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1.
Plant Physiol ; 191(3): 1803-1817, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36516417

RESUMO

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.


Assuntos
Arabidopsis , Complexo Citocromos b6f , Complexo Citocromos b6f/genética , Complexo Citocromos b6f/metabolismo , Prótons , Elétrons , NADP/metabolismo , Transporte de Elétrons/fisiologia , Fotossíntese/genética , Oxirredução , Arabidopsis/genética , Arabidopsis/metabolismo , Trifosfato de Adenosina/metabolismo , Oxigênio/metabolismo
2.
Plant Physiol ; 189(1): 329-343, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35157085

RESUMO

Linear electron flow (LEF) and cyclic electron flow (CEF) compete for light-driven electrons transferred from the acceptor side of photosystem I (PSI). Under anoxic conditions, such highly reducing electrons also could be used for hydrogen (H2) production via electron transfer between ferredoxin and hydrogenase in the green alga Chlamydomonas reinhardtii. Partitioning between LEF and CEF is regulated through PROTON-GRADIENT REGULATION5 (PGR5). There is evidence that partitioning of electrons also could be mediated via PSI remodeling processes. This plasticity is linked to the dynamics of PSI-associated light-harvesting proteins (LHCAs) LHCA2 and LHCA9. These two unique light-harvesting proteins are distinct from all other LHCAs because they are loosely bound at the PSAL pole. Here, we investigated photosynthetic electron transfer and H2 production in single, double, and triple mutants deficient in PGR5, LHCA2, and LHCA9. Our data indicate that lhca2 and lhca9 mutants are efficient in photosynthetic electron transfer, that LHCA2 impacts the pgr5 phenotype, and that pgr5/lhca2 is a potent H2 photo-producer. In addition, pgr5/lhca2 and pgr5/lhca9 mutants displayed substantially different H2 photo-production kinetics. This indicates that the absence of LHCA2 or LHCA9 impacts H2 photo-production independently, despite both being attached at the PSAL pole, pointing to distinct regulatory capacities.


Assuntos
Elétrons , Complexo de Proteína do Fotossistema I , Transporte de Elétrons , Hidrogênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Prótons , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo
3.
Biochem J ; 479(1): 111-127, 2022 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-34981811

RESUMO

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.


Assuntos
Antimicina A/farmacologia , Chlamydomonas reinhardtii/enzimologia , Complexo Citocromos b6f/metabolismo , Elétrons , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tilacoides/enzimologia , Antimicina A/metabolismo , Complexo Citocromos b6f/antagonistas & inibidores , Transporte de Elétrons/efeitos dos fármacos , Ativação Enzimática , Ferredoxinas/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Fosforilação/efeitos dos fármacos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Plastoquinona/metabolismo , Quinona Redutases/metabolismo
4.
Plant J ; 103(3): 1140-1154, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32365245

RESUMO

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.


Assuntos
Cloroplastos/metabolismo , Glutationa Redutase/metabolismo , Fotossíntese , Espécies Reativas de Oxigênio/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Bryopsida/enzimologia , Bryopsida/metabolismo , Bryopsida/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Cloroplastos/enzimologia , Cloroplastos/fisiologia , Técnicas de Inativação de Genes , Glutationa/metabolismo , Glutationa Redutase/fisiologia , Oxirredução
5.
Biochem J ; 477(9): 1631-1650, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32267468

