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1.
Annu Rev Biochem ; 82: 577-606, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23527694

RESUMEN

Photosystem II (PSII) uses light energy to split water into chemical products that power the planet. The stripped protons contribute to a membrane electrochemical potential before combining with the stripped electrons to make chemical bonds and releasing O2 for powering respiratory metabolisms. In this review, we provide an overview of the kinetics and thermodynamics of water oxidation that highlights the conserved performance of PSIIs across species. We discuss recent advances in our understanding of the site of water oxidation based upon the improved (1.9-Å resolution) atomic structure of the Mn4CaO5 water-oxidizing complex (WOC) within cyanobacterial PSII. We combine these insights with recent knowledge gained from studies of the biogenesis and assembly of the WOC (called photoassembly) to arrive at a proposed chemical mechanism for water oxidation.


Asunto(s)
Calcio/química , Cianobacterias/metabolismo , Manganeso/química , Oxígeno/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Agua/química , Calcio/metabolismo , Cinética , Manganeso/metabolismo , Oxidación-Reducción , Oxígeno/química , Complejo de Proteína del Fotosistema II/química , Termodinámica , Agua/metabolismo
2.
Plant Physiol ; 191(4): 2229-2244, 2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36510848

RESUMEN

The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 µE m-2 s-1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.


Asunto(s)
Nicotiana , Complejo de Proteína del Fotosistema II , Complejo de Proteína del Fotosistema II/genética , Nicotiana/genética , Luz , Biomasa , Cloroplastos , Plantas
3.
Photosynth Res ; 2024 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-38329705

RESUMEN

The green algal genus Picochlorum is of biotechnological interest because of its robust response to multiple environmental stresses. We compared the metabolic performance of P. SE3 and P. oklahomense to diverse microbial phototrophs and observed exceptional performance of photosystem II (PSII) in light energy conversion in both Picochlorum species. The quantum yield (QY) for O2 evolution is the highest of any phototroph yet observed, 32% (20%) by P. SE3 (P. okl) when normalized to total PSII subunit PsbA (D1) protein, and 80% (75%) normalized per active PSII, respectively. Three factors contribute: (1) an efficient water oxidizing complex (WOC) with the fewest photochemical misses of any organism; (2) faster reoxidation of reduced (PQH2)B in P. SE3 than in P. okl. (period-2 Fourier amplitude); and (3) rapid reoxidation of the plastoquinol pool by downstream electron carriers (Cyt b6f/PETC) that regenerates PQ faster in P. SE3. This performance gain is achieved without significant residue changes around the QB site and thus points to a pull mechanism involving faster PQH2 reoxidation by Cyt b6f/PETC that offsets charge recombination. This high flux in P. SE3 may be explained by genomically encoded plastoquinol terminal oxidases 1 and 2, whereas P. oklahomense has neither. Our results suggest two distinct types of PSII centers exist, one specializing in linear electron flow and the other in PSII-cyclic electron flow. Several amino acids within D1 differ from those in the low-light-descended D1 sequences conserved in Viridiplantae, and more closely match those in cyanobacterial high-light D1 isoforms, including changes near tyrosine Yz and a water/proton channel near the WOC. These residue changes may contribute to the exceptional performance of Picochlorum at high-light intensities by increasing the water oxidation efficiency and the electron/proton flux through the PSII acceptors (QAQB).

4.
Photosynth Res ; 156(1): 113-128, 2023 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-36436152

RESUMEN

Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H+o/H+i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA-) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H+o/H+i) and the PQ pool redox poise via e-/H+ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q-H+)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P+QA-) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.


Asunto(s)
Complejo de Proteína del Fotosistema II , Plastoquinona , Complejo de Proteína del Fotosistema II/metabolismo , Transporte de Electrón , Plastoquinona/metabolismo , Electrones , Protones , Fotosíntesis/fisiología , Hidroquinonas , Oxidación-Reducción , Agua/química
5.
Photosynth Res ; 143(3): 287-299, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-31893333

