraries derived from a cyt c2- mutant of this species to complement a Ps- cyt c2- mutant of R.sphaeroides to Ps+ growth. The complementing 1.2 kbp DNA fragment contained a gene, cycY, encoding a novel membrane-associated c-type cytochrome, cyt cy, based on predicted amino acid sequence, optical difference spectra and SDS-PAGE analysis of chromatophore membranes. The predicted primary sequence of cyt cy is unusual in having two distinct domains, a hydrophobic amino-terminal region and a carboxyl-terminus with strong homology to cytochromes c. A cyt cy- mutant of R.capsulatus remains Ps+ as does the cyt c2- mutant. However, a mutant lacking both cyt c2 and cy is Ps-, and can be complemented to Ps+ by either cyt c2 or cyt cy. These findings demonstrate that each of the cytochromes c2 and cy is essential for photosynthesis only in the absence of the other. Thus, two distinct electron transfer pathways, unrecognized until now, operate during photosynthesis in R.capsulatus under appropriate conditions, one via the soluble cyt c2 and the other via the membrane-associated cyt cy.     

Electrochemical and spectroscopic investigations of the cytochrome bc1 complex from Rhodobacter capsulatus.

The cytochrome bc(1) complex from Rhodobacter capsulatus was investigated by protein electrochemistry and visible/IR spectroscopy. Infrared difference spectra, which represent redox-induced conformational changes of cofactors and their protein environments, show signals of the hemes, the quinone Q(i), and small conformational changes of the protein backbone. Furthermore, band features were tentatively assigned to protonated aspartic or glutamic acids involved in the redox transition of each of the b hemes, a proline in that of the [2Fe-2S] protein, and an arginine in that of cytochrome b(H). The midpoint potential of the [2Fe-2S] protein was determined for the first time at physiological temperature to be +290 mV at pH 8.7. The reduced minus oxidized difference extinction coefficients of the alpha-bands of the cytochromes were calculated as 11.5, 19, and 6.7 mM(-1) cm(-1) for cytochromes c(1), b(H), and b(L), respectively. A novel method has been developed to quantify protonation reactions of the complex during the redox reactions of its cofactors by evaluation of the buffer signals in the midinfrared region. Values will be discussed in relation to the pH dependence of the midpoint potentials.     

ATR-FTIR spectroscopy studies of iron-sulfur protein and cytochrome c1 in the Rhodobacter capsulatus cytochrome bc1 complex

Redox transitions in the Rhodobacter capsulatus cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a cytochrome c(1) point mutant, M183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bc(1) complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron-sulfur protein which in turn allowed the IR redox difference spectra of ISP and cytochrome c(1) to be deconvoluted at high signal/noise ratios, and features of these spectra are interpreted in light of structural and mechanistic information.     

Membrane-associated cytochrome cy of Rhodobacter capsulatus is an electron carrier from the cytochrome bc1 complex to the cytochrome c oxidase during respiration.

We have recently established that the facultative phototrophic bacterium Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center during photosynthesis (F.E. Jenney and F. Daldal, EMBO J. 12:1283-1292, 1993; F.E. Jenney, R.C. Prince, and F. Daldal, Biochemistry 33:2496-2502, 1994). One pathway is via the well-characterized, water-soluble cytochrome c2 (cyt c2), and the other is via a novel membrane-associated c-type cytochrome named cyt cy. In this work, we probed the role of cyt cy in respiratory electron transport by isolating a set of R. capsulatus mutants lacking either cyt c2 or cyt cy, in the presence or in the absence of a functional quinol oxidase-dependent alternate respiratory pathway. The growth and inhibitor sensitivity patterns of these mutants, their respiratory rates in the presence of specific inhibitors, and the oxidation-reduction kinetics of c-type cytochromes monitored under appropriate conditions demonstrated that cyt cy, like cyt c2, connects the bc1 complex and the cyt c oxidase during respiratory electron transport. Whether cyt c2 and cyt cy are the only electron carriers between these two energy-transducing membrane complexes of R. capsulatus is unknown.     

