RESUMEN
Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SIGNIFICANCE: A crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)-1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.
Asunto(s)
Oxidación-Reducción , Cinética , Ligandos , Transporte de Electrón , Fotosíntesis , Luz , Complejo III de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Bacterioclorofilas/metabolismo , Bacterioclorofilas/químicaRESUMEN
The development of chromophores that absorb in the near-infrared (NIR) region beyond 1000 nm underpins numerous applications in medical and energy sciences, yet also presents substantial challenges to molecular design and chemical synthesis. Here, the core bacteriochlorin chromophore of nature's NIR absorbers, bacteriochlorophylls, has been adapted and tailored by annulation in an effort to achieve absorption in the NIR-II region. The resulting bacteriochlorin, Phen2,1-BC, contains two annulated naphthalene groups spanning meso,ß-positions of the bacteriochlorin and the 1,2-positions of the naphthalene. Phen2,1-BC was prepared via a new synthetic route. Phen2,1-BC is an isomer of previously examined Phen-BC, which differs only in attachment via the 1,8-positions of the naphthalene. Despite identical π-systems, the two bacteriochlorins have distinct spectroscopic and photophysical features. Phen-BC has long-wavelength absorption maximum (912 nm), oscillator strength (1.0), and S1 excited-state lifetime (150 ps) much different than Phen2,1-BC (1292 nm, 0.23, and 0.4 ps, respectively). These two molecules and an analogue with intermediate characteristics bearing annulated phenyl rings have unexpected properties relative to those of non-annulated counterparts. Understanding the distinctions requires extending concepts beyond the four-orbital-model description of tetrapyrrole spectroscopic features. In particular, a reduction in symmetry resulting from annulation results in electronic mixing of x- and y-polarized transitions/states, as well as vibronic coupling that together reduce oscillator strength of the long-wavelength absorption manifold and shorten the S1 excited-state lifetime. Collectively, the results suggest a heuristic for the molecular design of tetrapyrrole chromophores for deep penetration into the relatively unutilized NIR-II region.
Asunto(s)
Porfirinas , Espectroscopía Infrarroja Corta , Porfirinas/química , Naftalenos/química , Estructura Molecular , Bacterioclorofilas/químicaRESUMEN
The rates, yields, mechanisms and directionality of electron transfer (ET) are explored in twelve pairs of Rhodobacter (R.) sphaeroides and R. capsulatus mutant RCs designed to defeat ET from the excited primary donor (P*) to the A-side cofactors and re-direct ET to the normally inactive mirror-image B-side cofactors. In general, the R. sphaeroides variants have larger P+HB- yields (up to â¼90%) than their R. capsulatus analogs (up to â¼60%), where HB is the B-side bacteriopheophytin. Substitution of Tyr for Phe at L-polypeptide position L181 near BB primarily increases the contribution of fast P* â P+BB- â P+HB- two-step ET, where BB is the "bridging" B-side bacteriochlorophyll. The second step (â¼6-8 ps) is slower than the first (â¼3-4 ps), unlike A-side two-step ET (P* â P+BA- â P+HA-) where the second step (â¼1 ps) is faster than the first (â¼3-4 ps) in the native RC. Substitutions near HB, at L185 (Leu, Trp or Arg) and at M-polypeptide site M133/131 (Thr, Val or Glu), strongly affect the contribution of slower (20-50 ps) P* â P+HB- one-step superexchange ET. Both ET mechanisms are effective in directing electrons "the wrong way" to HB and both compete with internal conversion of P* to the ground state (â¼200 ps) and ET to the A-side cofactors. Collectively, the work demonstrates cooperative amino-acid control of rates, yields and mechanisms of ET in bacterial RCs and how A- vs. B-side charge separation can be tuned in both species.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética , Rhodobacter capsulatus , Rhodobacter sphaeroides , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/genética , Transporte de Electrón , Rhodobacter capsulatus/metabolismo , Rhodobacter capsulatus/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Mutación , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Bacterioclorofilas/metabolismo , Bacterioclorofilas/química , FotosíntesisRESUMEN
Chlorosomes of green photosynthetic bacteria are large light-harvesting complexes enabling these organisms to survive at extremely low-light conditions. Bacteriochlorophylls found in chlorosomes self-organize and are ideal candidates for use in biomimetic light-harvesting in artificial photosynthesis and other applications for solar energy utilization. Here we report on the construction and characterization of an artificial antenna consisting of bacteriochlorophyll c co-aggregated with ß-carotene, which is used to extend the light-harvesting spectral range, and bacteriochlorophyll a, which acts as a final acceptor for excitation energy. Efficient energy transfer between all three components was observed by means of fluorescence spectroscopy. The efficiency varies with the ß-carotene content, which increases the average distance between the donor and acceptor in both energy transfer steps. The efficiency ranges from 89 to 37% for the transfer from ß-carotene to bacteriochlorophyll c, and from 93 to 69% for the bacteriochlorophyll c to bacteriochlorophyll a step. A significant part of this study was dedicated to a development of methods for determination of energy transfer efficiency. These methods may be applied also for study of chlorosomes and other pigment complexes.