RESUMO

Proton gradient regulation 5 (PGR5) is involved in the control of photosynthetic electron transfer, but its mechanistic role is not yet clear. Several models have been proposed to explain phenotypes such as a diminished steady-state proton motive force (pmf) and increased photodamage of photosystem I (PSI). Playing a regulatory role in cyclic electron flow (CEF) around PSI, PGR5 contributes indirectly to PSI protection by enhancing photosynthetic control, which is a pH-dependent down-regulation of electron transfer at the cytochrome b6f complex (b6f). Here, we re-evaluated the role of PGR5 in the green alga Chlamydomonas reinhardtii and conclude that pgr5 possesses a dysfunctional b6f. Our data indicate that the b6f low-potential chain redox activity likely operated in two distinct modes - via the canonical Q cycle during linear electron flow and via an alternative Q cycle during CEF, which allowed efficient oxidation of the low-potential chain in the WT b6f. A switch between the two Q cycle modes was dependent on PGR5 and relied on unknown stromal electron carrier(s), which were a general requirement for b6f activity. In CEF-favoring conditions, the electron transfer bottleneck in pgr5 was the b6f, in which insufficient low-potential chain redox tuning might account for the mutant pmf phenotype. By attributing a ferredoxin-plastoquinone reductase activity to the b6f and investigating a PGR5 cysteine mutant, a current model of CEF is challenged.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Complexo Citocromos b6f/metabolismo , Transporte de Elétrons/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Oxirredução , Fotossíntese/fisiologia , Força Próton-Motriz
6.
Plant Physiol ; 179(2): 630-639, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30498023

RESUMO

Whereas photosynthetic function under steady-state light conditions has been well characterized, little is known about its changes that occur in response to light fluctuations. Chlororespiration, a simplified respiratory chain, is widespread across all photosynthetic lineages, but its role remains elusive. Here, we show that chlororespiration plays a crucial role in intermittent-light conditions in the green alga Chlamydomonas reinhardtii Chlororespiration, which is localized in thylakoid membranes together with the photosynthetic electron transfer chain, involves plastoquinone reduction and plastoquinol oxidation by a Plastid Terminal Oxidase (PTOX). We show that PTOX activity is critical for growth under intermittent light, with severe growth defects being observed in a mutant lacking PTOX2, the major plastoquinol oxidase. We demonstrate that the hampered growth results from a major change in the kinetics of redox relaxation of the photosynthetic electron transfer chain during the dark periods. This change, in turn, has a dramatic effect on the physiology of photosynthesis during the light periods, notably stimulating cyclic electron flow at the expense of the linear electron flow.


Assuntos
Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Chlamydomonas reinhardtii/genética , Complexo Citocromos b6f/metabolismo , Escuridão , Transporte de Elétrons , Luz , Mutação , Oxirredução , Oxirredutases/genética , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/genética , Plastoquinona/análogos & derivados , Plastoquinona/metabolismo , Tilacoides/metabolismo , Regulação para Cima
7.
J Biol Chem ; 293(45): 17559-17573, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30228184

RESUMO

The supramolecular organization of membrane proteins (MPs) is sensitive to environmental changes in photosynthetic organisms. Isolation of MP supercomplexes from the green algae Chlamydomonas reinhardtii, which are believed to contribute to cyclic electron flow (CEF) between the cytochrome b6f complex (Cyt-b6f) and photosystem I (PSI), proved difficult. We were unable to isolate a supercomplex containing both Cyt-b6f and PSI because in our hands, most of Cyt-b6f did not comigrate in sucrose density gradients, even upon using chemical cross-linkers or amphipol substitution of detergents. Assisted by independent affinity purification and MS approaches, we utilized disintegrating MP assemblies and demonstrated that the algae-specific CEF effector proteins PETO and ANR1 are bona fide Cyt-b6f interactors, with ANR1 requiring the presence of an additional, presently unknown, protein. We narrowed down the Cyt-b6f interface, where PETO is loosely attached to cytochrome f and to a stromal region of subunit IV, which also contains phosphorylation sites for the STT7 kinase.