RESUMEN

Lichens are a symbiosis between a fungus and one or more photosynthetic microorganisms that enables the symbionts to thrive in places and conditions they could not compete independently. Exchanges of water and sugars between the symbionts are the established mechanisms that support lichen symbiosis. Herein, we present a new linkage between algal photosynthesis and fungal respiration in lichen Flavoparmelia caperata that extends the physiological nature of symbiotic co-dependent metabolisms, mutually boosting energy conversion rates in both symbionts. Measurements of electron transport by oximetry show that photosynthetic O2 is consumed internally by fungal respiration. At low light intensity, very low levels of O2 are released, while photosynthetic electron transport from water oxidation is normal as shown by intrinsic chlorophyll variable fluorescence yield (period-4 oscillations in flash-induced Fv/Fm). The rate of algal O2 production increases following consecutive series of illumination periods, at low and with limited saturation at high light intensities, in contrast to light saturation in free-living algae. We attribute this effect to arise from the availability of more CO2 produced by fungal respiration of photosynthetically generated sugars. We conclude that the lichen symbionts are metabolically coupled by energy conversion through exchange of terminal electron donors and acceptors used in both photosynthesis and fungal respiration. Algal sugars and O2 are consumed by the fungal symbiont, while fungal delivered CO2 is consumed by the alga.


Asunto(s)
Dióxido de Carbono/metabolismo , Líquenes/metabolismo , Oxígeno/metabolismo , Fotosíntesis , Simbiosis , Aerobiosis/efectos de la radiación , Anaerobiosis/efectos de la radiación , Respiración de la Célula/efectos de la radiación , Clorofila/metabolismo , Electrodos , Transporte de Electrón/efectos de la radiación , Fluorescencia , Luz , Oxidación-Reducción , Fotosíntesis/efectos de la radiación , Simbiosis/efectos de la radiación , Factores de Tiempo , Agua/metabolismo
6.
Plant Cell Environ ; 43(4): 945-964, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31759337

RESUMEN

Algal-fungal symbionts share water, nutrients, and gases via an architecture unique to lichens. Because lichen activity is controlled by moisture dynamics, understanding water transport is prerequisite to understand their fundamental biology. We propose a model of water distributions within foliose lichens governed by laws of fluid motion. Our model differentiates between water stored in symbionts, on extracellular surfaces, and in distinct morphological layers. We parameterize our model with hydraulic properties inverted from laboratory measurements of Flavoparmelia caperata and validate for wetting and drying. We ask: (1) Where is the bottleneck to water transport? (2) How do hydration and dehydration dynamics differ? and (3) What causes these differences? Resistance to vapor flow is concentrated at thallus surfaces and acts as the bottleneck for equilibrium, while internal resistances are small. The model captures hysteresis in hydration and desiccation, which are shown to be controlled by nonlinearities in hydraulic capacitance. Muting existing nonlinearities slowed drying and accelerated wetting, while exaggerating nonlinearities accelerated drying and slowed wetting. The hydraulic nonlinearity of F. caperata is considerable, which may reflect its preference for humid and stable environments. The model establishes the physical foundation for future investigations of transport of water, gas, and sugar between symbionts.


Asunto(s)
Parmeliaceae/metabolismo , Deshidratación , Modelos Biológicos , Agua/metabolismo
7.
Physiol Plant ; 166(1): 165-180, 2019 May.
Artículo en Inglés | MEDLINE | ID: mdl-30693529

RESUMEN

High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water-oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane-inlet mass spectrometry and O2 -polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these 'PSII birth defects' in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de-etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O2 -polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, QB -inhibitor binding, and thermoluminescence studies indicate that the decline of the high-light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability QA - → QB during de-etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer-range energy transfer.


Asunto(s)
Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Cloroplastos/ultraestructura , Espectrometría de Masas , Microscopía Electrónica , Biogénesis de Organelos , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/ultraestructura
8.
Adv Exp Med Biol ; 1080: 171-213, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30091096

RESUMEN

With the demand for renewable energy growing, hydrogen (H2) is becoming an attractive energy carrier. Developing H2 production technologies with near-net zero carbon emissions is a major challenge for the "H2 economy." Certain cyanobacteria inherently possess enzymes, nitrogenases, and bidirectional hydrogenases that are capable of H2 evolution using sunlight, making them ideal cell factories for photocatalytic conversion of water to H2. With the advances in synthetic biology, cyanobacteria are currently being developed as a "plug and play" chassis to produce H2. This chapter describes the metabolic pathways involved and the theoretical limits to cyanobacterial H2 production and summarizes the metabolic engineering technologies pursued.


