Enhanced activity of galactono-1,4-lactone dehydrogenase and ascorbate–glutathione cycle in mitochondria from complex III deficient Arabidopsis

The mitochondrial antioxidant homeostasis was investigated in Arabidopsis ppr40-1 mutant, which presents a block of electron flow at complex III. The activity of the ascorbate biosynthetic enzyme, l-galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) (GLDH) was elevated in mitochondria isolated from mutant plants. In addition increased activities of the enzymes of Foyer–Halliwell–Asada cycle and elevated glutathione (GSH) level were observed in the mutant mitochondria. Lower ascorbate and ascorbate plus dehydroascorbate contents were detected at both cellular and mitochondrial level. Moreover, the more oxidized mitochondrial redox status of ascorbate in the ppr40-1 mutant indicated that neither the enhanced activity of GLDH nor Foyer–Halliwell–Asada cycle could compensate for the enhanced ascorbate consumption in the absence of a functional respiratory chain.     

Impact of diabetes-associated lipoproteins on oxygen consumption and mitochondrial enzymes in porcine aortic endothelial cells

Impairments in mitochondrial function have been proposed to play an important role in the pathogenesis of diabetes. Atherosclerotic coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated low density lipoproteins (glyLDL) and oxidized LDL (oxLDL) were detected in patients with diabetes. Our previous studies demonstrated that oxLDL and glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). The present study examined the effects of glyLDL and oxLDL on mitochondrial respiration, membrane potential and the activities and proteins of key enzymes in mitochondrial electron transport chain (mETC) in cultured porcine aortic EC (PAEC). The results demonstrated that glyLDL or oxLDL significantly reduced oxygen consumption in Complex I, II/III and IV of mETC in PAEC compared to LDL or vehicle control using oxygraphy. Incubation with glyLDL or oxLDL significantly reduced mitochondrial membrane potential, the activities of mitochondrial ETC enzymes - NADH dehydrogenase (Complex I), succinate cytochrome c reductase (Complex II + III), ubiquinol cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV) in PAEC compared to LDL or control. Treatment with oxLDL or glyLDL reduced the abundance of subunits of Complex I, ND1 and ND6 in PAEC. However, the effects of oxLDL on mitochondrial activity and proteins were not significantly different from glyLDL. The findings suggest that the glyLDL or oxLDL impairs mitochondrial respiration, as a result from the reduction of the abundance of several key enzymes in mitochondria of vascular EC, which potentially may lead to oxidative stress in vascular EC, and the development of diabetic vascular complications.     

Oxidative stress caused by blocking of mitochondrial complex I H(+) pumping as a link in aging/disease vicious cycle

Vulnerability of mitochondrial Complex I to oxidative stress determines an organism's lifespan, pace of aging, susceptibility to numerous diseases originating from oxidative stress and certain mitopathies. The mechanisms involved, however, are largely unknown. We used confocal microscopy and fluorescent probe MitoSOX to monitor superoxide production due to retarded forward electron transport in HEPG2 cell mitochondrial Complex I in situ. Matrix-released superoxide production, the un-dismuted surplus (J(m)) was low in glucose-cultivated cells, where an uncoupler (FCCP) reduced it to half. Rotenone caused a 5-fold J(m) increase (AC(50) 2 microM), which was attenuated by uncoupling, membrane potential (DeltaPsi(m)), and DeltapH-collapse, since addition of FCCP (IC(50) 55 nM), valinomycin, and nigericin prevented this increase. J(m) doubled after cultivation with galactose/glutamine (i.e. at obligatory oxidative phosphorylation). A hydrophobic amiloride that acts on the ND5 subunit and inhibits Complex I H(+) pumping enhanced J(m) and even countered the FCCP effect (AC(50) 0.3 microM). Consequently, we have revealed a new principle predicting that Complex I produces maximum superoxide only when both electron transport and H(+) pumping are retarded. H(+) pumping may be attenuated by high protonmotive force or inhibited by oxidative stress-related mutations of ND5 (ND2, ND4) subunit. We predict that in a vicious cycle, when oxidative stress leads to higher fraction of, e.g. mutated ND5 subunits, it will be accelerated more and more. Thus, inhibition of Complex I H(+) pumping, which leads to oxidative stress, appears to be a missing link in the theory of mitochondrial aging and in the etiology of diseases related to oxidative stress.     