Assuntos
Chlamydomonas reinhardtii/enzimologia , Complexo Citocromos b6f/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Chlamydomonas reinhardtii/genética , Complexo Citocromos b6f/genética , Complexo de Proteína do Fotossistema I/genética
8.
Biochim Biophys Acta Bioenerg ; 1858(12): 966-974, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28890176

RESUMO

The chloroplast F1Fo-ATP synthase (CF1Fo) drives ATP synthesis and the reverse reaction of ATP hydrolysis. The enzyme evolved in a cellular environment where electron transfer processes and molecular oxygen are abundant, and thiol modulation in the γ-subunit via thioredoxin is important for its ATPase activity regulation. Especially under high light, oxygen can be reduced and forms reactive oxygen species (ROS) which can oxidize CF1Fo among various other biomolecules. Mutation of the conserved ROS targets resulted in a tolerant enzyme, suggesting that ROS might play a regulatory role. The mutations had several side effects in vitro, including disturbance of the ATPase redox regulation [F. Buchert et al., Biochim. Biophys. Acta, 1817 (2012) 2038-2048]. This would prevent disentanglement of thiol- and ROS-specific modes of regulation. Here, we used the F1 catalytic core in vitro to identify a point mutant with a functional ATPase redox regulation and increased H2O2 tolerance. In the next step, the mutation was introduced into Chlamydomonas reinhardtii CF1Fo, thereby allowing us to study the physiological role of ROS regulation of the enzyme in vivo. We demonstrated in high light experiments that CF1Fo ROS targets were involved in the significant inhibition of ATP synthesis rates. Molecular events upon modification of CF1Fo by ROS will be considered.


Assuntos
Chlamydomonas reinhardtii/enzimologia , Cloroplastos/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Translocadoras de Prótons/genética , ATPases de Cloroplastos Translocadoras de Prótons/química , ATPases de Cloroplastos Translocadoras de Prótons/genética , Peróxido de Hidrogênio/química , ATPases Mitocondriais Próton-Translocadoras/química , Oxirredução , Mutação Puntual/genética , ATPases Translocadoras de Prótons/química , Espécies Reativas de Oxigênio/metabolismo , Tiorredoxinas/genética
9.
Biochim Biophys Acta ; 1847(4-5): 441-450, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25660164

RESUMO

The soluble F1 complex of ATP synthase (FoF1) is capable of ATP hydrolysis, accomplished by the minimum catalytic core subunits α3ß3γ. A special feature of cyanobacterial F1 and chloroplast F1 (CF1) is an amino acid sequence inserted in the γ-subunit. The insertion is extended slightly into the CF1 enzyme containing two additional cysteines for regulation of ATPase activity via thiol modulation. This molecular switch was transferred to a chimeric F1 by inserting the cysteine-containing fragment from spinach CF1 into a cyanobacterial γ-subunit [Y. Kim et al., redox regulation of rotation of the cyanobacterial F1-ATPase containing thiol regulation switch, J Biol Chem, 286 (2011) 9071-9078]. Under oxidizing conditions, the obtained F1 tends to lapse into an ADP-inhibited state, a common regulation mechanism to prevent wasteful ATP hydrolysis under unfavorable circumstances. However, the information flow between thiol modulation sites on the γ-subunit and catalytic sites on the ß-subunits remains unclear. Here, we clarified a possible interplay for the CF1-ATPase redox regulation between structural elements of the ßDELSEED-loop and the γ-subunit neck region, i.e., the most convex part of the α-helical γ-termini. Critical residues were assigned on the ß-subunit, which received the conformation change signal produced by disulfide/dithiol formation on the γ-subunit. Mutant response to the ATPase redox regulation ranged from lost to hypersensitive. Furthermore, mutant cross-link experiments and inversion of redox regulation indicated that the γ-redox state might modulate the subunit interface via reorientation of the ßDELSEED motif region.


Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/química , Cloroplastos/enzimologia , Cianobactérias/enzimologia , ATPases Translocadoras de Prótons/química , Proteínas Recombinantes de Fusão/química , Spinacia oleracea/química , Compostos de Sulfidrila/química , Trifosfato de Adenosina/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/genética , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Escherichia coli/genética , Hidrólise , Mutação/genética , Oxirredução , Conformação Proteica , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spinacia oleracea/genética , Spinacia oleracea/metabolismo , Relação Estrutura-Atividade
10.
Plants (Basel) ; 13(15)2024 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-39124221