Asunto(s)
Cianobacterias , Hidrógeno/metabolismo , Ingeniería Metabólica/métodos , Biología Sintética/métodos , Cianobacterias/genética , Cianobacterias/metabolismo
9.
Biochim Biophys Acta Bioenerg ; 1858(11): 873-883, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28734933

RESUMEN

The desert microalga Chlorella ohadii was reported to grow at extreme light intensities with minimal photoinhibition, tolerate frequent de/re-hydrations, yet minimally employs antenna-based non-photochemical quenching for photoprotection. Here we investigate the molecular mechanisms by measuring Photosystem II charge separation yield (chlorophyll variable fluorescence, Fv/Fm) and flash-induced O2 yield to measure the contributions from both linear (PSII-LEF) and cyclic (PSII-CEF) electron flow within PSII. Cells grow increasingly faster at higher light intensities (µE/m2/s) from low (20) to high (200) to extreme (2000) by escalating photoprotection via shifting from PSII-LEF to PSII-CEF. This shifts PSII charge separation from plastoquinone reduction (PSII-LEF) to plastoquinol oxidation (PSII-CEF), here postulated to enable proton gradient and ATP generation that powers photoprotection. Low light-grown cells have unusually small antennae (332 Chl/PSII), use mainly PSII-LEF (95%) and convert 40% of PSII charge separations into O2 (a high O2 quantum yield of 0.06mol/mol PSII/flash). High light-grown cells have smaller antenna and lower PSII-LEF (63%). Extreme light-grown cells have only 42 Chl/PSII (no LHCII antenna), minimal PSII-LEF (10%), and grow faster than any known phototroph (doubling time 1.3h). Adding a synthetic quinone in excess to supplement the PQ pool fully uncouples PSII-CEF from its natural regulation and produces maximum PSII-LEF. Upon dark adaptation PSII-LEF rapidly reverts to PSII-CEF, a transient protection mechanism to conserve water and minimize the cost of antenna biosynthesis. The capacity of the electron acceptor pool (plastoquinone pool), and the characteristic times for exchange of (PQH2)B with PQpool and reoxidation of (PQH2)pool were determined.


Asunto(s)
Chlorella/metabolismo , Electrones , Microalgas/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Adenosina Trifosfato/biosíntesis , Chlorella/crecimiento & desarrollo , Chlorella/efectos de la radiación , Clorofila/metabolismo , Transporte de Electrón , Cinética , Luz , Microalgas/crecimiento & desarrollo , Microalgas/efectos de la radiación , Oxidación-Reducción , Oxígeno/metabolismo , Plastoquinona/análogos & derivados , Plastoquinona/metabolismo , Termodinámica , Tilacoides/metabolismo , Tilacoides/efectos de la radiación
10.
Biochim Biophys Acta ; 1857(9): 1550-1560, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27317268

RESUMEN

Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5-8× slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45°C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S=5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S=1/2 form.


Asunto(s)
Complejo de Proteína del Fotosistema II/metabolismo , Estroncio/farmacología , Agua/química , Transporte de Electrón , Compuestos de Manganeso/farmacología , Mutación , Oxidación-Reducción , Óxidos/farmacología , Oxígeno/análisis , Termodinámica
11.
Biochim Biophys Acta ; 1857(9): 1380-1391, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27117512

RESUMEN

We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB(-) and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB(-) acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.


Asunto(s)
Cianobacterias/metabolismo , Electrones , Microalgas/metabolismo , Oxígeno/análisis , Complejo de Proteína del Fotosistema II/metabolismo , Fotosíntesis , Plastoquinona/análogos & derivados , Plastoquinona/química
12.
Plant J ; 81(6): 947-60, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25645872

RESUMEN

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.


Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/genética , Almidón/metabolismo , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Biomasa , Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Transporte de Electrón , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Glucofosfatos/metabolismo , Luz , Metabolómica , Mutación , NADP/metabolismo , Oxidación-Reducción , Fenotipo , Fotosíntesis , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo
13.
Photosynth Res ; 128(2): 141-50, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26687161

RESUMEN

Oxygenic photosynthesis efficiency at increasing solar flux is limited by light-induced damage (photoinhibition) of Photosystem II (PSII), primarily targeting the D1 reaction center subunit. Some cyanobacteria contain two natural isoforms of D1 that function better under low light (D1:1) or high light (D1:2). Herein, rates and yields of photoassembly of the Mn4CaO5 water-oxidizing complex (WOC) from the free inorganic cofactors (Mn(2+), Ca(2+), water, electron acceptor) and apo-WOC-PSII are shown to differ significantly: D1:1 apo-WOC-PSII exhibits a 2.3-fold faster rate-limiting step of photoassembly and up to seven-fold faster rate to the first light-stable Mn(3+) intermediate, IM1*, but with a much higher rate of photoinhibition than D1:2. Conversely, D1:2 apo-WOC-PSII assembles slower but has up to seven-fold higher yield, achieved by a higher quantum yield of charge separation and slower photoinhibition rate. These results confirm and extend previous observations of the two holoenzymes: D1:2-PSII has a greater quantum yield of primary charge separation, faster [P680 (+) Q A (-) ] charge recombination and less photoinhibition that results in a slower rate and higher yield of photoassembly of its apo-WOC-PSII complex. In contrast, D1:1-PSII has a lower quantum yield of primary charge separation, a slower [P680 (+) Q A (-) ] charge recombination rate, and faster photoinhibition that together result in higher rate but lower yield of photoassembly at higher light intensities. Cyanobacterial PSII reaction centers that contain the high- and low-light D1 isoforms can tailor performance to optimize photosynthesis at varying light conditions, with similar consequences on their photoassembly kinetics and yield. These different efficiencies of photoassembly versus photoinhibition impose differential costs for biosynthesis as a function of light intensity.