Asunto(s)
Bacterioclorofila A , Bacterioclorofilas , Bacterioclorofilas/química , Bacterioclorofila A/química , beta Caroteno , Complejos de Proteína Captadores de Luz/química , Proteínas Bacterianas/metabolismo , Transferencia de Energía , FotosíntesisRESUMEN
Chlorosomes, the photosynthetic antenna complexes of green sulfur bacteria, are paradigms for light-harvesting elements in artificial designs, owing to their efficient energy transfer without protein participation. We combined magic angle spinning (MAS) NMR, optical spectroscopy and cryogenic electron microscopy (cryo-EM) to characterize the structure of chlorosomes from a bchQ mutant of Chlorobaculum tepidum. The chlorosomes of this mutant have a more uniform composition of bacteriochlorophyll (BChl) with a predominant homolog, [8Ethyl, 12Ethyl] BChl c, compared to the wild type (WT). Nearly complete 13C chemical shift assignments were obtained from well-resolved homonuclear 13C-13C RFDR data. For proton assignments heteronuclear 13C-1H (hCH) data sets were collected at 1.2 GHz spinning at 60 kHz. The CHHC experiments revealed intermolecular correlations between 132/31, 132/32, and 121/31, with distance constraints of less than 5 Å. These constraints indicate the syn-anti parallel stacking motif for the aggregates. Fourier transform cryo-EM data reveal an axial repeat of 1.49 nm for the helical tubular aggregates, perpendicular to the inter-tube separation of 2.1 nm. This axial repeat is different from WT and is in line with BChl syn-anti stacks running essentially parallel to the tube axis. Such a packing mode is in agreement with the signature of the Qy band in circular dichroism (CD). Combining the experimental data with computational insight suggests that the packing for the light-harvesting function is similar between WT and bchQ, while the chirality within the chlorosomes is modestly but detectably affected by the reduced compositional heterogeneity in bchQ.
Asunto(s)
Bacterioclorofilas , Chlorobi , Chlorobi/genética , Chlorobi/metabolismo , Bacterioclorofilas/química , Mutación , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Complejos de Proteína Captadores de Luz/genética , Microscopía por Crioelectrón , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
To capture weak light fluxes, green photosynthetic bacteria have unique structures - chlorosomes, consisting of 104-5 molecules of bacteriochlorophyll (BChl) c, d, e. Chlorosomes are attached to the cytoplasmic membrane through the baseplate, a paracrystalline protein structure containing BChl a and carotenoids (Car). The most important function of Car is the quenching of triplet states of BChl, which prevents the formation of singlet oxygen and thereby provides photoprotection. In our work, we studied the dynamics of the triplet states of BChl a and Car in the baseplate of Chloroflexus aurantiacus chlorosomes using picosecond differential spectroscopy. BChl a of the baseplate was excited into the Qy band at 810 nm, and the corresponding absorption changes were recorded in the range of 420-880 nm. It was found that the formation of the Car triplet state occurs in â¼1.3 ns, which is â¼3 times faster than the formation of this state in the peripheral antenna of C. aurantiacus according to literature data. The Car triplet state was recorded by the characteristic absorption band T1 â Tn at â¼550 nm. Simultaneously with the appearance of absorption T1 â Tn, there was a bleaching of the singlet absorption of Car in the region of 400-500 nm. Theoretical modeling made it possible to estimate the characteristic time of formation of the triplet state of BChl a as â¼0.5 ns. It is shown that the experimental data are well described by the sequential scheme of formation and quenching of the BChl a triplet state: BChl a* â BChl aT â CarT. Thus, carotenoids from green bacteria effectively protect the baseplate from possible damage by singlet oxygen.