A relationship between humidity response, growth form and photosynthetic operating point in C3 plants

Gas exchange experiments were performed with 13 plant species that differ from each other in growth-form and natural habitat. These comprised three herbaceous species, two ferns, two temperate deciduous trees, five rainforest trees and one liana from wet tropical forest. The aims were to investigate whether plants of similar growth-form and from similar habitats tended to respond similarly to a change in leaf-to-air vapour pressure difference (VPD), and to compare their ratio of intercellular to ambient partial pressures of CO₂ for given conditions. Leaves were subjected to a step change in VPD and the initial and final steady rates of transpiration were used to calculate an index of sensitivity, φ , which enabled comparison of species. The results suggest that species of similar growth-form and habitat respond similarly to increasing VPD, with the temperate deciduous trees undergoing a greater reduction in stomatal conductance than the herbaceous plants in well-watered soil. Also, for these experimental conditions, the ratio of leaf internal to ambient CO₂ partial pressure (p_i/p_a) was positively correlated with both CO₂ assimilation rate and stomatal insensitivity to VPD, across the 13 species. The results are discussed in terms of growth strategies and possible advantages and limitations of hydraulic systems in different plants.     

A functional hybrid between the cytochrome bc1 complex and its physiological membrane-anchored electron acceptor cytochrome cy in Rhodobacter capsulatus

The membrane integral ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductase (or the cyt bc1 complex) and its physiological electron acceptor, the membrane-anchored cytochrome cy (cyt cy), are discrete components of photosynthetic and respiratory electron transport chains of purple non-sulfur, facultative phototrophic bacteria of Rhodobacter species. In Rhodobacter capsulatus, it has been observed previously that, depending on the growth condition, absence of the cyt bc1 complex is often correlated with a similar lack of cyt cy (Jenney, F. E., et al. (1994) Biochemistry 33, 2496-2502), as if these two membrane integral components form a non-transient larger structure. To probe whether such a structural super complex can exist in photosynthetic or respiratory membranes, we attempted to genetically fuse cyt cy to the cyt bc1 complex. Here, we report successful production, and initial characterization, of a functional cyt bc1-cy fusion complex that supports photosynthetic growth of an appropriate R. capsulatus mutant strain. The three-subunit cyt bc1-cy fusion complex has an unprecedented bis-heme cyt c1-cy subunit instead of the native mono-heme cyt c1, is efficiently matured and assembled, and can sustain cyclic electron transfer in situ. The remarkable ability of R. capsulatus cells to produce a cyt bc1-cy fusion complex supports the notion that structural super complexes between photosynthetic or respiratory components occur to ensure efficient cellular energy production.     

Sinorhizobium meliloti rpoE2 is necessary for H2O2 stress resistance during the stationary growth phase

RpoE2 is an extracytoplasmic σ factor produced by Sinorhizobium meliloti during stationary growth phase. Its inactivation affected the synthesis of the superoxide dismutase, SodC, and catalase, KatC. The absence of SodC within the cell did not result in an increased sensitivity to extracellular superoxides. In contrast, the absence of KatC affected the resistance of S. meliloti to H₂O₂ during the stationary growth phase. A katC strain behaved as an rpoE2 strain during an H₂O₂ challenge, suggesting that the H₂O₂ sensitivity of the rpoE2 strain resulted only from the lack of KatC in this strain.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

Evidence that cytochrome b is the antimycin-binding component of the yeast mitochondrial cytochrome bc1 complex