SLO2, a mitochondrial pentatricopeptide repeat protein affecting several RNA editing sites, is required for energy metabolism

Pentatricopeptide repeat (PPR) proteins belong to a family of approximately 450 members in Arabidopsis, of which few have been characterized. We identified loss of function alleles of SLO2, defective in a PPR protein belonging to the E+ subclass of the P-L-S subfamily. slo2 mutants are characterized by retarded leaf emergence, restricted root growth, and late flowering. This phenotype is enhanced in the absence of sucrose, suggesting a defect in energy metabolism. The slo2 growth retardation phenotypes are largely suppressed by supplying sugars or increasing light dosage or the concentration of CO(2) . The SLO2 protein is localized in mitochondria. We identified four RNA editing defects and reduced editing at three sites in slo2 mutants. The resulting amino acid changes occur in four mitochondrial proteins belonging to complex I of the electron transport chain. Both the abundance and activity of complex I are highly reduced in the slo2 mutants, as well as the abundance of complexes III and IV. Moreover, ATP, NAD+, and sugar contents were much lower in the mutants. In contrast, the abundance of alternative oxidase was significantly enhanced. We propose that SLO2 is required for carbon energy balance in Arabidopsis by maintaining the abundance and/or activity of complexes I, III, and IV of the mitochondrial electron transport chain.     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

Functional analysis of SLO2 provides new insight into the role of plant PPR proteins

PPR (Pentatricopeptide repeat) proteins are mainly involved in RNA metabolism. In Arabidopsis, the PPR family is composed of more than 450 members; however, only few of them were functionally characterized. In a previous report,1 we identified a novel mitochondrial PPR RNA editing factor, named SLO2, which is responsible for 7 editing events in Arabidopsis. Loss-of-function mutation in SLO2 results in plant growth retardation, and delayed development, and leads to the dysfunction of mitochondrial complex I, III and IV. slo2 is the first example of a single gene mutation affecting 3 complexes of the mitochondrial electron transport chain. This Short Communication discusses the conservation of upstream regions of editing sites affected by SLO2 and illustrates the effect of mutation of SLO2 on activation of the alternative pathway. We also reflect upon the implications and perspectives of these findings.     

Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation

L5178Y-R mouse lymphoma (LY-R) cells undergo rapid apoptosis when treated with photodynamic therapy (PDT) sensitized with the silicon phthalocyanine Pc 4. In this study we show that cytochrome c is released into the cytosol within 10 min of an LD99.9 dose of PDT. Cellular respiration is inhibited by 42% at 15 min, and 60% at 30 min after PDT treatment, and caspase 3-like protease activity is elevated by 15 min post-PDT. In digitonin-permeabilized cells addition of cytochrome c to the respiration buffer reverses PDT-induced inhibition of state 3 respiration via Complex I by 40-60%, and via Complex III by 50-90%. In contrast, extramitochondrial cytochrome c does not stimulate respiration in permeabilized control cells, and catalyzes only a low rate of oxygen consumption via electron transfer to cytochrome b5 on the outer mitochondrial membrane. These results demonstrate that PDT-induced inhibition of respiration is primarily due to leakage of cytochrome c into the cytosol rather than to damage to the major enzyme complexes of the electron transport chain. Whether or not inhibition of respiration influences the time course or extent of Pc 4-PDT-induced apoptosis in LY-R cells is not clear at the present time.     