Asunto(s)
Bacterioclorofila A , Carotenoides , Chloroflexus , Carotenoides/metabolismo , Oxígeno Singlete , Bacterias/metabolismo , Proteínas Bacterianas/química , Bacterioclorofilas/químicaRESUMEN
The exciton states on the smallest type-I photosynthetic reaction center complex of a green sulfur bacterium Chlorobaculum tepidum (GsbRC) consisting of 26 bacteriochlorophylls a (BChl a) and four chlorophylls a (Chl a) located on the homodimer of two PscA reaction center polypeptides were investigated. This analysis involved the study of exciton states through a combination of theoretical modeling and the genetic removal of BChl a pigments at eight sites. (1) A theoretical model of the pigment assembly exciton state on GsbRC was constructed using Poisson TrESP (P-TrESP) and charge density coupling (CDC) methods based on structural information. The model reproduced the experimentally obtained absorption spectrum, circular dichroism spectrum, and excitation transfer dynamics, as well as explained the effects of mutation. (2) Eight BChl a molecules at different locations on the GsbRC were selectively removed by genetic exchange of the His residue, which ligates the central Mg atom of BChl a, with the Leu residue on either one or two PscAs in the RC. His locations are conserved among all type-I RC plant polypeptide, cyanobacteria, and bacteria amino acid sequences. (3) Purified mutant-GsbRCs demonstrated distinct absorption and fluorescence spectra at 77 K, which were different from each other, suggesting successful pigment removal. (4) The same mutations were applied to the constructed theoretical model to analyze the outcomes of these mutations. (5) The combination of theoretical predictions and experimental mutations based on structural information is a new tool for studying the function and evolution of photosynthetic reaction centers.
Asunto(s)
Chlorobi , Cianobacterias , Proteínas del Complejo del Centro de Reacción Fotosintética , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Chlorobi/química , Mutación , Cianobacterias/metabolismo , Azufre/metabolismo , Bacterioclorofilas/química , Proteínas Bacterianas/químicaRESUMEN
The light harvesting-reaction center complex (LH-RC) of Roseiflexus castenholzii binds bacteriochlorophylls a (BChls a), B800 and B880, absorbing around 800 and 880 nm, respectively. We comparatively investigated the interband excitation energy transfer (EET) dynamics of the wild-type LH-RC (wt-LH-RC) of Rfl. castenholzii and its carotenoid (Car)-less mutant (m-LH-RC) and found that Car can boost the B800 â B880 EET rate from (2.43 ps)-1 to (1.75 ps)-1, accounting for 38% acceleration of the EET process. Interestingly, photoexcitation of wt-LH-RC at 800 nm induced pronounced excitation dynamics of Car despite the insufficient photon energy for direct Car excitation, a phenomenon which is attributed to the BChl-Car exciplex 1[B800(↑↑)···Car(↓↓)]*. Such an exciplex is suggested to play an essential role in promoting the B800 â B880 EET process, as corroborated by the recently reported cryo-EM structures of wt-LH-RC and m-LH-RC. The mechanism of Car-mediated EET will be helpful to deepen the understanding of the role of Car in bacterial photosynthesis.
Asunto(s)
Chloroflexi , Fotosíntesis , