RESUMEN
The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 fâ occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 fâ also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 fâ monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 fâ structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis.
Asunto(s)
Microscopía por Crioelectrón , Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/ultraestructura , Spinacia oleracea/química , Spinacia oleracea/ultraestructura , Sitios de Unión , Clorofila/química , Hemo/química , Lípidos/química , Modelos Moleculares , Oxidación-Reducción , Fotosíntesis , Plastoquinona/química , Relación Estructura-ActividadRESUMEN
The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and ß-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris, each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αß-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αß-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.
Asunto(s)
Bacterioclorofilas , Rhodopseudomonas , Proteínas Bacterianas/metabolismo , Bacterioclorofilas/metabolismo , Microscopía por Crioelectrón , Complejos de Proteína Captadores de Luz/metabolismo , Péptidos/metabolismo , Rhodopseudomonas/genéticaRESUMEN
The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 ß-polypeptides sandwiched between 17 α- and 16 γ-polypeptides. Tight packing of the γ-apoproteins between ß-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th γ-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q P, which adopts a compact, export-ready conformation before passage through the pore and eventual diffusion to the cytochrome bc 1 complex.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Hyphomicrobiaceae/química , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/ultraestructura , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestructura , Proteínas Bacterianas/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Benzoquinonas/metabolismo , Sitios de Unión , Complejos de Proteína Captadores de Luz/metabolismo , Magnesio/química , Magnesio/metabolismo , Modelos Moleculares , Fotosíntesis , Conformación Proteica , Estabilidad ProteicaRESUMEN
The RNA polymerase inhibitor favipiravir is currently in clinical trials as a treatment for infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite limited information about the molecular basis for its activity. Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure shows clear evidence for the inhibitor at the catalytic site of the enzyme, and resolves the conformation of key side chains and ions surrounding the binding pocket. Polymerase activity assays indicate that the inhibitor is weakly incorporated into the RNA primer strand, and suppresses RNA replication in the presence of natural nucleotides. The structure reveals an unusual, nonproductive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp, which explains its low rate of incorporation into the RNA primer strand. Together, these findings inform current and future efforts to develop polymerase inhibitors for SARS coronaviruses.
Asunto(s)
Amidas/farmacología , ARN Polimerasa Dependiente de ARN de Coronavirus/metabolismo , Inhibidores Enzimáticos/farmacología , Pirazinas/farmacología , SARS-CoV-2/ultraestructura , Amidas/química , ARN Polimerasa Dependiente de ARN de Coronavirus/antagonistas & inhibidores , ARN Polimerasa Dependiente de ARN de Coronavirus/química , Microscopía por Crioelectrón/métodos , Inhibidores Enzimáticos/química , Pirazinas/química , Ribonucleótidos/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Imagen Individual de Molécula/métodosRESUMEN
The combination of achiral Cp*Rh(III) with chiral carboxylic acids (CCAs) represents an efficient catalytic system in transition metal-catalyzed enantioselective C-H activation. However, this hybrid catalysis is limited to redox-neutral C-H activation reactions and the adopt to oxidative enantioselective C-H activation remains elusive and pose a significant challenge. Herein, we describe the development of an electrochemical Cp*Rh(III)-catalyzed enantioselective C-H annulation of sulfoximines with alkynes enabled by chiral carboxylic acid (CCA) in an operationally friendly undivided cell at room temperature. A broad range of enantioenriched 1,2-benzothiazines are obtained in high yields with excellent enantioselectivities (up to 99 % yield and 98 : 2â er). The practicality of this method is demonstrated by scale-up reaction in a batch reactor with external circulation. A crucial chiral Cp*Rh(III) intermediate is isolated, characterized, and transformed, providing rational support for a Rh(III)/Rh(I) electrocatalytic cycle.
