BBA - Bioenergetics (v.1817, #7)
Editorial Board (i).
Assessing the photoprotective effectiveness of non-photochemical chlorophyll fluorescence quenching: A new approach by Alexander V. Ruban; Erik H. Murchie (977-982).
The photoprotective nature of non-photochemical quenching (NPQ) has not been effectively quantified and the major reason is the inability to quantitatively separate NPQ that acts directly to prevent photoinhibition of photosystem II (PSII). Here we describe a technique in which we use the values of the PSII yield and qP measured in the dark following illumination. We expressed the quantum yield of PSII (ΦPSII) via NPQ as: ΦPSII = qP × (Fv/Fo) / (1 + Fv/Fo + NPQ). We then tested this theoretical relationship using Arabidopsis thaliana plants that had been exposed to gradually increasing irradiance. The values of qP in the dark immediately after the illumination period (here denoted qPd) were determined using a previously described technique for Fo′ calculation: Fo′calc. = 1 / (1/Fo − 1/Fm − 1/Fm′). We found that in every case the actual ΦPSII deviated from theoretical values at the same point that qPd deviated from a value of 1.0. In an increasing series of irradiance levels, WT leaves tolerated 1000 μmol m− 2 s− 1 of light before qPd declined. Leaves treated with the uncoupler nigericin, leaves of the mutant lacking PsbS protein and leaves overexpressing PsbS showed a qPd reduction at 100, 600 and 2000 μmol m− 2 s− 1 respectively, each at an increasing value of NPQ. Therefore we suggest that this simple and timely technique will be instrumental for identifying photoprotective NPQ (pNPQ) and that it is more appropriate than the qE component. Its applications should be broad: for example it will be useful in physiology-based studies to define the optimal level of nonphotochemical quenching for plant protection and productivity.► Effectiveness of non-photochemical fluorescence quenching (NPQ) is assessed. ► The method measures dark levels of PSII yield and qP after illumination. ► The relationships between PSII yield/qP and NPQ display the onset of photoinhibition. ► The level of protective NPQ (pNPQ) when reaction centres remain intact is determined. ► The maximum light intensity which is “safe” for plants to grow at can be evaluated.
Keywords: Protective NPQ; Thylakoid membrane; Photosystem II; LHCII; Xanthophyll; PsbS;
Characterization of the peridinin–chlorophyll a-protein complex in the dinoflagellate Symbiodinium by Jing Jiang; Hao Zhang; Yisheng Kang; David Bina; Cynthia S. Lo; Robert E. Blankenship (983-989).
► The PCP in the dinoflagellate Symbiodinium was characterized. ► PCP genes were cloned and are organized in intronless tandem arrays. ► The amino acid sequence of PCP was determined by mass spectrometry.
Keywords: Photosynthetic antenna; Peridinin–chlorophyll a-protein; Symbiodinium; Peptide sequencing; Absorption spectroscopy; Fluorescence spectroscopy;
Lack of cytochrome c in Arabidopsis decreases stability of Complex IV and modifies redox metabolism without affecting Complexes I and III by Elina Welchen; Tatjana M. Hildebrandt; Dagmar Lewejohann; Daniel H. Gonzalez; Hans-Peter Braun (990-1001).
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.► Knock-out of cytochrome c causes embryo lethality in plants. ► Plants with low cytochrome c levels have increased alternative respiration. ► Cytochrome c may participate in the assembly/stabilization of Complex IV in plants. ► Mutants show reduced ROS levels and induction of genes encoding antioxidant enzymes. ► A small amount of cytochrome c is enough to support normal ascorbic acid synthesis.
Keywords: Cytochrome c; Arabidopsis thaliana; Mitochondrion; Embryo lethal; Respiratory chain complex;
Mitochondrial DNA metabolism in early development of zebrafish (Danio rerio) by Lucia Artuso; Alessandro Romano; Tiziano Verri; Alice Domenichini; Francesco Argenton; Filippo Maria Santorelli; Vittoria Petruzzella (1002-1011).