Asunto(s)
Chlamydomonas reinhardtii/fisiología , Oxígeno/metabolismo , Fotosíntesis/efectos de la radiación , Complejo de Proteína del Fotosistema II/metabolismo , Agua/metabolismo , Chlamydomonas reinhardtii/efectos de la radiación , Luz , Oxidación-Reducción , Complejo de Proteína del Fotosistema II/efectos de la radiación , Isoformas de Proteínas
14.
Biotechnol Bioeng ; 113(5): 979-88, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26479976

RESUMEN

To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations--both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))--were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.


Asunto(s)
Proteínas Bacterianas/metabolismo , Fermentación , Nitrato-Reductasa/metabolismo , Synechococcus/metabolismo , Proteínas Bacterianas/genética , Técnicas de Inactivación de Genes , Hidrógeno/metabolismo , NAD/metabolismo , NADP/metabolismo , Nitrato-Reductasa/genética , Nitratos/metabolismo , Nitritos/metabolismo , Synechococcus/genética
15.
Proc Natl Acad Sci U S A ; 109(44): 17765-9, 2012 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-23071342

RESUMEN

Synchronization of the circadian clock in cyanobacteria with the day/night cycle proceeds without an obvious photoreceptor, leaving open the question of its specific mechanism. The circadian oscillator can be reconstituted in vitro, where the activities of two of its proteins, KaiA and KaiC, are affected by metabolites that reflect photosynthetic activity: KaiC phosphorylation is directly influenced by the ATP/ADP ratio, and KaiA stimulation of KaiC phosphorylation is blocked by oxidized, but not reduced, quinones. Manipulation of the ATP/ADP ratio can reset the timing of KaiC phosphorylation peaks in the reconstituted in vitro oscillator. Here, we show that pulses of oxidized quinones reset the cyanobacterial circadian clock both in vitro and in vivo. Onset of darkness causes an abrupt oxidation of the plastoquinone pool in vivo, which is in contrast to a gradual decrease in the ATP/ADP ratio that falls over the course of hours until the onset of light. Thus, these two metabolic measures of photosynthetic activity act in concert to signal both the onset and duration of darkness to the cyanobacterial clock.


Asunto(s)
Relojes Circadianos , Cianobacterias/fisiología , Oscuridad , Quinonas/metabolismo , Transducción de Señal , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Péptidos y Proteínas de Señalización del Ritmo Circadiano/química , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Modelos Moleculares , Oxidación-Reducción , Fosforilación
16.
J Biol Chem ; 288(8): 5451-62, 2013 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-23271739

RESUMEN

Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn(4)CaO(5) core of the water-oxidizing complex (WOC). Most cyanobacteria have 3-5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O(2) yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O(2) than either cyanobacterial D1 isoform. D1:2-PSII makes more O(2) per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S(3) state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.


Asunto(s)
Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/fisiología , Biomasa , Chlamydomonas/metabolismo , Clorofila/metabolismo , Cianobacterias/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Evolución Molecular , Análisis de Fourier , Variación Genética , Cinética , Luz , Mutación , Oxidación-Reducción , Oxígeno/química , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Isoformas de Proteínas , Energía Solar , Tilacoides/metabolismo
17.
Biochim Biophys Acta ; 1827(7): 861-8, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23643726