The effect of glucagon on hepatic respiratory capacity

Data from numerous laboratories show that mitochondria isolated from livers treated acutely with glucagon have higher rates of state 3 respiration than control mitochondria. The purpose of the present study was to learn whether this phenomenon is an isolation artifact resulting from a stabilization of the mitochondrial membrane or whether it represents a real increase in the maximal respiratory capacity of liver cells due to glucagon treatment. Electron transport was measured through different spans of the electron transport chain by using ferricyanide as an alternate electron acceptor to O2. With isolated intact liver mitochondria, pretreatment with glucagon was found to cause an increase in electron flow, through both Complex I and Complex III, suggesting that the effect of glucagon was not specific for a single site in the electron transport chain. Using intact isolated hepatocytes, different results are obtained. Respiration was measured in isolated hepatocytes after quantitation of the hepatocyte mitochondrial content by assay of citrate synthase. Hepatocyte respiration could therefore be reported per mg of mitochondrial protein. By providing durohydroquinone to the cells, it was possible to measure electron flow from coenzyme Q to O2 in the absence of the physiological regulation of substrate supply. Likewise, the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone released the in situ mitochondria from control by the cytosolic ATP/ADP ratio and it was possible to measure maximal electron flow rates through Complex III. In the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, electron flow was higher in mitochondria in the cell than in isolated mitochondria. Glucagon caused no increase in mitochondrial respiration in situ either in the presence of the physiological substrates or in the presence of durohydroquinone. The data obtained do not support a role for the electron transport chain as a target of glucagon action in hepatocytes.     

A Link between Mitochondrial Dysregulation and Idiopathic Autism Spectrum Disorder (ASD): Alterations in Mitochondrial Respiratory Capacity and Membrane Potential

Autism spectrum disorder (ASD) is a neurological disorder triggered by various factors through complex mechanisms. Research has been done to elucidate the potential etiologic mechanisms in ASD, but no single cause has been confirmed. The involvement of oxidative stress is correlated with ASD and possibly affects mitochondrial function. This study aimed to elucidate the link between mitochondrial dysregulation and idiopathic ASD by focusing on mitochondrial respiratory capacity and membrane potential. Our findings showed that mitochondrial function in the energy metabolism pathway was significantly dysregulated in a lymphoblastoid cell line (LCL) derived from an autistic child (ALCL). Respiratory capacities of oxidative phosphorylation (OXPHOS), electron transfer of the Complex I and Complex II linked pathways, membrane potential, and Complex IV activity of the ALCL were analyzed and compared with control cell lines derived from a developmentally normal non-autistic sibling (NALCL). All experiments were performed using high-resolution respirometry. Respiratory capacities of OXPHOS, electron transfer of the Complex I- and Complex II-linked pathways, and Complex IV activity of the ALCL were significantly higher compared to healthy controls. Mitochondrial membrane potential was also significantly higher, measured in the Complex II-linked pathway during LEAK respiration and OXPHOS. These results indicate the abnormalities in mitochondrial respiratory control linking mitochondrial function with autism. Correlating mitochondrial dysfunction and autism is important for a better understanding of ASD pathogenesis in order to produce effective interventions.     

A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression

Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation.     

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.     

Interaction of positively charged ubiquinone analog (MitoQ10) with DT-diaphorase from liver mitochondria

Effects of the coenzyme Q analog (MitoQ₁₀) carrying a positively charged decyltetraphenylphosphonium group on functional activity of phosphorylating liver mitochondria were studied. Using inhibitory analysis it was found that at micromolar concentrations this quinone is reduced by NADH-dependent DT-diaphorase. Under conditions of malate oxidation, MitoQ₁₀ stimulates electron transfer from NADH to oxygen by shunting the block of rotenone-induced electron transport in Complex I. Steady-state mitochondrial respiration induced by rotenone and MitoQ₁₀ (1 μM), as well as K3 shunt are both blocked by the DT-diaphorase inhibitor dicumarol, the Complex III inhibitor myxothiazole, and the cytochrome oxidase inhibitor cyanide. The electron transport chain induced in liver mitochondria by MitoQ₁₀ in the presence of rotenone appears as follows: NADH → DT-diaphorase → MitoQ₁₀ → Complex III → Complex IV → O₂. Under conditions of malate (but not succinate) oxidation, MitoQ₁₀ and high concentrations of vitamin K3 induce in mitochondria cyanide-resistant respiration and opening of the nonspecific pore eventually resulting in inhibition of oxidative phosphorylation. It is concluded that MitoQ₁₀ should be regarded as an analog of hydrophilic quinones (vitamin K3, duroquinone, etc.) widely known as substrates for mitochondrial DT-diaphorase not interacting with CoQ₁₀ rather than as a natural CoQ₁₀ analog.     

