Cytochrome b Mutations That Modify the Ubiquinol-binding Pocket of the Cytochrome bc 1 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

Molecular Basis for Atovaquone Binding to the Cytochrome bc 1 Complex

Atovaquone is a substituted 2-hydroxynaphthoquinone that is used therapeutically to treat Plasmodium falciparum malaria, Pneumocystis carinii pneumonia, and Toxoplasma gondii toxoplasmosis. It is thought to act on these organisms by inhibiting the cytochrome bc1 complex. We have examined the interaction of atovaquone with the bc1 complex isolated from Saccharomyces cerevisiae, a surrogate, nonpathogenic fungus. Atovaquone inhibits the bc1 complex competitively with apparent Ki = 9 nm, raises the midpoint potential of the Rieske iron-sulfur protein from 285 to 385 mV, and shifts the g values in the EPR spectrum of the Rieske center. These results indicate that atovaquone binds to the ubiquinol oxidation pocket of the bc1 complex, where it interacts with the Rieske iron-sulfur protein. A computed energy-minimized structure for atovaquone liganded to the yeast bc1 complex suggests that a phenylalanine at position 275 of cytochrome b in the bovine bc1 complex, as opposed to leucine at the equivalent position in the yeast enzyme, is responsible for the decreased sensitivity of the bovine bc1 complex (Ki = 80 nm) to atovaquone. When a L275F mutation was introduced into the yeast cytochrome b, the sensitivity of the yeast enzyme to atovaquone decreased (Ki = 100 nm) with no loss in activity, confirming that the L275F exchange contributes to the differential sensitivity of these two species to atovaquone. These results provide the first molecular description of how atovaquone binds to the bc1 complex and explain the differential inhibition of the fungal versus mammalian enzymes

Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

The bc 1 complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochrome c oxidoreductase ( bc 1 complex). In both Rhodobacter sphaeroides and Rhodobacter capsulatus , the bc 1 complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because the bc 1 complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochrome b subunit, in the Rieske iron-sulfur subunit, and in the cytochrome c 1 subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q o ) site or the quinol reductase (Q i ) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44795/1/10863_2004_Article_BF00762582.pd

Bidirectional fluxes of spermine across the mitochondrial membrane.

The polyamine spermine is transported into the mitochondrial matrix by an electrophoretic mechanism having as driving force the negative electrical membrane potential (DW). The presence of phosphate increases spermine uptake by reducingDpH and enhancingDW. The transport system is a specific uniporter constituted by a protein channel exhibiting two asymmetric energy barriers with the spermine binding site located in the energy well between the two barriers. Although spermine transport is electrophoretic in origin, its accumulation does not follow the Nernst equation for the presence of an efflux pathway. Spermine efflux may be induced by different agents, such as FCCP, antimycin A and mersalyl, able to completely or partially reduce theDWvalue and, consequently, suppress or weaken the force necessary to maintain spermine in the matrix. However this efflux may also take place in normal conditions when the electrophoretic accumulation of the polycationic polyamine induces a sufficient drop inDWable to trigger the efflux pathway. The release of the polyamine is most probably electroneutral in origin and can take place in exchange with protons or in symport with phosphate anion. The activity of both the uptake and efflux pathways induces a continuous cycling of spermine across the mitochondrial membrane, the rate of which may be prominent in imposing the concentrations of spermine in the inner and outer compartment. Thus, this event has a significant role on mitochondrial permeability transition modulation and consequently on the triggering of intrinsic apoptosis

Heterologous complementation reveals that mutant alleles of QSR1 render 60S ribosomal subunits unstable and translationally inactive.

QSR1 is a highly conserved gene which encodes a 60S ribosomal subunit protein that is required for joining of large and small ribosomal subunits. In this report we demonstrate heterologous complementation of a yeast QSR1 deletion strain with both the human and corn homologs and show that the human and corn proteins are assembled into hybrid yeast/human and yeast/corn ribosomes. While the homologous genes complement lethality of the QSR1 deletion, they also result in a diminished growth rate. Analyses of the translation rates of ribosomes containing the human and corn proteins reveal a partial loss of function. Velocity gradient analyses of the hybrid ribosomes after exposure to high concentrations of salt indicate that the decreased activity is due to lability of the hybrid 60S subunits

Strain-dependent variation in carbon source regulation of nucleus-encoded mitochondrial proteins of Saccharomyces cerevisiae.

Nuclear genes encoding mitochondrial proteins are regulated by carbon source with significant heterogeneity among four Saccharomyces cerevisiae strains. This strain-dependent variation is seen both in respiratory capacity of the cells and in the expression of beta-galactosidase reporter fusions to the promoters of CYB2, CYC1, CYC3, MnSOD, and RPO41