The binding of antimycin was studied in several mutant strains of yeast that have specific defects in cytochrome b. The strains have mutations in a part of the mitochondrial DNA that contains the structural gene for the apoprotein of cytochome b. Two of the mutants lack this protein and have no spectral cytochrome b. These mutants also lack the strong antimycin-binding site that is present in wild-type yeast mitochondria in the ratio of one site per two cytochrome b molecules. A third mutant which contains normal levels of spectral cytochrome b, but shows an altered absorption maximum for cytochrome b at 77 °K, was found to bind normal amounts of antimycin. However, the fluorescence of antimycin bound to mitochondria of this strain was found to be less efficiently quenched than in the case of the wild-type strain. In another mutant which contains only 20% of the normal spectral level of cytochrome b, the number of antimycin-binding sites was proportionately less. In an antimycin-resistant mutant, the binding of antimycin was too weak to be detected. The simultaneous modification of the structure of cytochrome b and the alteration of the antimycin-binding site in these mutants suggests that the antimycin-binding site is located on the apoprotein of cytochrome b.     

Genetic analysis of the cytochrome c-aa3 branch of the Bradyrhizobium japonicum respiratory chain

Further genetic evidence is provided here that Bradyrhizobium japonicum possesses a mitochondria-like electron-transport pathway: 2[H]----UQ----bc1----c----aa3----O2. Two Tn5-induced mutants, COX122 and COX132, having cytochrome c oxidase-negative phenotypes, were obtained and characterized. Mutant COX122 was defective in a novel gene, named cycM, which was responsible for the synthesis of a c-type cytochrome with an Mr of 20,000 (20K). This 20K cytochrome c appeared to catalyse electron transport from the cytochrome bc1 complex to the aa3-type terminal oxidase and, unlike mitochondrial cytochrome c, was membrane-bound in B. japonicum. The Tn5 insertion of mutant COX132 was localized in coxA, the structural gene for subunit I of cytochrome aa3. This finding also led to the cloning and sequencing of the corresponding wild-type coxA gene that encoded a 541-amino-acid protein with a predicted Mr of 59,247. The CoxA protein shared about 60% sequence identity with the cytochrome aa3 subunit I of mitochondria. The B. japonicum cycM and coxA mutants were able to fix nitrogen in symbiosis with soybean (Fix+). In contrast, mutants described previously which lacked the bc1 complex did not develop into endosymbiotic bacteroids and were thus Fix-. The data suggest that a symbiosis-specific respiratory chain exists in B. japonicum in which the electrons branch off at the bc1 complex.     

Germination of cocklebur seed and growth of their axial and cotyledonary tissues in response to C2H4, CO2 and/or O2 under water stress

Abstract. Germination modes of lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) under different water stresses, prepared with mannitol solution, were examined in relation to gaseous factors. As the concentration of mannitol increased, germination was increasingly inhibited at a mode which was drawn by two straight lines having different slopes and meeting at an angle. One is a sharp line occurring at the lower concentrations of mannitol; the other is a gentle line occurring at higher concentrations of mannitol. The former reflected the growth response of axial tissues to mild water stress, whereas the latter reflected the growth response of cotyledonary tissues to severe water stress. The germination potential of cocklebur seeds increased with increasing temperature. Thus, the seeds were more resistant to water stress at higher than al lower temperatures. This increased germination potential under water stress resulted from the greater growth potential of axial tissues, but not cotyledonary tissues, at higher temperature. Increased O₂ levels improved both the reduced axial and cotyledonary growth under water stress. Carbon dioxide predominantly enhanced axial growth under water stress, whereas C₂H₄ exclusively enhanced cotyledonary growth. Thus, these gases were effective in potentiating germination under water stress. When combined with each other, these gases caused more pronounced growth of the axial and cotyledonary tissues, leading to germination under more severe water stresses. Maximal axial and cotyledonary growth under water stress occurred in the simultaneous presence of CO₂, C₂H₄ and O₂, which allowed the germination at higher mannitol concentrations above 0.6 kmol m^?3 From these results, it was suggested that cocklebur seeds would override water stress by depending upon both the Corresponding axial growth and the C₂H₄-responding cotyledonary growth.