RESUMO
The redox state of the plastoquinone (PQ) pool is a known sensor for retrograde signaling. In this paper, we asked, "does the redox state of the PQ pool modulate the saturation state of thylakoid lipids?" Data from fatty acid composition and mRNA transcript abundance analyses suggest a strong connection between these two aspects in a model marine diatom. Fatty acid profiles of Phaeodactylum tricornutum exhibited specific changes when the redox state of the PQ pool was modulated by light and two chemical inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)]. Data from liquid chromatography with tandem mass spectrometry (LC-MS/MS) indicated a ca. 7-20% decrease in the saturation state of all four conserved thylakoid lipids in response to an oxidized PQ pool. The redox signals generated from an oxidized PQ pool in plastids also increased the mRNA transcript abundance of nuclear-encoded C16 fatty acid desaturases (FADs), with peak upregulation on a timescale of 6 to 12 h. The connection between the redox state of the PQ pool and thylakoid lipid saturation suggests a heretofore unrecognized retrograde signaling pathway that couples photosynthetic electron transport and the physical state of thylakoid membrane lipids.
Assuntos
Diatomáceas , Plastoquinona , Benzoquinonas , Cromatografia Líquida , Diatomáceas/metabolismo , Dibromotimoquinona/metabolismo , Diurona/farmacologia , Transporte de Elétrons , Ácidos Graxos Dessaturases/análise , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/análise , Luz , Lipídeos , Oxirredução , Plastoquinona/metabolismo , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Tilacoides/metabolismoRESUMO
Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Ligação a DNA/genética , Galactosiltransferases/genética , Proteínas de Choque Térmico/genética , Estresse Oxidativo , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Dibromotimoquinona/metabolismo , Dissacarídeos/biossíntese , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Fatores de Transcrição de Choque Térmico , Peróxido de Hidrogênio/farmacologia , Plantas Geneticamente Modificadas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/fisiologia , Ativação TranscricionalRESUMO
The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na(+)-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na(+)-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na(+)-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na(+)-NQR.
Assuntos
Proteínas de Bactérias/química , Dibromotimoquinona/química , Hidroxiquinolinas/química , Quinona Redutases/química , Vibrio cholerae/enzimologia , Domínio Catalítico , Dibromotimoquinona/metabolismo , Hidroxiquinolinas/metabolismo , Espectroscopia de Ressonância Magnética , NAD/química , NAD/metabolismo , Subunidades Proteicas , Quinona Redutases/antagonistas & inibidores , Quinona Redutases/metabolismo , Ubiquinona/química , Ubiquinona/metabolismoRESUMO
The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism. Here, we report that KaiA also binds the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the oxidized form of DBMIB, but not its reduced form, decreases the stability of KaiA in vivo, causes multimerization in vitro, and blocks KaiA stimulation of KaiC phosphorylation, which is central to circadian oscillation. Our data suggest that KaiA directly senses environmental signals as changes in redox state and modulates the circadian clock.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Synechococcus/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Ritmo Circadiano/fisiologia , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Dibromotimoquinona/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Fosforilação , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Synechococcus/genéticaRESUMO
Circadian rhythms are endogenous cellular programs that time metabolic and behavioral events to occur at optimal times in the daily cycle. Light and dark cycles synchronize the endogenous clock with the external environment through a process called entrainment. Previously, we identified the bacteriophytochrome-like circadian input kinase CikA as a key factor for entraining the clock in the cyanobacterium Synechococcus elongatus PCC 7942. Here, we present evidence that CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms, varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC during resetting of circadian phase by a dark pulse. The abundance of CikA varies inversely with light intensity, and its stability decreases in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The pseudo-receiver domain of CikA is crucial for sensitivity to DBMIB, and it binds the quinone directly, a demonstration of a previously unrecognized ligand-binding role for the receiver fold. Our results suggest that resetting the clock in S. elongatus is metabolism-dependent and that it is accomplished through the interaction of the circadian oscillator with CikA.
