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

Hydroxy-naphthoquinones are competitive inhibitors of the cytochrome bc₁ 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 bc₁ 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 bc₁ complexes as surrogates to model the interaction of atovaquone with the bc₁ complexes of the target pathogens and human host. As a first step to identify new cytochrome bc₁ 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 bc₁ complex. Molecular modeling of these into the crystal structure of the yeast cytochrome bc₁ 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.     

Protonmotive pathways and mechanisms in the cytochrome bc1 complex

The cytochrome bc(1) complex catalyzes electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which electron transfer is linked to proton translocation across the inner mitochondrial membrane. In the Q cycle mechanism proton translocation is the net result of topographically segregated reduction of quinone and reoxidation of quinol on opposite sides of the membrane, with protons being carried across the membrane as hydrogens on the quinol. The linkage of proton chemistry to electron transfer during quinol oxidation and quinone reduction requires pathways for moving protons to and from the aqueous phase and the hydrophobic environment in which the quinol and quinone redox reactions occur. Crystal structures of the mitochondrial cytochrome bc(1) complexes in various conformations allow insight into possible proton conduction pathways. In this review we discuss pathways for proton conduction linked to ubiquinone redox reactions with particular reference to recently determined structures of the yeast bc(1) complex.     

Molecular basis of resistance to cytochrome bc1 inhibitors.

Inhibitors of the mitochondrial respiratory chain enzyme cytochrome bc1 (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.     

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain

The binding of cytochrome c to the cytochrome bc1 complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc1 complex were measured. Mixtures of bc1 complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dual-wavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 12 derivative produced a diminution of the polypeptide of the bc1 coplex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c1, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc1 complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.     

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.     

Biogenesis of the cytochrome bc1 complex in isolated rat hepatocytes

Isolated rat hepatocytes were labeled with [³⁵S]methionine in the absence or presence of cycloheximide or chloramphenicol. The cytochrome $\text{bc}$₁ complex was isolated from labeled cells by a micromethod and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. All subunits except the two smallest, subunits VII and VIII, were labeled in the absence of translational inhibitors. In the presence of cycloheximide only subunit III (molecular weight, 30 000) was labeled. This polypeptide, identified as an apo-cytochrome b, was weakly labeled with [³⁵S]methionine in the presence of cycloheximide, indicating a strict dependence of cytoplasmically synthesized products for its assembly. In the presence of chloramphenicol, labeling was inhibited only in subunit III.     

Anti-cooperative oxidation of ubiquinol by the yeast cytochrome bc1 complex

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc(1) complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c(1) reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is approximately 200 mV and in a bc(1) complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c(1) reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc(1) complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc(1) complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b(H) hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinol-cytochrome c reductase activity of the bc(1) complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc(1) dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc(1) complex dimer.     

Structural basis of multifunctional bovine mitochondrial cytochrome bc1 complex

The mitochondrial cytochrome bc1 complex is a multifunctional membrane protein complex. It catalyzes electron transfer, proton translocation, peptide processing, and superoxide generation. Crystal structure data at 2.9 A resolution not only establishes the location of the redox centers and inhibitor binding sites, but also suggests a movement of the head domain of the iron-sulfur protein (ISP) during bc1 catalysis and inhibition of peptide-processing activity during complex maturation. The functional importance of the movement of extramembrane (head) domain of ISP in the bc1 complex is confirmed by analysis of the Rhodobacter sphaeroides bc1 complex mutants with increased rigidity in the ISP neck and by the determination of rate constants for acid/base-induced intramolecular electron transfer between [2Fe-2S] and heme c1 in native and inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovine heart mitochondrial bc1 complex by nonionic detergent at concentrations that inactivate electron transfer activity. This peptide-processing activity is shown to be associated with subunits I and II by cloning, overexpression and in vitro reconstitution. The superoxide-generation site of the cytochrome bc1 complex is located at reduced bL and Q*-. The reaction is membrane potential-, and cytochrome c-dependent.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

Observations concerning the quinol oxidation site of the cytochrome bc1 complex

A direct hydrogen bond between ubiquinone/quinol bound at the QO site and a cluster-ligand histidine of the iron-sulfur protein (ISP) is described as a major determining factor explaining much experimental data on position of the ISP ectodomain, electron paramagnetic resonance (EPR) lineshape and midpoint potential of the iron-sulfur cluster, and the mechanism of the bifurcated electron transfer from ubiquinol to the high and low potential chains of the bc1 complex.     

Two distinct quinone-modulated modes of antimycin-sensitive cytochrome b reduction in the cytochrome bc1 complex

Reduction of cytochrome b-560 (analogous to cyt b-562 of mitochondria) via an antimycin-sensitive route has been revealed in chromatophores of the photosynthetic bacterium, Rhodopseudomonas sphaeroides Ga. Indeed, the results suggest that two reductive mechanisms can be operative. One is consistent with the idea that the quinol generated at the reaction center QB site enters the Q pool and, via the Qc site, equilibrates with cytochrome b-560. The other reductive mode circumvents redox equilibrium with the pool; we consider that this could result from a direct encounter of the reaction center with the bc1 complex perhaps involving a direct QB-Qc site interaction. This latter reaction is suppressed by occupancy of the Qc site, not only by antimycin but by ubiquinol and ubiquinone.     

Biogenesis of the cytochrome bc1 complex and role of assembly factors

The cytochrome bc₁ complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc₁ complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc₁ complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc₁ complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc₁ complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc₁ complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Resolution of the circular dichroism spectra of the mitochondrial cytochrome bc1 complex

The circular dichroic spectrum of the mitochondrial cytochrome bc1 complex isolated from bovine heart has been resolved into the contributions from the prosthetic groups: cytochrome c1, the 'Rieske' iron-sulphur centre and the two b cytochromes. It is apparent that firstly, the circular dichroism (CD) properties of cytochrome c1 within the bc1 complex differ from those found in the isolated cytochrome c1 and secondly, both the oxidized and reduced b cytochromes exhibit an intense spectrum of bilobic shape, with the wavelengths of the cross-over points closely corresponding to those of the maxima in the optical absorbance spectra. These latter CD features are discussed in relation to the proposed structure of cytochrome b.     

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.     

Oxygen dependent electron transfer in the cytochrome bc(1) complex

The effect of molecular oxygen on the electron transfer activity of the cytochrome bc(1) complex was investigated by determining the activity of the complex under the aerobic and anaerobic conditions. Molecular oxygen increases the activity of Rhodobacter sphaeroides bc(1) complex up to 82%, depending on the intactness of the complex. Since oxygen enhances the reduction rate of heme b(L), but shows no effect on the reduction rate of heme b(H), the effect of oxygen in the electron transfer sequence of the cytochrome bc(1) complex is at the step of heme b(L) reduction during bifurcated oxidation of ubiquinol.     

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.