Inactivation of mitochondrial electron transport by photosensitization of a pheophorbide a derivative

We examined the damage to mitochondrial electron transport caused by photosensitization of a pheophorbide a derivative, DH-I-180-3, shown recently to induce necrosis of lung carcinoma cells with low dark toxicity. Confocal microscopy showed that DH-I-180-3 co-localized with dihydrorhodamine-123 suggesting that it mainly accumulates in mitochondria. The photosensitizer alone in the dark did not affect mitochondrial electron transport. Illumination of isolated mitochondria in the presence of DH-I-180-3 resulted in inhibition of both NADH- and succinate-dependent respiration. Measurement of the activity of each component of the electron transport chain revealed that Complex I and III were very susceptible to the treatment whereas Complex IV was resistant. We conclude that the photosensitizer is localized in mitochondria and, upon illumination, produces reactive oxygen species that inactivate Complexes I and III.     

Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III

We studied the role of cytochrome c (CYTc), which mediates electron transfer between Complexes III and IV, in cellular events related with mitochondrial respiration, plant development and redox homeostasis. We analyzed single and double homozygous mutants in both CYTc-encoding genes from Arabidopsis: CYTC-1 and CYTC-2. While individual mutants were similar to wild-type, knock-out of both genes produced an arrest of embryo development, showing that CYTc function is essential at early stages of plant development. Mutants in which CYTc levels were extremely reduced respective to wild-type had smaller rosettes with a pronounced decrease in parenchymatic cell size and an overall delay in development. Mitochondria from these mutants had lower respiration rates and a relative increase in alternative respiration. Furthermore, the decrease in CYTc severely affected the activity and the amount of Complex IV, without affecting Complexes I and III. Reactive oxygen species levels were reduced in these mutants, which showed induction of genes encoding antioxidant enzymes. Ascorbic acid levels were not affected, suggesting that a small amount of CYTc is enough to support its normal synthesis. We postulate that, in addition to its role as an electron carrier between Complexes III and IV, CYTc influences Complex IV levels in plants, probably reflecting a role of this protein in Complex IV stability. This double function of CYTc most likely explains why it is essential for plant survival.     

Evidence for a relationship between mitochondrial Complex I activity and mitochondrial aldehyde dehydrogenase during nitroglycerin tolerance: Effects of mitochondrial antioxidants

The medical use of nitroglycerin (GTN) is limited by patient tolerance. The present study evaluated the role of mitochondrial Complex I in GTN biotransformation and the therapeutic effect of mitochondrial antioxidants. The development of GTN tolerance (in rat and human vessels) produced a decrease in mitochondrial O(2) consumption. Co-incubation with the mitochondria-targeted antioxidant mitoquinone (MQ, 10(-6)mol/L) or with glutathione ester (GEE, 10(-4)mol/L) blocked GTN tolerance and the effects of GTN on mitochondrial respiration and aldehyde dehydrogenase 2 (ALDH-2) activity. Biotransformation of GTN depended on the mitochondria being functionally active, particularly mitochondrial Complex I. Tolerance induced mitochondrial ROS production and oxidative stress, though these effects were not detected in HUVECρ(0) cells or Complex I mutant cells. Experiments performed to evaluate Complex I-dependent respiration demonstrated that its inhibition by GTN was prevented by the antioxidants in control samples. These results point to a key role for mitochondrial Complex I in the adequate functioning of ALDH-2. In addition, we have identified mitochondrial Complex I as one of the targets at which the initial oxidative stress responsible for GTN tolerance takes place. Our data also suggest a role for mitochondrial-antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.