Changes in the mitochondrial DNA (mtDNA) population, together with the expression of a set of genes involved in mtDNA replication and transcription and genes encoding for components of OxPhos complexes, were studied during zebrafish development from early embryo to larval stages. The mtDNA copy number, measured from 1 h post-fertilization to the adult stage, significantly decreased over time, suggesting that mtDNA replication is not active in early zebrafish embryos and that, as in mammals, there occurs partition of the maternal mtDNA copies. Zebrafish genes involved in mtDNA replication (i.e. catalytic subunit of the mtDNA polymerase γ, mitochondrial deoxyribonucleoside kinase) are expressed late in embryo development, further supporting the notion that there is no replication of mtDNA in the early stages of zebrafish development. Notably, as from 4 days post-fertilization, marked expression of “replication genes” was observed in the exocrine pancreas. Interestingly, the mtDNA helicase, also involved in mtDNA replication, was detected early in development, suggesting diverse regulation of this gene. On the other hand, zebrafish mtDNA transcription genes (i.e. mtDNA-directed RNA polymerase, mitochondrial transcription factor A) were ubiquitously expressed in the early stages of development, suggesting that mitochondrial transcription is already active before mtDNA replication. This hypothesis of early activation of mtDNA transcription fits in with the high early expression of structural OxPhos genes, suggesting that an active OxPhos system is necessary during early embryogenesis. As well as providing the first description of mtDNA distribution during zebrafish development, the present study also represents a step toward the use of Danio rerio as a model for investigation of mitochondrial metabolism and disease.► mtDNA replication is inactive in zebrafish embryos but in the exocrine pancreas. ► mtDNA helicase shows different regulation respect to replicative genes. ► mtDNA transcription is already active before mtDNA replication. ► An active OxPhos system is necessary during early zebrafish embryogenesis. ► Danio rerio is a good model for investigation of mitochondrial disease.
Keywords: Danio rerio; Mitochondrial DNA; Respiratory chain complex; Embryo development; Mitochondrial transcription; Mitochondrial replication;
Investigation of OCP-triggered dissipation of excitation energy in PSI/PSII-less Synechocystis sp. PCC 6803 mutant using non-linear laser fluorimetry by F.I. Kuzminov; N.V. Karapetyan; M.G. Rakhimberdieva; I.V. Elanskaya; M.Y. Gorbunov; V.V. Fadeev (1012-1021).
In order to prevent photodestruction by high light, photosynthetic organisms have evolved a number of mechanisms, known as non-photochemical quenching (NPQ), that deactivate the excited states of light harvesting pigments. Here we investigate the NPQ mechanism in the cyanobacterium Synechocystis sp. PCC 6803 mutant deficient in both photosystems. Using non-linear laser fluorimetry, we have determined molecular photophysical characteristics of phycocyanin and spectrally distinct forms of allophycocyanin for the cells in non-quenched and quenched states. Our analysis of non-linear fluorescence characteristics revealed that NPQ activation leads to an ~ 2-fold decrease in the relaxation times of both allophycocyanin fluorescence components, F660 and F680, and a 5-fold decrease in the effective excitation cross-section of F680, suggesting an emergence of a pathway of energy dissipation for both types of allophycocyanin. In contrast, NPQ does not affect the rates of singlet–singlet exciton annihilation. This indicates that, upon NPQ activation, the excess excitation energy is transferred from allophycocyanins to quencher molecules (presumably 3′hydroxyechinenone in the orange carotenoid protein), rather than being dissipated due to conformational changes of chromophores within the phycobilisome core. Kinetic measurements of fluorescence quenching in the Synechocystis mutant revealed the presence of several stages in NPQ development, as previously observed in the wild type. However, the lack of photosystems in the mutant enhanced the magnitude of NPQ as compared to the wild type, and allowed us to better characterize this process. Our results suggest a more complex kinetics of the NPQ process, thus clarifying a multistep model for the formation of the quenching center.► OCP-induced NPQ originates from both long- and short wavelength allophycocyanins. ► NPQ has no effect on the rates of singlet–singlet annihilation of the phycobilins. ► Studies on Synechocystis PSI/PSII-less mutant revealed new stages in the NPQ process.
Keywords: Cyanobacterium; Energy dissipation; Fluorescence quenching; Non-linear laser fluorimetry; Non-photochemical quenching; Phycobilisome;
Metal cations modulate the bacteriochlorophyll–protein interaction in the light-harvesting 1 core complex from Thermochromatium tepidum by Yukihiro Kimura; Yuta Inada; Tomoko Numata; Teruhisa Arikawa; Yong Li; Jian-Ping Zhang; Zheng-Yu Wang; Takashi Ohno (1022-1029).
The light-harvesting 1 reaction center (LH1-RC) complex from Thermochromatium (Tch.) tepidum exhibits unusual Q y absorption by LH1 bacteriochlorophyll-a (BChl-a) molecules at 915 nm, and the transition energy is finely modulated by the binding of metal cations to the LH1 polypeptides. Here, we demonstrate the metal-dependent interactions between BChl-a and the polypeptides within the intact LH1-RC complexes by near-infrared Raman spectroscopy. The wild-type LH1-RC (B915) exhibited Raman bands for the C3-acetyl and C13-keto C＝O stretching modes at 1637 and 1675 cm− 1, respectively. The corresponding bands appeared at 1643 and 1673 cm− 1 when Ca2 + was biosynthetically replaced with Sr2 + (B888) or at 1647 and 1669 cm− 1 in the mesophilic counterpart, Allochromatium vinosum. These results indicate the significant difference in the BChl–polypeptide interactions between B915 and B888 and between B915 and the mesophilic counterpart. The removal of the original metal cations from B915 and B888 resulted in marked band shifts of the C3-acetyl/C13-carbonyl νC＝O modes to ~ 1645/~ 1670 cm− 1, supporting a model in which the metal cations are involved in the fine-tuning of the hydrogen bonding between the BChl-a and LH1-polypeptides. Interestingly, the interaction modes were almost identical between the Ca2 +-depleted B915 and Sr2 +-depleted B888 and between B915 and Ca2 +-substituted B888, despite the significant differences in their LH1 Q y peak positions and the denaturing temperatures, as revealed by differential scanning calorimetry. These results suggest that not only the BChl–polypeptide interactions but some structural origin may be involved in the unusual Q y red-shift and the enhanced thermal stability of the LH1-RC complexes from Tch. tepidum.► We examine BChl–protein interactions in the intact LH1-RC from Tch. tepidum. ► Metal cations modulate hydrogen-bonding interactions between BChl and LH1 protein. ► Some structural origins are responsible for unusual properties of Tch. tepidum. ► BChl–protein interactions are different between Tch. tepidum and Alc. vinosum.
Keywords: Thermochromatium tepidum; Light-harvesting 1 complex; Bacteriochlorophyll-a; Calcium; Near-infrared Raman spectroscopy;
A minimal phycobilisome: Fusion and chromophorylation of the truncated core-membrane linker and phycocyanin by Kun Tang; Xiao-Li Zeng; Yi Yang; Zhi-Bin Wang; Xian-Jun Wu; Ming Zhou; Dror Noy; Hugo Scheer; Kai-Hong Zhao (1030-1036).
Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610–660 nm, and fluoresced at ~ 670 nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540–575 nm, and fluorescing at ~ 585 nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.► We solubilize phycobilisome core-membrane linker by selective truncation. ► We fuse the truncated core-membrane linker with the minimal chromophore domain of phycocyanin. ► The fused constructs can be bi-chromophorylated with phycocyanobilin, phycoviolobilin, phycoerythrobilin and/or phycourobilin. ► The bi-chromophorylated constructs have broad absorptions, efficient energy transfer and large Stokes shifts.
