The effect of pH on the alternative oxidase activity in isolated Acanthamoeba castellanii mitochondria

Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence.     

A type-1 metacaspase from Acanthamoeba castellanii

The complete sequence of a type-1 metacaspase from Acanthamoeba castellanii is reported comprising 478 amino acids. The metacaspase was recovered from an expression library using sera specific for membrane components implicated in stimulating encystation. A central domain of 155 amino acid residues contains the Cys/His catalytic dyad and is the most conserved region containing at least 30 amino acid identities in all metacaspases. The Acanthamoeba castellanii metacaspase has the most proline-rich N-terminus so far reported in type-1 metacaspases with over 40 prolines in the first 150 residues. Ala–Pro–Pro is present 11 times. Phylogenies constructed using only the conserved proteolytic domains or the complete sequences show identical branching patterns, differing only in the rates of change.     

The Alternative Oxidase of Yarrowia lipolytica Mitochondria Is Unable To Compete with the Cytochrome Pathway for Electrons

The activity of the cyanide-resistant alternative oxidase (pathway) of Yarrowia lipolytica mitochondria was studied as a function of the activity of the major, cyanide-sensitive, cytochrome pathway. The contribution of the alternative oxidase to the total respiration of mitochondria was evaluated by measuring the rate of oxygen consumption in the presence of cyanide (an inhibitor of the cytochrome pathway). The potential activity of the cytochrome pathway was evaluated spectrophotometrically, by measuring the oxidation rate of cytochrome c by ferricyanide, which accepts electrons from complex III (cytochrome c) of this pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was accompanied by oxygen consumption due to the transfer of electrons through the alternative pathway. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation in the cytochrome pathway) completely inhibited the consumption of oxygen by the mitochondria. Under these conditions, the inhibition of the alternative pathway by benzohydroxamic acid failed to affect the transfer of electrons from cytochrome c to ferricyanide. Benzohydroxamic acid did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These findings indicate that the alternative pathway is unable to compete with the cytochrome respiratory chain for electrons. The alternative pathway transfers only electrons that are superfluous for the cytochrome chain.     

Acanthamoeba castellanii: structural basis of the cytopathic mechanisms

In this study we report observations on the structural mechanisms of the cytopathic effect of Acanthamoeba castellanii trophozoites on cultured MDCK cell monolayers. Co-incubations were carried out for a maximum of 24h. The first evidence of damage to the cell monolayer was detected by measuring the transepithelial resistance of cell monolayers that interacted with the amoebae. At 6h, transepithelial resistance diminished to 51% and amoebae required 5-6h to produce evidence of structural injury at the light microscopy level. Following 12h of incubation, the cell monolayer was severely damaged. After making intimate contact with the surface of target cells, trophozoites detached cells from the substrate, lysed and by means of food-cups ingested the damaged cells. There was no morphological evidence of modifications in MDCK cell membranes, membrane fusion or junction formation between the amoeba and host plasma membrane. The lytic capacity of the amoebas appears to be the result of cytotoxic factors secreted by the amoebae since, when monolayers were incubated with conditioned medium, there was also a decrease in the transepithelial resistance. Besides, mechanical injury produced by the attachment and movement of the trophozoites may contribute to the disruption of the cell monolayer. As in other pathogenic amoebae, the cytopathic action of A. castellanii on the cell monolayers can subjectively be separated into four stages: adhesion, cytolysis, phagocytosis, and intracellular degradation.     

Acanthamoeba castellanii: identification and distribution of actin cytoskeleton

The presence of the cytoskeleton of Acanthamoeba castellanii was observed by means of cryo-electronmicroscopy and immunofluorescence techniques. This structure is formed largely by fibers and networks of actin located mainly in cytoplasmic locomotion structures as lamellipodia and as well as in various endocytic structures. In addition, the comparision between total actin content in whole extracts among different amoebae was made. The molecular weight of actin in A. castellanii was 44 kDa, and 45 kDa for Naegleria fowleri and Entamoeba histolytica.     

An Inception Report on the TOM Complex of the Amoeba <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis>, a Simple Model Protozoan in Mitochondria Studies

It is suggested that in the course of the TOM complex evolution at least two lineages have appeared: the animal–fungal and green plant ones. The latter involves also the TOM complexes of algae and protozoans. The amoeba Acanthamoeba castellanii is a free-living nonphotosynthetic soil protozoan, whose mitochondria share many bioenergetic properties with mitochondria of plants, animals and fungi. Here, we report that a protein complex, identified electrophysiologically as the A. castellanii TOM complex, contains a homologue of yeast/animal Tom70. Further, molecular weight of the complex (about 500 kDa) also points to A. castellanii evolutionary relation with fungi and animal. Thus, the data indicates that the TOM complex of A. castellanii is not a typical example of the protozoan TOM complex.     

Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.     

The Role of Electrostatic Interactions for Cytochrome c Oxidase Function

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a ₃–Cu_B binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.     

Structural Study of the Interaction Between the Mitochondrial Presequence of Cytochrome c Oxidase Subunit IV and Model Membranes

The structural effect of the presequence of cytochrome oxidase subunit IV (p25) on multilamellar liposomes with different lipid compositions has been investigated using X-ray diffraction and electron microscopy. The presequence causes the disordering of the liposomes containing negatively charged lipids, without destabilizing the bilayer structure or destroying the multilamellar nature of the liposomes. In the systems containing only zwitterionic lipids, a small increase in the d-spacing (lamellar stacking spacing) is observed without any disorder effect suggesting a weaker interaction of the peptide and lipid. Circular Dichroism measurements of the peptide, in the presence and absence of the different lipid systems studied, show that the secondary structure of the peptide is modulated by the lipid environment. Considerable amounts of -helix in the presequence is only observed in the systems containing negatively charged lipids. These are the same systems for which the disordering effect is observed with X-ray diffraction. It is proposed that p25 disorders the bilayer stacking by corrugating the membranes. The results are discussed in terms of the relevance of the specific lipid properties (e.g., electric charge and ability to form inverted phases) in determining how the peptide interacts with the lipid and affects its structural organization. It is suggested that the lipid properties relevant for the disordering effect induced by the peptide are the same as those involved in the formation of contact sites between mitochondrial membranes during the import of nuclear coded proteins.     

