RESUMEN
Iron porphyrins are among the most studied molecular catalysts for carbon dioxide (CO2 ) reduction and their reactivity is constantly being enhanced through the implementation of chemical functionalities in the second coordination sphere inspired by the active sites of enzymes. In this study, we were intrigued to observe that a multipoint hydrogen bonding scheme provided by embarked urea groups could also shift the redox activation step of CO2 from the well-admitted Fe(0) to the Fe(I) state. Using EPR, resonance Raman, IR and UV-Visible spectroscopies, we underpinned a two-electron activation step of CO2 starting from the Fe(I) oxidation state to form, after protonation, an Fe(III)-COOH species. The addition of another electron and a proton to the latter species converged to the cleavage of a C-O bond with the loss of water molecule resulting in an Fe(II)-CO species. DFT analyses of these postulated intermediates are in good agreement with our collected spectroscopic data, allowing us to propose an alternative pathway in the catalytic CO2 reduction with iron porphyrin catalyst. Such a remarkable shift opens new lines of research in the design of molecular catalysts to reach low overpotentials in performing multi-electronic CO2 reduction catalysis.
RESUMEN
Small CAB-like proteins (SCPs) are single-helix light-harvesting-like proteins found in all organisms performing oxygenic photosynthesis. We investigated the effect of growth in moderate salt stress on these stress-induced proteins in the cyanobacterium Synechocystis sp. PCC 6803 depleted of Photosystem I (PSI), which expresses SCPs constitutively, and compared these cells with a PSI-less/ScpABCDE- mutant. SCPs, by stabilizing chlorophyll-binding proteins and Photosystem II (PSII) assembly, protect PSII from photoinhibitory damages, and in their absence electrons accumulate and will lead to ROS formation. The presence of 0.2 M NaCl in the growth medium increased the respiratory activity and other PSII electron sinks in the PSI-less/ScpABCDE- strain. We postulate that this salt-induced effect consumes the excess of PSII-generated electrons, reduces the pressure of the electron transport chain, and thereby prevents 1O2 production.
Asunto(s)
Complejos de Proteína Captadores de Luz/metabolismo , Luz , Complejo de Proteína del Fotosistema II/metabolismo , Estrés Fisiológico/efectos de la radiación , Synechocystis/metabolismo , Synechocystis/efectos de la radiación , Carbono/metabolismo , Color , Concentración de Iones de Hidrógeno , Mutación/genética , Nitrógeno/metabolismo , Concentración Osmolar , Complejo de Proteína del Fotosistema I/metabolismo , Pigmentación/efectos de los fármacos , Pigmentos Biológicos/metabolismo , Oxígeno Singlete/metabolismo , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In the family of chlorophyll binding proteins, single helix small CAB-like proteins (SCPs) are found in all organisms performing oxygenic photosynthesis. Here, we investigated the function of these stress-inducible proteins in the cyanobacterium Synechocystis sp. PCC 6803. We compared physiological, proteome and transcriptome traits of a Photosystem I (PSI) deletion strain, which constitutively induces SCPs, and a PSI-less/ScpABCDE(-) without SCPs. The SCP mutant cells were larger in size, showed irregular thylakoid structure and differed in cell-surface morphology. Deletion of scp genes strongly affected the carbon (C) and nitrogen (N) balance, resulting in accumulation of carbohydrates and a decrease in N-rich compounds (proteins and chlorophyll). Data from transcriptomic and metabolomic experiments revealed a role of SCPs in the control of chlorophyll biosynthesis. Additionally, SCPs diminished formation of reactive oxygen species, thereby preventing damage within Photosystem II. We conclude that the lack of SCP-function to remove free chlorophyll under stress conditions has a large impact on the metabolism of the entire cell.