RESUMEN

Forty-three years ago, Kok and coworkers introduced a phenomenological model describing period-four oscillations in O2 flash yields during photosynthetic water oxidation (WOC), which had been first reported by Joliot and coworkers. The original two-parameter Kok model was subsequently extended in its level of complexity to better simulate diverse data sets, including intact cells and isolated PSII-WOCs, but at the expense of introducing physically unrealistic assumptions necessary to enable numerical solutions. To date, analytical solutions have been found only for symmetric Kok models (inefficiencies are equally probable for all intermediates, called "S-states"). However, it is widely accepted that S-state reaction steps are not identical and some are not reversible (by thermodynamic restraints) thereby causing asymmetric cycles. We have developed a mathematically more rigorous foundation that eliminates unphysical assumptions known to be in conflict with experiments and adopts a new experimental constraint on solutions. This new algorithm termed STEAMM for S-state Transition Eigenvalues of Asymmetric Markov Models enables solutions to models having fewer adjustable parameters and uses automated fitting to experimental data sets, yielding higher accuracy and precision than the classic Kok or extended Kok models. This new tool provides a general mathematical framework for analyzing damped oscillations arising from any cycle period using any appropriate Markov model, regardless of symmetry. We illustrate applications of STEAMM that better describe the intrinsic inefficiencies for photon-to-charge conversion within PSII-WOCs that are responsible for damped period-four and period-two oscillations of flash O2 yields across diverse species, while using simpler Markov models free from unrealistic assumptions.


Asunto(s)
Cadenas de Markov , Modelos Teóricos , Oxígeno/metabolismo , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/química , Agua/química , Algoritmos , Catálisis , Modelos Químicos , Termodinámica , Agua/metabolismo
18.
J Am Chem Soc ; 136(10): 4048-55, 2014 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-24548276

RESUMEN

The D1 protein of Photosystem II (PSII) provides most of the ligating amino acid residues for the Mn4CaO5 water-oxidizing complex (WOC) and half of the reaction center cofactors, and it is present as two isoforms in the cyanobacterium Synechococcus elongatus PCC 7942. These isoforms, D1:1 and D1:2, confer functional advantages for photosynthetic growth at low and high light intensities, respectively. D1:1, D1:2, and seven point mutations in the D1:2 background that are native to D1:1 were expressed in the green alga Chlamydomonas reinhardtii. We used these nine strains to show that those strains that confer a higher yield of PSII charge separation under light-limiting conditions (where charge recombination is significant) have less efficient photochemical turnover, measured in terms of both a lower WOC turnover probability and a longer WOC cycle period. Conversely, these same strains under light saturation (where charge recombination does not compete) confer a correspondingly faster O2 evolution rate and greater protection against photoinhibition. Taken together, the data clearly establish that PSII primary charge separation is a trade-off between photochemical productivity (water oxidation and plastoquinone reduction) and charge recombination (photoprotection). These trade-offs add up to a significant growth advantage for the two natural isoforms. These insights provide fundamental design principles for engineering of PSII reaction centers with optimal photochemical efficiencies for growth at low versus high light intensities.


Asunto(s)
Proteínas Bacterianas/genética , Chlamydomonas reinhardtii/genética , Complejo de Proteína del Fotosistema II/genética , Ingeniería de Proteínas , Synechococcus/genética , Proteínas Bacterianas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Expresión Génica , Luz , Oxígeno/metabolismo , Fotoquímica , Complejo de Proteína del Fotosistema II/metabolismo , Mutación Puntual , Synechococcus/metabolismo
19.
J Biol Chem ; 287(4): 2777-86, 2012 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-22128188

RESUMEN

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.


Asunto(s)
Proteínas Bacterianas/metabolismo , Cyanothece/metabolismo , Hidrógeno/metabolismo , Hidrogenasas/metabolismo , Nitrogenasa/metabolismo , Proteínas Bacterianas/genética , Cyanothece/genética , Hidrogenasas/genética , Nitrogenasa/genética , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema I/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo
20.
Biochim Biophys Acta Bioenerg ; 1864(4): 148998, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37499962

RESUMEN

Chloride (Cl-) is essential for O2 evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* â†’ P+QA-), rates of water oxidation steps (S-states), rate of proton evolution, and flash O2 yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O2 evolution rate (-20 %) and proton release (-36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S2 â†’ S3 â†’ S0') slow with increasing light intensity and during O2/water exchange (S0' â†’ S0 â†’ S1), while the non-protonogenic S1 â†’ S2 transition is kinetically unaffected. As flash rate increases in Cl- cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br-. The Cl- advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H3O+Cl- in dry water channels vs. separated Br- and H+ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br- cultures for both proton evolution and less PSII charge recombination. In Br- cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br- with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.


Asunto(s)
Cloruros , Protones , Bromuros , Cinética , Agua/química , Complejo de Proteína del Fotosistema II/metabolismo
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