Assuntos
Proteínas de Bactérias/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Cianobactérias/efeitos dos fármacos , Cianobactérias/metabolismo , Dibromotimoquinona/farmacologia , Proteínas Quinases/metabolismo , Proteínas de Bactérias/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Cianobactérias/genética , Dibromotimoquinona/química , Dibromotimoquinona/metabolismo , Regulação Bacteriana da Expressão Gênica , Luz , Espectroscopia de Ressonância Magnética , Peso Molecular , Oxirredução , Fosforilação , Ligação Proteica , Proteínas Quinases/química , Sensibilidade e EspecificidadeRESUMO
Details are presented of the structural analysis of the cytochrome b(6)f complex from the thermophilic cyanobacterium, Mastigocladus laminosus, in the presence of the electrochemically positive (p)-side quinone analogue inhibitor, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). One DBMIB binding site was found. This site is peripheral to the quinone binding space defined by the binding sites of other p-side inhibitors previously resolved in cytochrome bc(1)/b(6)f complexes. This high-affinity site resides in a p-side interfacial niche bounded by cytochrome f, subunit IV, and cytochrome b(6), is close (8 A) to the p-side heme b, but distant (19 A) from the [2Fe-2S] cluster. No significant electron density associated with the DBMIB was found elsewhere in the structure. However, the site at which DBMIB can inhibit light-induced redox turnover is within a few A of the [2Fe-2S] cluster, as shown by the absence of inhibition in mutants of Synechococcus sp. PCC 7002 at iron sulfur protein-Leu-111 near the cluster. The ability of a minimum amount of initially oxidized DBMIB to inhibit turnover of WT complex after a second light flash implies that there is a light-activated movement of DBMIB from the distal peripheral site to an inhibitory site proximal to the [2Fe-2S] cluster. Together with the necessary passage of quinone/quinol through the small Q(p) portal in the complex, it is seen that transmembrane traffic of quinone-like molecules through the core of cytochrome bc complexes can be labyrinthine.
Assuntos
Cianobactérias/química , Complexo Citocromos b6f/química , Dibromotimoquinona/química , Modelos Químicos , Modelos Moleculares , Sítios de Ligação/genética , Cristalografia , Complexo Citocromos b6f/metabolismo , Dibromotimoquinona/metabolismo , Eletroquímica , Luz , Transporte Proteico/fisiologia , Análise EspectralRESUMO
Light modulation of the ability of three artificial quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duroquinone), to quench chlorophyll (Chl) fluorescence photochemically or non-photochemically was studied to simulate the functions of endogenous plastoquinones during the thermal phase of fast Chl fluorescence induction kinetics. DBMIB was found to suppress by severalfold the basal level of Chl fluorescence (F(o)) and to markedly retard the light-induced rise of variable fluorescence (F(v)). After irradiation with actinic light, Chl fluorescence rapidly dropped down to the level corresponding to F(o) level in untreated thylakoids and then slowly declined to the initial level. DBMIB was found to be an efficient photochemical quencher of energy in Photosystem II (PSII) in the dark, but not after prolonged irradiation. Those events were owing to DBMIB reduction under light and its oxidation in the dark. At high concentrations, DCBQ exhibited quenching behaviours similar to those of DBMIB. In contrast, duroquinone demonstrated the ability to quench F(v) at low concentration, while F(o) was declined only at high concentrations of this artificial quinone. Unlike for DBMIB and DCBQ, quenched F(o) level was attained rapidly after actinic light had been turned off in the presence of high duroquinone concentrations. That finding evidenced that the capacity of duroquinone to non-photochemically quench excitation energy in PSII was maintained during irradiation, which is likely owing to the rapid electron transfer from duroquinol to Photosystem I (PSI). It was suggested that DBMIB and DCBQ at high concentration, on the one hand, and duroquinone, on the other hand, mimic the properties of plastoquinones as photochemical and non-photochemical quenchers of energy in PSII under different conditions. The first model corresponds to the conditions under which the plastoquinone pool can be largely reduced (weak electron release from PSII to PSI compared to PSII-driven electron flow from water under strong light and weak PSI photochemical capacity because of inactive electron transport on its reducing side), while the second one mimics the behaviour of the plastoquinone pool when it cannot be filled up with electrons (weak or moderate light and high photochemical competence of PSI).
Assuntos
Cloroplastos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Quinonas/metabolismo , Benzoquinonas/metabolismo , Clorofila/química , Cloroplastos/química , Cloroplastos/efeitos da radiação , Escuridão , Dibromotimoquinona/metabolismo , Metabolismo Energético , Fluorescência , Membranas Intracelulares/metabolismo , Cinética , Luz , Complexos de Proteínas Captadores de Luz , Fotoquímica , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/efeitos da radiação , Complexo de Proteína do Fotossistema I , Complexo de Proteína do Fotossistema II , Plastoquinona/metabolismoRESUMO
The interaction of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with the Rieske protein of the chloroplast b6f complex has been studied by EPR. All three redox states of DBMIB were found to interact with the iron-sulphur cluster. The presence of the oxidised form of DBMIB altered the equilibrium distribution of the Rieske protein's conformational substates, strongly favouring the proximal position close to heme bL. In addition to this conformational effect, DBMIB shifted the pK-value of the redox-linked proton involved in the iron-sulphur cluster's redox transition by about 1.5 pH units towards more acidic values. The implications of these results with respect to the interaction of the native quinone substrate and the Rieske cluster in cytochrome bc complexes are discussed.
Assuntos
Cloroplastos/metabolismo , Grupo dos Citocromos b/metabolismo , Dibromotimoquinona/metabolismo , Complexo III da Cadeia de Transporte de Elétrons , Proteínas Ferro-Enxofre/metabolismo , Ácido Ascórbico/metabolismo , Sítios de Ligação , Grupo dos Citocromos b/antagonistas & inibidores , Complexo Citocromos b6f , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas Ferro-Enxofre/antagonistas & inibidores , Proteínas Ferro-Enxofre/química , Oxirredução , PrótonsRESUMO
When the isolated reaction centre of Photosystem II, reconstituted with the quinone, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), is exposed to photoinhibitory illumination, a D1-polypeptide breakdown product of 24 kDa is detected by immunoblotting. In addition, weaker bands are also detected at 17, 13 and 10 kDa. It is suggested that the 24 kDa D1-polypeptide breakdown product is the same as that first observed in vivo by Greenberg et al. (1987) EMBO J. 6, 2865-2869. Its appearance in isolated Photosystem II reaction centres requires the presence of an electron acceptor, but occurs under both aerobic and anaerobic conditions. In our in vitro experimental system the photoinduced degradation of the D1-polypeptide to the 24 kDa fragment was related to the functional activity of the reaction centre and the enzymatic nature of the proteolysis was characterised by a pH optimum of about 8.0 and by inhibition with proteinase inhibitors, especially the serine-type soybean trypsin inhibitor. The results support our earlier findings (Shipton and Barber (1991) Proc. Natl. Acad. Sci. USA 88, 6691-6695) that the appearance of the light-induced D1-polypeptide breakdown pattern of fragments occurs as a consequence of donor side photoinhibition when highly oxidising species accumulate in the reaction centre and bring about pigment oxidation and degradation. We suggest that it is this selective loss of pigments that induces a conformational change in the D1-polypeptide which triggers its autoproteolytic cleavage.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Western Blotting , Grupo dos Citocromos b/metabolismo , Dibromotimoquinona/metabolismo , Eletroforese em Gel de Poliacrilamida , Fabaceae/enzimologia , Fabaceae/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Oxirredução , Fotoquímica , Complexo de Proteína do Fotossistema II , Plantas MedicinaisRESUMO
The binding of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to chloroplast thylakoid membranes was investigated by analyzing the inhibition of electron transfer by DBMIB according to a steady-state rate relationship for enzyme-catalyzed reactions in the presence of tightly binding reversible inhibitors. DBMIB interacts with the cytochrome b6f complex in a manner best described by an apparent dissociation constant near 6 nM. The binding site titer is 1 mmol X mol chlorophyll-1. This number of DBMIB binding sites approaches one-half the number of cytochrome b6f complexes present in the membrane. These data suggest that the cytochrome b6f complex may function in electron transfer as a dimer, plastoquinol oxidation being totally inhibited by the binding of a single DBMIB molecule to the dimer.
Assuntos
Cloroplastos/metabolismo , Grupo dos Citocromos b/metabolismo , Dibromotimoquinona/metabolismo , Quinonas/metabolismo , Cloroplastos/efeitos dos fármacos , Grupo dos Citocromos b/antagonistas & inibidores , Complexo Citocromos b6f , Dibromotimoquinona/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Oxirredução/efeitos dos fármacos , PlantasRESUMO
We have investigated in detail the effects of dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on the ubiquinol-cytochrome c reductase (cytochrome bc1 complex) from bovine heart mitochondria. The inhibitory action of DBMIB on the steady-state activity of the bc1 complex is related to the specific binding of the quinone to the purified enzymatic complex. At concentrations higher than 10 mol per mol of the enzyme, DBMIB is able to stimulate an antimycin-insensitive reduction of cytochrome c catalyzed by the bc1 complex. In accordance with kinetic data showing a competition by endogenous ubiquinone in the inhibitory action, DBMIB can be considered as a product-like inhibitor of the ubiquinol-cytochrome c reductase activity. The site of specific binding of dibromothymoquinone in the bc1 complex enables it to interact with the iron-sulphur center of the enzyme, as indicated by changes induced in the EPR spectrum of the center. However, the inhibitor also directly interacts with cytochrome b, promoting a fast chemical oxidation of the reduced heme center. In spite of these effects, DBMIB has been found not to exert significant effects on the first turnover of the fully oxidized bc1 complex, as monitored by the rapid reduction of both cytochromes b and c1 by ubiquinol-1. In the presence of antimycin, only a stimulation of cytochrome c1 reduction, in parallel to an enhanced cytochrome b reoxidation, is observed. Moreover, DBMIB does not affect the oxidant-induced extra cytochrome b reduction in the presence of antimycin. On the basis of the evidences suggesting a competition with the endogenous ubiquinone in the redox cycle of the bc1 complex, a model is proposed for the mechanism of DBMIB inhibition. Such model can also explain at the molecular level the redox bypass induced by dibromothymoquinone in the whole respiratory chain (Degli Esposti, M., Rugolo, M. and Lenaz, G. (1983) FEBS Lett. 156, 15-19).