Claudine M. Sluse-Goffart

Respiratory chain network in mitochondria of Candida parapsilosis: ADP/O appraisal of the multiple electron pathways

In this study we demonstrated that mitochondria of Candida parapsilosis contain a constitutive ubiquinol alternative oxidase (AOX) in addition to a classical respiratory chain (CRC) and a parallel respiratory chain (PAR) both terminating by two different cytochrome c oxidases. The C. parapsilosis AOX is characterized by a fungi‐type regulation by GMP (as a stimulator) and linoleic acid (as an inhibitor). Inhibitor screening of the respiratory network by the ADP/O ratio and state 3 respiration determinations showed that (i) oxygen can be reduced by the three terminal oxidases through four paths implying one bypass between CRC and PAR and (ii) the sum of CRC, AOX and PAR capacities is higher than the overall respiration (no additivity) and that their engagement could be progressive according to the redox state of ubiquinone, i.e. first cytochrome pathway, then AOX and finally PAR.

EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation

The present study investigated the protective effects of Ginkgo biloba extract (EGb 761) on rat liver mitochondrial damage induced by in vitro anoxia/reoxygenation. Anoxia/reoxygenation was known to impair respiratory activities and mitochondrial oxidative phosphorylation efficiency. ADP/O (2.57 +/- 0.11) decreased after anoxia/reoxygenation (1.75 +/- 0.09, p < .01), as well as state 3 and uncoupled respiration (-20%, p < .01), but state 4 respiration increased (p < .01). EGb 761 (50-200 microg/ml) had no effect on mitochondrial functions before anoxia, but had a specific dose-dependent protective effect after anoxia/reoxygenation. When mitochondria were incubated with 200 microg/ml EGb 761, they showed an increase in ADP/O (2.09 +/- 0.14, p < .05) and a decrease in state 4 respiration (-22%) after anoxia/reoxygenation. In EPR spin-trapping measurement, EGb 761 decreased the EPR signal of superoxide anion produced during reoxygenation. In conclusion, EGb 761 specially protects mitochondrial ATP synthesis against anoxia/reoxygenation injury by scavenging the superoxide anion generated by mitochondria.

Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

Kinetic study of the aspartate/glutamate carrier in intact rat heart mitochondria and comparison with a reconstituted system.

The homologous exchange of external [14C] aspartate/internal aspartate catalyzed by the aspartate/glutamate carrier of rat heart mitochondria was investigated using aspartate-loaded, glutamate-depleted mitochondria. An inhibitor-stop technique was developed for kinetic studies by applying pyridoxal phosphate. Direct initial rate determinations from the linear phase of [14C] aspartate uptake were insufficiently accurate at high external and/or low internal substrate concentrations. Therefore, the full time-course of [14C] aspartate uptake until reaching isotope equilibrium was fitted by a single exponential function and was used to calculate reliable initial steady-state rates. This method was applied in bisubstrate analyses of the antiport reaction for different external and internal aspartate concentrations. The kinetic patterns obtained in double reciprocal plots showed straight lines converging on the abscissa. This result is consistent with a sequential antiport mechanism. It implies the existence of a catalytic ternary complex that is formed by the translocator and substrate molecules bound from both sides of the membrane. The Km values for aspartate were clearly different for the external and the internal sides of the membrane, 216 +/- 23 microM and 2.4 +/- 0.5 mM, respectively. These values indicated a definite transmembrane asymmetry of the carrier. The same asymmetry became evident when investigating the isolated protein from bovine heart mitochondria after reconstitution into liposomes. In this case the Km values for external and internal aspartate were determined to be 123 +/- 11 microM and 2.8 +/- 0.6 mM, respectively. This comparison demonstrates a right-side out orientation of the carrier after insertion into liposomal membranes. The sequential transport mechanism of the aspartate/glutamate carrier, elucidated both in proteoliposomes and in mitochondria, also seems to be a common characteristic of other mitochondrial antiport carriers.

