naphthoquinones and myxothiazol

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.

Topics: Antimycin A; Bacteria; Bacterial Physiological Phenomena; Benzoquinones; Cytochrome b Group; Cytochrome c Group; Electron Transport Complex III; Electrons; Enzyme Inhibitors; Ferricyanides; Kinetics; Light; Methacrylates; Naphthoquinones; Oxidation-Reduction; Phenylenediamines; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Proteobacteria; Thiazoles; Time Factors; Titrimetry

Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc(1) (CYT bc(1)) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYT b.

Topics: Amino Acid Sequence; Animals; Antimalarials; Atovaquone; Binding Sites; Cattle; Chickens; Conserved Sequence; Cytochrome b Group; Drug Resistance; Eukaryota; Methacrylates; Models, Molecular; Molecular Sequence Data; Mutation; Naphthoquinones; Plasmodium falciparum; Polyenes; Sequence Homology, Amino Acid; Thiazoles

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.

Topics: Amino Acid Sequence; Animals; Antimalarials; Atovaquone; Base Sequence; Chickens; Cytochrome b Group; DNA, Mitochondrial; Drug Resistance; Electron Transport; Membrane Potentials; Methacrylates; Mice; Mice, Inbred BALB C; Mitochondria; Models, Molecular; Molecular Sequence Data; Naphthoquinones; Plasmodium yoelii; Sequence Analysis, DNA; Thiazoles; Ubiquinone

Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase.

Topics: 2,6-Dichloroindophenol; Animals; Antimycin A; Coenzymes; Cytochrome b Group; Cytochrome c Group; Electron Transport; Electron Transport Complex III; Methacrylates; Mitochondria, Liver; Multienzyme Complexes; Naphthoquinones; Quinone Reductases; Rats; Succinates; Succinic Acid; Tetramethylphenylenediamine; Thiazoles; Ubiquinone