Keywords: Photosynthesis; Phycobilisome; Core–membrane linker; Phycocyanin; Phycocyanobilin; Phycoerythrobilin;
Compensatory upregulation of respiratory chain complexes III and IV in isolated deficiency of ATP synthase due to TMEM70 mutation by Vendula Havlíčková Karbanová; Alena Čížková Vrbacká; Kateřina Hejzlarová; Hana Nůsková; Viktor Stránecký; Andrea Potocká; Stanislav Kmoch; Josef Houštěk (1037-1043).
Early onset mitochondrial encephalo-cardiomyopathy due to isolated deficiency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding enzyme-specific ancillary factor. Diminished ATP synthase results in low ATP production, elevated mitochondrial membrane potential and increased ROS production. To test whether the patient cells may react to metabolic disbalance by changes in oxidative phosphorylation system, we performed a quantitative analysis of respiratory chain complexes and intramitochondrial proteases involved in their turnover. SDS- and BN-PAGE Western blot analysis of fibroblasts from 10 patients with TMEM70 317-2A > G homozygous mutation showed a significant 82–89% decrease of ATP synthase and 50–162% increase of respiratory chain complex IV and 22–53% increase of complex III. The content of Lon protease, paraplegin and prohibitins 1 and 2 was not significantly changed. Whole genome expression profiling revealed a generalized upregulation of transcriptional activity, but did not show any consistent changes in mRNA levels of structural subunits, specific assembly factors of respiratory chain complexes, or in regulatory genes of mitochondrial biogenesis which would parallel the protein data. The mtDNA content in patient cells was also not changed. The results indicate involvement of posttranscriptional events in the adaptive regulation of mitochondrial biogenesis that allows for the compensatory increase of respiratory chain complexes III and IV in response to deficiency of ATP synthase.► Isolated deficiency of ATP synthase causes a severe mitochondrial disease. ► Metabolic disbalance upregulates respiratory chain complexes IV and III. ► Adaptive changes in mitochondrial biogenesis result from posttranscriptional events.
Keywords: ATP synthase; TMEM70; Disease; Gene expression profiling; Oxidative phosphorylation; Mitochondrial biogenesis;
Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms by Kathi Gundermann; Claudia Büchel (1044-1052).
Fucoxanthin-chlorophyll complexes (FCP) from the centric diatom Cyclotella meneghiniana were isolated and the trimeric FCPa complex was reconstituted into liposomes at different lipid to Chl a ratios. The fluorescence yield of the complexes in different environments was calculated from room temperature fluorescence emission spectra and compared to the aggregated state of FCPa. FCPa surrounded by high amounts of lipids resembled detergent solubilised complexes and with decreasing lipid levels, i.e. in a situation where protein contacts were increasingly favoured, the fluorescence yield of FCPa gradually decreased. In addition, the yield displayed a strong pH-dependency in case of lower lipid contents. The further reduction in fluorescence yield brought about by the conversion of diadinoxanthin to diatoxanthin was pH independent and only depended on the amount of diatoxanthin synthesised. The implications of these data for non-photochemical quenching in centric diatoms are discussed.► Fluorescence yield of FCPa depends on protein-protein interactions in lipids. ► Lower pH decreases the yield the more the lower the amount of lipids present. ► pH sensitive amino acid residues are found in both constitutive polypeptides. ► Diatoxanthin acts independently of the pH. ► Diatoxanthin acts in addition to the effects induced by protein interaction.
Keywords: Diatoms; Fucoxanthin-chlorophyll complexes; Non-photochemical quenching; Light harvesting; Xanthophyll cycle; Diatoxanthin;
Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc 1 complex by Sangjin Hong; Doreen Victoria; Antony R. Crofts (1053-1062).
The homodimeric bc 1 complexes are membrane proteins essential in respiration and photosynthesis. The ~ 11 Å distance between the two b L-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc 1 complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined.Display Omitted► Linear titration of myxothiazol inhibition is diagnostic of a monomeric mechanism. ► Heterodimeric expression systems generate wild-type homodimer by recombination. ► Selection for survival of functional bc 1 complex variants occurs during growth. ► Intermonomer electron transfer is not competitive with monomeric turnover.
Keywords: Inter-monomer electron transfer; Crossover recombination; Q-cycle; Inhibitor titration; bc 1 complex;
Structural backgrounds for the formation of a catalytically competent complex with NADP(H) during hydride transfer in ferredoxin–NADP+ reductases by Ana Sánchez-Azqueta; Matías A. Musumeci; Marta Martínez-Júlvez; Eduardo A. Ceccarelli; Milagros Medina (1063-1071).
The role of the highly conserved C266 and L268 of pea ferredoxin–NADP+ reductase (FNR) in formation of the catalytically competent complex of the enzyme with NADP(H) was investigated. Previous studies suggest that the volume of these side-chains, situated facing the side of the C-terminal Y308 catalytic residue not stacking the flavin isoalloxazine ring, may be directly involved in the fine-tuning of the catalytic efficiency of the enzyme. Wild-type pea FNR as well as single and double mutants of C266 and L268 residues were analysed by fast transient-kinetic techniques and their midpoint reduction potentials were determined. For the C266A, C266M and C266A/L268A mutants a significant reduction in the overall hydride transfer (HT) rates was observed along with the absence of charge-transfer complex formation. The HT rate constants for NADPH oxidation were lower than those for NADP+ reduction, reaching a 30-fold decrease in the double mutant. In agreement, these variants exhibited more negative midpoint potentials with respect to the wild-type enzyme. The three-dimensional structures of C266M and L268V variants were solved. The C266M mutant shows a displacement of E306 away from the relevant residue S90 to accommodate the bulky methionine introduced. The overall findings indicate that in FNR the volume of the residue at position 266 is essential to attain the catalytic architecture between the nicotinamide and isoalloxazine rings at the active site and, therefore, for an efficient HT process. In addition, flexibility of the 268–270 loop appears to be critical for FNR to achieve catalytically competent complexes with NADP(H).► C266 modulates stabilisation and architecture of the catalytically competent complex. ► C266 contributes to the efficient hydride transfer between FNR and NADP(H). ► The 268–270 loop flexibility contributes to the competent complex architecture. ► The FNR–NADP(H) competent complex architecture is finely tuned for catalysis.
Keywords: Ferredoxin–NADP+ reductase; Stopped-flow; X-ray structure; Protein–ligand interaction; Catalytically competent complex; Hydride transfer;
Substrate-dependent modulation of the enzymatic catalytic activity: Reduction of nitrate, chlorate and perchlorate by respiratory nitrate reductase from Marinobacter hydrocarbonoclasticus 617 by Jacopo Marangon; Patrícia M. Paes de Sousa; Isabel Moura; Carlos D. Brondino; José J.G. Moura; Pablo J. González (1072-1082).
The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.► Respiratory nitrate reductases catalyze the reduction of nitrate to nitrite. ► Its properties were studied by in-solution kinetics, PFV and redox titration. ► PFV showed at least two catalytically distinct forms of the enzyme. ► Substrate, pH and inhibitors affect these two forms differentially. ► A revised catalytic mechanism is proposed.
Keywords: Nitrate reductase; Molybdenum; Denitrification; Protein film voltammetry; Enzyme catalysis;
A unique regulation of the expression of the psbA, psbD, and psbE genes, encoding the D1, D2 and cytochrome b559 subunits of the Photosystem II complex in the chlorophyll d containing cyanobacterium Acaryochloris marina by Éva Kiss; Péter B. Kós; Min Chen; Imre Vass (1083-1094).
Photosynthetic electron transport, chromatic photoacclimation and expression of the genes encoding the D1, D2, and cytochrome b559 subunits of the Photosystem II complex were studied in the chlorophyll d containing cyanobacterium Acaryochloris marina MBIC11017 under various environmental conditions. During oxygen deprivation and inhibition of photosynthetic electron transport by dibromothymoquinone the psbA1 gene encoding a D1′ isoform was induced. All of the three psbA and one of the three psbD (psbD2) genes, encoding two different isoforms of the D1 and the abundant isoform of the D2 proteins, respectively were induced under exposure to UV-B radiation and high intensity visible light. Under far red light the amount of Photosystem II complexes increased, and expression of the psbE2 gene encoding the alpha-subunit of cytochrome b559 was enhanced. However, the psbF and psbE1 genes encoding the beta- and another isoform of alpha-cytochrome b559, respectively remained lowly expressed under all conditions. Far red light also induced the psbD3 gene encoding a D2′ isoform whose primary structure is different from the abundant D2 isoform. psbD3 was also induced under low intensity visible light, when chromatic photoacclimation was indicated by a red-shifted absorption of chlorophyll d. Our results show that differential expression of multigene families encoding different isoforms of D1 and D2 plays an important role in the acclimation of A. marina to contrasting environmental conditions. Moreover, the disproportionate quantity of transcripts of the alpha and beta subunits of cytochrome b559 implies the existence of an alpha–alpha homodimer organization of cytochrome b559 in Photosystem II complexes.
Keywords: Adaptation; Cyanobacteria; Acaryochloris marina; D1, D2, and cytochrome b559 proteins; psbA, psbD, psbE, and psbF genes;
Cytotoxicity of a mitochondriotropic quercetin derivative: Mechanisms by Nicola Sassi; Lucia Biasutto; Andrea Mattarei; Massimo Carraro; Valentina Giorgio; Anna Citta; Paolo Bernardi; Spiridione Garbisa; Ildikò Szabò; Cristina Paradisi; Mario Zoratti (1095-1106).
The mitochondriotropic compound 7-O-(4-triphenylphosphoniumbutyl)quercetin iodide (Q-7BTPI) in the μM concentration range caused necrotic death of cultured cells by acting as a prooxidant, with generation of superoxide anion in the mitochondria. Externally added membrane-permeating superoxide dismutase or catalase largely prevented death. Rescue by permeant catalase indicates that the toxicant is H2O2, or reactive species derived from it. Rescue by permeant dismutase suggests the possibility of a chain mechanism of H2O2 production, in which dismutation of superoxide constitutes a termination step. Oxidative stress was due to the presence of free phenolic hydroxyls and to accumulation in mitochondria, since the analogous mitochondriotropic per-O-methylated compound -3,3′,4′,5-tetra-O-methyl,7-O-(4-triphenylphosphoniumbutyl) quercetin iodide (QTM-7BTPI)—or Quercetin itself induced no or little superoxide production and cell death. Q-7BTPI did not cause a significant perturbation of the mitochondrial transmembrane potential or of respiration in cells. On the other hand its presence led to inhibition of glutathione peroxidase, an effect expected to accentuate oxidative stress by interfering with the elimination of H2O2. An exogenous permeable glutathione precursor determined a strong increase of cellular glutathione levels but did not rescue the cells. Death induction was selective for fast-growing C-26 tumoral cells and mouse embryonic fibroblasts (MEFs) while sparing slow-growing MEFs. This suggests a possible use of Q-7BTPI as a chemotherapeutic agent.► A mitochondriotropic quercetin derivative induces necrosis of cultured cells. ► Cancerous / fast-growing cells are selectively killed. ► Cytotoxicity is associated with accumulation of the compound in mitochondria. ► Cell death is caused by the generation of Reactive Oxygen Species (ROS). ► The compound inhibits enzymes of the Glutathione system.
Keywords: Mitochondriotropic compounds; Mitocans; Quercetin; Necrosis; Reactive oxygen species; Glutathione;