Asunto(s)
Carbono/metabolismo , Proteínas de Unión a Clorofila/fisiología , Nitrógeno/metabolismo , Synechocystis/metabolismo , Proteínas de Unión a Clorofila/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Homeostasis , Metabolómica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Photosynthetic organisms must respond to excess light in order to avoid photo-oxidative stress. In plants and green algae the fastest response to high light is non-photochemical quenching (NPQ), a process that allows the safe dissipation of the excess energy as heat. This phenomenon is triggered by the low luminal pH generated by photosynthetic electron transport. In vascular plants the main sensor of the low pH is the PsbS protein, while in the green alga Chlamydomonas reinhardtii LhcSR proteins appear to be exclusively responsible for this role. Interestingly, Chlamydomonas also possesses two PsbS genes, but so far the PsbS protein has not been detected and its biological function is unknown. Here, we reinvestigated the kinetics of gene expression and PsbS and LhcSR3 accumulation in Chlamydomonas during high light stress. We found that, unlike LhcSR3, PsbS accumulates very rapidly but only transiently. In order to determine the role of PsbS in NPQ and photoprotection in Chlamydomonas, we generated transplastomic strains expressing the algal or the Arabidopsis psbS gene optimized for plastid expression. Both PsbS proteins showed the ability to increase NPQ in Chlamydomonas wild-type and npq4 (lacking LhcSR3) backgrounds, but no clear photoprotection activity was observed. Quantification of PsbS and LhcSR3 in vivo indicates that PsbS is much less abundant than LhcSR3 during high light stress. Moreover, LhcSR3, unlike PsbS, also accumulates during other stress conditions. The possible role of PsbS in photoprotection is discussed.
Asunto(s)
Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Luz , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crecimiento & desarrollo , Chlamydomonas reinhardtii/efectos de la radiación , Clorofila/metabolismo , Medios de Cultivo , Fluorescencia , Regulación de la Expresión Génica/efectos de la radiación , Cinética , Complejos de Proteína Captadores de Luz/genética , Nitrógeno/deficiencia , Fenotipo , Complejo de Proteína del Fotosistema II/metabolismo , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de la radiaciónRESUMEN
State transitions are a phenomenon that maintains the excitation balance between photosystem II (PSII) and photosystem I (PSI-LHCI) by controlling their relative absorption cross-sections. Under light conditions exciting PSII preferentially, a trimeric LHCII antenna moves from PSII to PSI-LHCI to form the PSI-LHCI-LHCII supercomplex. In this work, the excited state dynamics in the PSI-LHCI and PSI-LHCI-LHCII supercomplexes isolated from Arabidopsis have been investigated by picosecond time-resolved fluorescence spectroscopy. The excited state decays were analysed using two approaches based on either (i) a sum of discrete exponentials or (ii) a continuous distribution of lifetimes. The results indicate that the energy transfer from LHCII to the bulk of the PSI antenna occurs with an average macroscopic transfer rate in the 35-65 ns-1 interval. Yet, the most satisfactory description of the data is obtained when considering a heterogeneous population containing two PSI-LHCI-LHCII supercomplexes characterised by a transfer time of â¼15 and â¼60 ns-1, likely due to the differences in the strength and orientation of LHCII harboured to PSI. Both these values are of the same order of magnitude of those estimated for the average energy transfer rates from the low energy spectral forms of LHCI to the bulk of the PSI antenna (15-40 ns-1), but they are slower than the transfer from the bulk antenna of PSI to the reaction centre (>150 ns-1), implying a relatively small kinetics bottleneck for the energy transfer from LHCII. Nevertheless, the kinetic limitation imposed by excited state diffusion has a negligible impact on the photochemical quantum efficiency of the supercomplex, which remains about 98% in the case of PSI-LHCI.