Growth Phase and the Number of Phosphorylation Sites in the Mitochondrial Electron Transport Chain of Acanthamoeba castellanii

SYNOPSIS. Mitochondria isolated from the soil ameba Acanthamoeba castellanii growing exponentially on complex medium have rotenone-insensitive oxygen uptake and ADP:O ratios which indicate the presence of only 2 phosphorylation sites in the electron transport chain. Stationary phase amebae yield mitochondria which are sensitive to inhibition by rotenone when respiring NAD⁺-Minked substrates and have 3 sites of phosphorylation. The levels of cytochromes (a + a ₃), b , and c are similar in mitochondria isolated from log or stationary phase amebae, and, with the exception of succinate, the respiratory rates obtained with different substrates do not change significantly from log to stationary growth phase.     

Structure and Function of the Plant Alternative Oxidase: Its Putative Role in the Oxygen Defence Mechanism

Current understanding of the structure and function of the plant alternative oxidase is reviewed. In particular, the role of the oxidase in the protection of tissues against oxidative stress is developed.     

Cytochrome c Effect on Respiration of Heart Mitochondria: Influence of Various Factors

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.     

The Effect of Cytochrome Oxidase Inhibitors on the Thermotolerance of Yeasts

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubraand D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.     

Activation of alternative oxidase and uncoupling protein lowers hydrogen peroxide formation in amoeba Acanthamoeba castellanii mitochondria

Mitochondria of amoeba Acanthamoeba castellanii were used to determine the role of two energy-dissipating systems, i.e., a free fatty acid (FFA)-activated, purine nucleotide-inhibited uncoupling protein (AcUCP) and a FFA-insensitive, purine nucleotide-activated ubiquinol alternative oxidase (AcAOX), in decreasing reactive oxygen species production in unicellular organisms. It is shown that the activation of AcUCP by externally added FFA resulted in a strong decrease in H2O2 production, whilst the inhibition of the FFA acid-induced AcUCP activity by GDP or addition of bovine serum albumin (BSA) enhanced production of H2O2. Similarly, the activation of antimycin-resistant AcAOX-mediated respiration by GMP significantly lowered H2O2 production, while inhibition of the oxidase by benzohydroxamate cancelled the GMP-induced effect on H2O2 production. When active together, both energy-dissipating systems revealed a cumulative effect on decreasing H2O2 formation. The results suggest that protection against mitochondrial oxidative stress may be a physiological role of AOX and UCP in unicellulars, such as A. castellanii.     

Lethal Effects of Helianthemum lippii (L.) on Acanthamoeba castellanii Cysts in Vitro

Acanthamoeba spp. commonly cause Acanthamoeba keratitis which is typically associated with the wear of contact lenses. Therefore, finding an economic, efficient, and safe therapy of natural origin is of outmost importance. This study examined the in vitro lethal potential of ethyl acetate and methanol extracts of Helianthemum lippii (L.) (sun roses) against Acanthamoeba castellanii cysts isolated from patients with amoebic keratitis. Both extracts proved to be potent as regard to their lethal effects on A. castellanii cysts with comparable results to chlorhexidine. The ethyl acetate was more promising with cumulative lethality. It showed a highly significant lethal percentage along the duration of treatment. The analysis of the more potent ethyl acetate extract revealed the presence of 2.96 mg/100 g of total phenolics, 0.289 mg/100 ml of total flavonoids and 37 mg/100 mg of total tannins which highlighted their phytomedicinal role.     

Increase of Lipid Hydroperoxides in Liver Mitochondria and Inhibition of Cytochrome Oxidase by Carbon Tetrachloride -Intoxication in Rats

The cellular localization of lipid hydroperoxides was determined for the first time in mitochondria, microsomes and cytosol of rat liver using a specific method involving chemical derivatization and HPLC. Mitochondria contained the highest level of hydroperoxides. After 6h of intragastric administration of carbon tetrachloride (CCl₄) to rats (2 ml/kg body weight), the concentration of lipid hydroperoxides increased significantly in liver mitochondria and cytochrome oxidase activity was inhibited to 35% of the control rats. The mitochondrial content of haem a decreased to 60% of the control at 12 h of CCl₄ administration. In vitro reaction of mitochondria with CCl₄ caused inactivation of cytochrome oxidase. These observations suggested that cytochrome oxidase and haem a in mitochondria were targets of CCl₄.     

Molecular Phylogeny of the Chipmunks Inferred from Mitochondrial Cytochrome b and Cytochrome Oxidase II Gene Sequences

There are currently 25 recognized species of the chipmunk genus Tamias. In this study we sequenced the complete mitochondrial cytochrome b (cyt b) gene of 23 Tamias species. We analyzed the cyt b sequence and then analyzed a combined data set of cyt b along with a previous data set of cytochrome oxidase subunit II (COII) sequence. Maximum-likelihood was used to further test the fit of models of evolution to the cyt b data. Other sciurid cyt b sequence was added to examine the evolution of Tamias in the context of other sciurids. Relationships among Tamias species are discussed, particularly the possibility of a current sorting event among taxa of the southwestern United States and the extreme divergences among the three subgenera (Neotamias, Eutamias, and Tamias).     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.