RESUMEN
The light-harvesting complex 2 (LH2) of purple bacteria is one of the most studied photosynthetic antenna complexes. Its symmetric structure and ring-like bacteriochlorophyll arrangement make it an ideal system for theoreticians and spectroscopists. LH2 complexes from most bacterial species are thought to have eightfold or ninefold symmetry, but recently a sevenfold symmetric LH2 structure from the bacterium Mch. purpuratum was solved by Cryo-Electron microscopy. This LH2 also possesses unique near-infrared absorption and circular dichroism (CD) spectral properties. Here we use an atomistic strategy to elucidate the spectral properties of Mch. purpuratum LH2 and understand the differences with the most commonly studied LH2 from Rbl. acidophilus. Our strategy exploits a combination of molecular dynamics simulations, multiscale polarizable quantum mechanics/molecular mechanics calculations, and lineshape simulations. Our calculations reveal that the spectral properties of LH2 complexes are tuned by site energies and exciton couplings, which in turn depend on the structural fluctuations of the bacteriochlorophylls. Our strategy proves effective in reproducing the absorption and CD spectra of the two LH2 complexes, and in uncovering the origin of their differences. This work proves that it is possible to obtain insight into the spectral tuning strategies of purple bacteria by quantitatively simulating the spectral properties of their antenna complexes.
Asunto(s)
Complejos de Proteína Captadores de Luz , Proteínas del Complejo del Centro de Reacción Fotosintética , Complejos de Proteína Captadores de Luz/metabolismo , Microscopía por Crioelectrón , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Bacterioclorofilas/química , Simulación de Dinámica Molecular , Proteobacteria/metabolismoRESUMEN
Image contrast is often limited by background autofluorescence in steady-state bioimaging microscopy. Upconversion bioimaging can overcome this by shifting the emission lifetime and wavelength beyond the autofluorescence window. Here we demonstrate the first example of triplet-triplet annihilation upconversion (TTA-UC) based lifetime imaging microscopy. A new class of ultra-small nanoparticle (NP) probes based on TTA-UC chromophores encapsulated in an organic-inorganic host has been synthesised. The NPs exhibit bright UC emission (400-500â nm) in aerated aqueous media with a UC lifetime of ≈1â µs, excellent colloidal stability and little cytotoxicity. Proof-of-concept demonstration of TTA-UC lifetime imaging using these NPs shows that the long-lived anti-Stokes emission is easily discriminable from typical autofluorescence. Moreover, fluctuations in the UC lifetime can be used to map local oxygen diffusion across the subcellular structure. Our TTA-UC NPs are highly promising stains for lifetime imaging microscopy, affording excellent image contrast and potential for oxygen mapping that is ripe for further exploitation.
RESUMEN
The reaction centre light-harvesting 1 (RC-LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC-LH1 complex from Rhodospirillum rubrum at 2.5â Å resolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between the RC and LH1 complexes. The LH1 complex comprises a circular array of 16 αß-polypeptide subunits that completely surrounds the RC, with a preferential binding site for a quinone, designated QP, on the inner face of the encircling LH1 complex. Quinols, initially generated at the RC QB site, are proposed to transiently occupy the QP site prior to traversing the LH1 barrier and diffusing to the cytochrome bc1 complex. Thus, the QP site, which is analogous to other such sites in recent cryo-EM structures of RC-LH1 complexes, likely reflects a general mechanism for exporting quinols from the RC-LH1 complex.
Asunto(s)
Proteínas Bacterianas/química , Microscopía por Crioelectrón/métodos , Complejos de Proteína Captadores de Luz/química , Rhodospirillum rubrum/química , Proteínas Bacterianas/aislamiento & purificación , Bacterioclorofilas/química , Benzoquinonas/química , Sitios de Unión , Cristalización , Complejo III de Transporte de Electrones/química , Enlace de Hidrógeno , Hidroquinonas/química , Ligandos , Complejos de Proteína Captadores de Luz/aislamiento & purificación , Fosfolípidos/química , Conformación Proteica en Hélice alfaRESUMEN
Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5â Å resolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αß heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could impede passage of quinones through small, transient pores in the LH1 ring, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilising an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered quinone diffusion.