Asunto(s)
Arabidopsis/metabolismo , Transferencia de Energía , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Cinética , Luz , Complejo de Proteína del Fotosistema I/química , Espectrometría de Fluorescencia , Tilacoides/metabolismoRESUMEN
Cryptophytes like the cryptomonad Guillardia theta are part of the marine phytoplankton and therefore major players in global carbon and biogeochemical cycles. Despite the importance for the cell in being able to cope with large changes in illumination on a daily basis, very little is known about photoprotection mechanisms in cryptophytes. Here, we show that Guillardia theta is able to perform non-photochemical quenching, although none of the usual xanthophyll cycle pigments (e.g., zeaxanthin, diadinoxanthin, diatoxanthin) are present at detectable levels. Instead, acclimation to high light intensity seems to involve an increase of alloxanthin. Guillardia theta has genes for 2 one-helix "light-harvesting-like" proteins, related to some cyanobacterial genes which are induced in response to high light stress. Both the plastid-encoded gene (hlipP) and the nucleomorph-encoded gene (HlipNm) are expressed, but transcript levels decrease rather than increase during high light exposure, suggesting that they are not involved in a high light stress response. The HlipNm protein was detected with a specific antibody; expression was constant, independent of the light exposure.
Asunto(s)
Criptófitas/química , Luz , Fitoplancton/química , Proteínas de Plantas/química , Estructura Secundaria de Proteína , Estrés Fisiológico , Secuencia de Aminoácidos , Clorofila/química , Criptófitas/citología , Fluorescencia , Datos de Secuencia Molecular , Fotoquímica , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Fitoplancton/citología , Proteínas de Plantas/genética , Plastidios/genética , Alineación de SecuenciaRESUMEN
The five small CAB-like proteins (ScpA-E) of the cyanobacterium Synechocystis sp. PCC 6803 belong to the family of stress-induced light-harvesting-like proteins, but are constitutively expressed in a mutant deficient of Photosystem I (PSI). Using absorption, fluorescence and thermoluminescence measurements this PSI-less strain was compared with a mutant, in which all SCPs were additionally deleted. Depletion of SCPs led to structural rearrangements in Photosystem II (PSII): less photosystems were assembled; and in these, the Q(B) site was modified. Despite the lower amount of PSII, the SCP-deficient cells contained the same amount of phycobilisomes (PBS) as the control. Although the excess PBS were functionally disconnected, their fluorescence was quenched under high irradiance by the activated Orange Carotenoid Protein (OCP). Additionally the amount of OCP, but not of the iron-stress induced protein (isiA), was higher in this SCP-depleted mutant compared with the control. As previously described, the lack of SCPs affects the chlorophyll biosynthesis (Vavilin, D., Brune, D. C., Vermaas, W. (2005) Biochim Biophys Acta 1708, 91-101). We demonstrate that chlorophyll synthesis is required for efficient PSII repair and that it is partly impaired in the absence of SCPs. At the same time, the amount of chlorophyll also seems to influence the expression of ScpC and ScpD.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clorofila/biosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Synechocystis/metabolismo , Electroforesis en Gel de Poliacrilamida , Espectrometría de FluorescenciaRESUMEN
In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. The V185 of the D1 protein has been shown to be an important amino acid in PSII function (Dilbeck et al. Biochemistry 52 (2013) 6824-6833). Here, we have studied its role by making a V185T site-directed mutant in the thermophilic cyanobacterium Thermosynechococcus elongatus. The properties of the V185T-PSII have been compared to those of the WT*3-PSII by using EPR spectroscopy, polarography, thermoluminescence and time-resolved UV-visible absorption spectroscopy. It is shown that the V185 and the chloride binding site very likely interact via the H-bond network linking TyrZ and the halide. The V185 contributes to the stabilization of S2 into the low spin (LS), Sâ¯=â¯1/2, configuration. Indeed, in the V185T mutant a high proportion of S2 exhibits a high spin (HS), Sâ¯=â¯5/2, configuration. By using bromocresol purple as a dye, a proton release was detected in the S1TyrZâ¯ââ¯S2HSTyrZ transition in the V185T mutant in contrast to the WT*3-PSII in which there is no proton release in this transition. Instead, in WT*3-PSII, a proton release kinetically much faster than the S2LSTyrZâ¯ââ¯S3TyrZ transition was observed and we propose that it occurs in the S2LSTyrZâ¯ââ¯S2HSTyrZ intermediate step before the S2HSTyrZâ¯ââ¯S3TyrZ transition occurs. The dramatic slowdown of the S3TyrZâ¯ââ¯S0TyrZ transition in the V185T mutant does not originate from a structural modification of the Mn4CaO5 cluster since the spin Sâ¯=â¯3â¯S3 EPR signal is not modified in the mutant. More probably, it is indicative of the strong implication of V185 in the tuning of an efficient relaxation processes of the H-bond network and/or of the protein.
Asunto(s)
Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Valina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Mediciones Luminiscentes , Modelos Moleculares , Synechococcus/metabolismo , Factores de TiempoRESUMEN
The study of biochemical dynamics by ultrafast spectroscopic methods is often restricted by the limited amount of liquid sample available, while the high repetition rate of light sources can induce photodamage. In order to overcome these limitations, we designed a high flux, sub-ml, capillary flow-cell. While the 0.1 mm thin window of the 0.5 mm cross-section capillary ensures an optimal temporal resolution and a steady beam deviation, the cell-pump generates flows up to â¼0.35 ml/s that are suitable to pump laser repetition rates up to â¼14 kHz, assuming a focal spot-diameter of 100 µm. In addition, a decantation chamber efficiently removes bubbles and allows, via septum, for the addition of chemicals while preserving the closed atmosphere. The minimal useable amount of sample is â¼250 µl.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Synechocystis/citología , Tilacoides/metabolismoRESUMEN
Oxygenic photosynthesis is indispensable both for the development and maintenance of life on earth by converting light energy into chemical energy and by producing molecular oxygen and consuming carbon dioxide. This latter process has been responsible for reducing the CO2 from its very high levels in the primitive atmosphere to the present low levels and thus reducing global temperatures to levels conducive to the development of life. Photosystem I and photosystem II are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyse the primary photosynthetic endergonic reactions producing high energy compounds. Both photosystems are highly organised membrane supercomplexes composed of a core complex, containing the reaction centre where electron transport is initiated, and of a peripheral antenna system, which is important for light harvesting and photosynthetic activity regulation. If on the one hand both the chemical reactions catalysed by the two photosystems and their detailed structure are different, on the other hand they share many similarities. In this review we discuss and compare various aspects of the organisation, functioning and regulation of plant photosystems by comparing them for similarities and differences as obtained by structural, biochemical and spectroscopic investigations.
Asunto(s)
Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Plantas/enzimología , Plantas/metabolismoRESUMEN
Heme is a cofactor for proteins participating in many important cellular processes, including respiration, oxygen metabolism and oxygen binding. The key enzyme in the heme biosynthesis pathway is ferrochelatase (protohaem ferrolyase, EC 4.99.1.1), which catalyzes the insertion of ferrous iron into protoporphyrin IX. In higher plants, the ferrochelatase enzyme is localized not only in mitochondria, but also in chloroplasts. The plastidic type II ferrochelatase contains a C-terminal chlorophyll a/b (CAB) motif, a conserved hydrophobic stretch homologous to the CAB domain of plant light harvesting proteins and light-harvesting like proteins. This type II ferrochelatase, found in all photosynthetic organisms, is presumed to have evolved from the cyanobacterial ferrochelatase. Here we describe a detailed enzymological study on recombinant, refolded and functionally active type II ferrochelatase (FeCh) from the cyanobacterium Synechocystis sp. PCC 6803. A protocol was developed for the functional refolding and purification of the recombinant enzyme from inclusion bodies, without truncation products or soluble aggregates. The refolded FeCh is active in its monomeric form, however, addition of an N-terminal His(6)-tag has significant effects on its enzyme kinetics. Strikingly, removal of the C-terminal CAB-domain led to a greatly increased turnover number, k(cat), compared to the full length protein. While pigments isolated from photosynthetic membranes decrease the activity of FeCh, direct pigment binding to the CAB domain of FeCh was not evident.