Dynamics of the Dictyostelium discoideum mitochondrial proteome during vegetative growth, starvation and early stages of development

In this study, a quantitative comparative proteomics approach has been used to analyze the Dictyostelium discoideum mitochondrial proteome variations during vegetative growth, starvation and the early stages of development. Application of 2‐D DIGE technology allowed the detection of around 2000 protein spots on each 2‐D gel with 180 proteins exhibiting significant changes in their expression level. In total, 96 proteins (51 unique and 45 redundant) were unambiguously identified. We show that the D. discoideum mitochondrial proteome adaptations mainly affect energy metabolism enzymes (the Krebs cycle, anaplerotic pathways, the oxidative phosphorylation system and energy dissipation), proteins involved in developmental and signaling processes as well as in protein biosynthesis and fate. The most striking observations were the opposite regulation of expression of citrate synthase and aconitase and the very large variation in the expression of the alternative oxidase that highlighted the importance of citrate and alternative oxidase in the physiology of the development of D. discoideum. Mitochondrial energy states measured in vivo with MitoTracker Orange CM™Ros showed an increase in mitochondrial membrane polarization during D. discoideum starvation and starvation‐induced development.

Secondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.

Energy conservation and dissipation in mitochondria isolated from developing tomato fruit of ethylene-defective mutants failing normal ripening: the effect of ethephon, a chemical precursor of ethylene.

Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.

Effects of cold and warm ischemia on the mitochondrial oxidative phosphorylation of swine lung.

BACKGROUND The aim of the study was to investigate the consequence of warm and cold ischemia on lung mitochondria in order to define bioenergetic limits within lung could be suitable for pulmonary transplantation. METHODS Twenty-two pigs underwent lung harvesting after lung flush with Euro-Collins solution. Mitochondria were isolated from fresh lungs, from lungs submitted to 24 or 48 hr of cold ischemia, to 30 or 45 min of warm ischemia, and to 30 min of warm ischemia followed by 24 or 48 hr of cold ischemia. Mitochondrial oxidative phosphorylation parameters were determined in isolated mitochondria by in vitro measurement of oxygen consumption. RESULTS Relative to controls, mitochondria submitted to cold ischemia showed an alteration in the oxidoreductase activities of the respiratory chain but no membrane permeability alteration. After 48 hr of cold ischemia, there was a decrease in the yield of the oxidative phosphorylation. Thirty minutes of warm ischemia did not alter the mitochondrial respiratory parameters. However, lung submitted to 45 min of warm ischemia showed mitochondrial damage as a decrease in the oxidative phosphorylation efficiency and ADP availability but no change in the oxidoreductase activities. Relative to cold ischemia alone, 30 min of warm ischemia preceding cold ischemia promoted no significant change in the respiratory parameters. CONCLUSIONS On bioenergetic basis, lung submitted to warm ischemia could be suitable for transplantation if the warm ischemia duration does not exceed 30 min. This could be a major concern in lung procurement from non-heart beating donors.

Bioenergetic peculiarity of heart mitochondria from the hemoglobin- and myoglobin-free antarctic icefish

Heart mitochondria of the hemoglobin and myoglobin-free antarctic icefish are specifically noncoupled with respect to succinate oxidation but not during NADH-dependent substrate oxidation. This observation suggests the coexistence of a noncoupled respiratory chain specialized in succinate oxidation and of the energy-conserving respiratory pathway. The alternative pathway of succinate would lead to the dissipation of the redox energy as heat and this thermogenic process could favour recovery after hypoxic stress periods, during which O 2 supply is severely impaired by the lack of both heme pigments.

Interactions Between the Cytochrome Pathway and the Alternative Oxidase in Isolated Acanthamoeba castellanii Mitochondria

The steady-state activity of the two quinol-oxidizing pathways of Acanthamoeba castellanii mitochondria, the phosphorylating cytochrome pathway (i.e. the benzohydroxamate(BHAM)-resistant respiration in state 3) and the alternative oxidase (i.e. the KCN-resistant respiration), is shown to be fixed by ubiquinone (Q) pool redox state independently of the reducing substrate (succinate or exogenous reduced nicotinamide adenine dinucleotide (NADH)), indicating that the active Q pool is homogenous. For both pathways, activity increases with the Q reduction level (up to 80%). However, the cytochrome pathway respiration partially inhibited (about 50%) by myxothiazol decreases when the Q reduction level increases above 80%. The decrease can be explained by the Q cycle mechanism of complex III. It is also shown that BHAM has an influence on the relationship between the rate of ADP phosphorylation and the Q reduction level when alternative oxidase is active, and that KCN has an influence on the relationship between the alternative oxidase activity and the Q reduction level. These unexpected effects of BHAM and KCN observed at a given Q reduction level are likely due to functional connections between the two pathways activities or to protein–protein interaction.