RESUMO

The global ecosystem relies on the metabolism of photosynthetic organisms, featuring the ability to harness light as an energy source. The most successful type of photosynthesis utilizes a virtually inexhaustible electron pool from water, but the driver of this oxidation, sunlight, varies on time and intensity scales of several orders of magnitude. Such rapid and steep changes in energy availability are potentially devastating for biological systems. To enable a safe and efficient light-harnessing process, photosynthetic organisms tune their light capturing, the redox connections between core complexes and auxiliary electron mediators, ion passages across the membrane, and functional coupling of energy transducing organelles. Here, microalgal species are the most diverse group, featuring both unique environmental adjustment strategies and ubiquitous protective mechanisms. In this review, we explore a selection of regulatory processes of the microalgal photosynthetic apparatus supporting smooth electron flow in variable environments.

11.
Biochim Biophys Acta ; 1817(11): 2038-48, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22727877

RESUMO

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.


Assuntos
Trifosfato de Adenosina/química , ATPases de Cloroplastos Translocadoras de Prótons/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tilacoides/enzimologia , Sequência de Aminoácidos , ATPases de Cloroplastos Translocadoras de Prótons/química , Peróxido de Hidrogênio/metabolismo , Hidrólise , Dados de Sequência Molecular , Subunidades Proteicas , Oxigênio Singlete/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Sulfitos/farmacologia
12.
Biochim Biophys Acta Bioenerg ; 1862(8): 148434, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33932368

RESUMO

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain that contains a cysteine couple which interacts with the torque-transmitting ßDELSEED-loop. This thiol modulation equips CF1Fo with an important environmental fine-tuning mechanism. In vitro, disulfide formation in the γ-redox domain slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient ( [Formula: see text] ), which agrees with its proposed role as chock based on recently solved structure. The γ-dithiol formation at the onset of light is crucial to maximize photosynthetic efficiency since it lowers the [Formula: see text] activation level for ATP synthesis in vitro. Here, we validate these findings in vivo by utilizing absorption spectroscopy in Arabidopsis thaliana. To do so, we monitored the [Formula: see text] present in darkness and identified its mitochondrial sources. By following the fate and components of light-induced extra [Formula: see text] , we estimated the ATP lifetime that lasted up to tens of minutes after long illuminations. Based on the relationship between [Formula: see text] and CF1Fo activity, we conclude that the dithiol configuration in vivo facilitates photosynthesis by driving the same ATP synthesis rate at a significative lower [Formula: see text] than in the γ-disulfide state. The presented in vivo findings are an additional proof of the importance of CF1Fo thiol modulation, reconciling biochemical in vitro studies and structural insights.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Força Próton-Motriz , Compostos de Sulfidrila/metabolismo , Arabidopsis/crescimento & desenvolvimento , Oxirredução , Folhas de Planta/crescimento & desenvolvimento
13.
FEBS Lett ; 584(1): 147-52, 2010 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-19925794

RESUMO

Singlet oxygen ((1)O(2)) produced in plants during photosynthesis has a strong damaging effect not only on both photosystems but also on the whole photosynthetic machinery. This is also applicable for the adenosine triphosphate (ATP) synthase. Here we describe the impact of (1)O(2) generated by the photosensitizer Rose Bengal on the ATP hydrolysis and ATP-driven proton translocation activity of CF1CFo. Both activities were reduced dramatically within 1min of exposure. Interestingly, it is shown that oxidized thylakoid ATP synthase is more susceptible to (1)O(2) than CF1CFo in its reduced state, a new insight on the mechanism of (1)O(2) interaction with the gamma subunit.


Assuntos
ATPases de Cloroplastos Translocadoras de Prótons/antagonistas & inibidores , Prótons , Oxigênio Singlete/metabolismo , Tilacoides/enzimologia , Trifosfato de Adenosina/metabolismo , Membrana Celular/enzimologia , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Cisteína/metabolismo , Hidrólise , Magnésio/metabolismo , Oxirredução , Rosa Bengala/farmacologia , Spinacia oleracea/enzimologia
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