Asunto(s)
Proteínas Bacterianas/química , Complejos de Proteína Captadores de Luz/química , Péptidos/química , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Sitios de Unión , Carotenoides/química , Carotenoides/metabolismo , Microscopía por Crioelectrón , Expresión Génica , Hidroquinonas/química , Hidroquinonas/metabolismo , Luz , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Modelos Moleculares , Péptidos/genética , Péptidos/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/efectos de la radiaciónRESUMEN
The dimeric reaction centre light-harvesting 1 (RC-LH1) core complex of Rhodobacter sphaeroides converts absorbed light energy to a charge separation, and then it reduces a quinone electron and proton acceptor to a quinol. The angle between the two monomers imposes a bent configuration on the dimer complex, which exerts a major influence on the curvature of the membrane vesicles, known as chromatophores, where the light-driven photosynthetic reactions take place. To investigate the dimerisation interface between two RC-LH1 monomers, we determined the cryogenic electron microscopy structure of the dimeric complex at 2.9â Å resolution. The structure shows that each monomer consists of a central RC partly enclosed by a 14-subunit LH1 ring held in an open state by PufX and protein-Y polypeptides, thus enabling quinones to enter and leave the complex. Two monomers are brought together through N-terminal interactions between PufX polypeptides on the cytoplasmic side of the complex, augmented by two novel transmembrane polypeptides, designated protein-Z, that bind to the outer faces of the two central LH1 ß polypeptides. The precise fit at the dimer interface, enabled by PufX and protein-Z, by C-terminal interactions between opposing LH1 αß subunits, and by a series of interactions with a bound sulfoquinovosyl diacylglycerol lipid, bring together each monomer creating an S-shaped array of 28 bacteriochlorophylls. The seamless join between the two sets of LH1 bacteriochlorophylls provides a path for excitation energy absorbed by one half of the complex to migrate across the dimer interface to the other half.
Asunto(s)
Proteínas Bacterianas , Complejos de Proteína Captadores de Luz , Rhodobacter sphaeroides/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dimerización , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Estructura MolecularRESUMEN
Light-harvesting 2 (LH2) antenna complexes augment the collection of solar energy in many phototrophic bacteria. Despite its frequent role as a model for such complexes, there has been no three-dimensional (3D) structure available for the LH2 from the purple phototroph Rhodobacter sphaeroides. We used cryo-electron microscopy (cryo-EM) to determine the 2.1 Å resolution structure of this LH2 antenna, which is a cylindrical assembly of nine αß heterodimer subunits, each of which binds three bacteriochlorophyll a (BChl) molecules and one carotenoid. The high resolution of this structure reveals all of the interpigment and pigment-protein interactions that promote the assembly and energy-transfer properties of this complex. Near the cytoplasmic face of the complex there is a ring of nine BChls, which absorb maximally at 800 nm and are designated as B800; each B800 is coordinated by the N-terminal carboxymethionine of LH2-α, part of a network of interactions with nearby residues on both LH2-α and LH2-ß and with the carotenoid. Nine carotenoids, which are spheroidene in the strain we analyzed, snake through the complex, traversing the membrane and interacting with a ring of 18 BChls situated toward the periplasmic side of the complex. Hydrogen bonds with C-terminal aromatic residues modify the absorption of these pigments, which are red-shifted to 850 nm. Overlaps between the macrocycles of the B850 BChls ensure rapid transfer of excitation energy around this ring of pigments, which act as the donors of energy to neighboring LH2 and reaction center light-harvesting 1 (RC-LH1) complexes.
Asunto(s)
Proteínas Bacterianas/ultraestructura , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/ultraestructura , Proteínas Bacterianas/metabolismo , Bacterioclorofila A/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Microscopía por Crioelectrón/métodos , Transferencia de Energía , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/ultraestructuraRESUMEN
Ru(II)-catalyzed enantioselective C-H functionalization involving an enantiodetermining C-H cleavage step remains undeveloped. Here we describe a Ru(II)-catalyzed enantioselective C-H activation/annulation of sulfoximines with α-carbonyl sulfoxonium ylides using a novel class of chiral binaphthyl monocarboxylic acids as chiral ligands, which can be easily and modularly prepared from 1,1'-binaphthyl-2,2'-dicarboxylic acid. A broad range of sulfur-stereogenic sulfoximines were prepared in high yields with excellent enantioselectivities (up to 99% yield and 99% ee) via desymmetrization, kinetic resolution, and parallel kinetic resolution. Furthermore, the resolution products can be easily transformed to chiral sulfoxides and key intermediates for kinase inhibitors.
RESUMEN
Genetic resources are important national strategic resources. Their preservation, protection and rational utilization form a solid foundation to guarantee national security and to build national competitiveness for the future. Due to a relatively late starting point, China is actively catching up with global peers in storing genetic samples and data. In view of this, in 2011 China approved a plan to build its first nation-level comprehensive gene bank, the China National GeneBank (CNGB), and entrusted BGI-Research to implement its construction and operation. It is China's first gene bank for "reading, writing and storing" bioresources. In this paper, we summarize the development of influential platforms at home and abroad, and focus on CNGB's position, mission, and its structure of "Three Banks and Two Platforms". CNGB launched its official operation in September 2016 and aims to develop a world-class, non-profit and strategic platform that supports science and technology development. It has built capacities to store tens of millions of traceable samples and to analyze handreds of thousanda of WGS each year. It has also set up China's first Pb-level digitalization platform and a high-efficient synthesis platform with a production rate of ten million bases per year. Based on such capacities, CNGB has established its open sharing mechanism for biological samples and data, provided public platform services for life science research, and achieved initial results in supporting innovation and development of the bio-industry.
Asunto(s)
Bases de Datos Genéticas , Investigación , China , Difusión de la InformaciónRESUMEN
The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.
Asunto(s)
Proteínas Bacterianas/química , Complejos de Proteína Captadores de Luz/química , Rhodopseudomonas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/metabolismo , Espectrometría de Masas , Rhodopseudomonas/genética , Rhodopseudomonas/metabolismoRESUMEN
Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.
Asunto(s)
Proteínas Bacterianas/química , Complejo de Citocromo b6f/química , Complejo III de Transporte de Electrones/química , Maleatos/química , Lípidos de la Membrana/química , Poliestirenos/química , Cromatóforos Bacterianos/química , Cromatóforos Bacterianos/metabolismo , Cromatóforos Bacterianos/ultraestructura , Proteínas Bacterianas/metabolismo , Complejo de Citocromo b6f/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Transferencia de Energía , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Maleatos/metabolismo , Lípidos de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Modelos Moleculares , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Poliestirenos/metabolismo , Rhodobacter sphaeroides/metabolismo , Solubilidad , Synechocystis/metabolismo , Tilacoides/química , Tilacoides/metabolismo , Tilacoides/ultraestructuraRESUMEN
In bacterial photosynthesis reaction center-light-harvesting 1 (RC-LH1) complexes trap absorbed solar energy by generating a charge separated state. Subsequent electron and proton transfers form a quinol, destined to diffuse to the cytochrome bc1 complex. In bacteria such as Rhodobacter (Rba.) sphaeroides and Rba. capsulatus the PufX polypeptide creates a channel for quinone/quinol traffic across the LH1 complex that surrounds the RC, and it is therefore essential for photosynthetic growth. PufX also plays a key role in dimerization of the RC-LH1-PufX core complex, and the structure of the Rba. sphaeroides complex shows that the PufX C-terminus, particularly the region from X49-X53, likely mediates association of core monomers. To investigate this putative interaction we analysed mutations PufX R49L, PufX R53L, PufX R49/53L and PufX G52L by measuring photosynthetic growth, fractionation of detergent-solubilised membranes, formation of 2-D crystals and electron microscopy. We show that these mutations do not affect assembly of PufX within the core or photosynthetic growth but they do prevent dimerization, consistent with predictions from the RC-LH1-PufX structure. We obtained low resolution structures of monomeric core complexes with and without PufX, using electron microscopy of negatively stained single particles and 3D reconstruction; the monomeric complex with PufX corresponds to one half of the dimer structure whereas LH1 completely encloses the RC if the gene encoding PufX is deleted. On the basis of the insights gained from these mutagenesis and structural analyses we propose a sequence for assembly of the dimeric RC-LH1-PufX complex.
Asunto(s)
Proteínas Bacterianas/fisiología , Complejos de Proteína Captadores de Luz/química , Rhodobacter sphaeroides/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Benzoquinonas/metabolismo , Cristalización , Dimerización , Hidroquinonas/metabolismo , Procesamiento de Imagen Asistido por Computador , Complejos de Proteína Captadores de Luz/genética , Complejos de Proteína Captadores de Luz/fisiología , Complejos de Proteína Captadores de Luz/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Mutación Missense , Mutación Puntual , Conformación Proteica , Dominios Proteicos , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/efectos de la radiaciónRESUMEN
Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated C = C bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.
Asunto(s)
Carotenoides/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/metabolismo , Bacterioclorofilas/metabolismo , Cromatografía Líquida de Alta Presión , Transferencia de Energía , Espectrometría de Masas , Oxidorreductasas/fisiología , Xantófilas/metabolismoRESUMEN
The photosynthetic membranes of the filamentous anoxygenic phototroph Roseiflexus castenholzii have been studied with electron microscopy, atomic force microscopy, and biochemistry. Electron microscopy of the light-harvesting reaction center complex produced a 3D model that aligns with the solved crystal structure of the RC-LH1 from Thermochromatium tepidum with the H subunit removed. Atomic force microscopy of the whole membranes yielded a picture of the supramolecular organization of the major proteins in the photosynthetic electron transport chain. The results point to a loosely packed membrane without accessory antenna proteins or higher order structure.
Asunto(s)
Membrana Celular/química , Chloroflexi/química , Complejos de Proteína Captadores de Luz/química , Proteínas Bacterianas/química , Chloroflexi/metabolismo , Chromatiaceae/química , Hemo/análisis , Imagenología Tridimensional , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Microscopía de Fuerza Atómica/métodos , Microscopía Electrónica de Transmisión/métodos , FotosíntesisRESUMEN
In the purple phototrophic bacterium Rhodobacter sphaeroides, many protein complexes congregate within the membrane to form operational photosynthetic units consisting of arrays of light-harvesting LH2 complexes and monomeric and dimeric reaction center (RC)-light-harvesting 1 (LH1)-PufX "core" complexes. Each half of a dimer complex consists of a RC surrounded by 14 LH1 αß subunits, with two bacteriochlorophylls (Bchls) sandwiched between each αß pair of transmembrane helices. We used atomic force microscopy (AFM) to investigate the assembly of single molecules of the RC-LH1-PufX complex using membranes prepared from LH2-minus mutants. When the RC and PufX components were also absent, AFM revealed a series of LH1 variants where the repeating α(1)ß(1)(Bchl)2 units had formed rings of variable size, ellipses, and spirals and also arcs that could be assembly products. The spiral complexes occur when the LH1 ring has failed to close, and short arcs are suggestive of prematurely terminated LH1 complex assembly. In the absence of RCs, we occasionally observed captive proteins enclosed by the LH1 ring. When production of LH1 units was restricted by lowering the relative levels of the cognate pufBA transcript, we imaged a mixture of complete RC-LH1 core complexes, empty LH1 rings, and isolated RCs, leading us to conclude that once a RC associates with the first α1ß1(Bchl)2 subunit, cooperative associations between subsequent subunits and the RC tend to drive LH1 ring assembly to completion.
Asunto(s)
Proteínas Bacterianas/metabolismo , Imagenología Tridimensional , Complejos de Proteína Captadores de Luz/metabolismo , Microscopía de Fuerza Atómica/métodos , Rhodobacter sphaeroides/metabolismo , Detergentes/farmacología , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Modelos Moleculares , Proteínas Mutantes/metabolismo , Subunidades de Proteína/metabolismo , Rhodobacter sphaeroides/efectos de los fármacos , UltracentrifugaciónRESUMEN
Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll-protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.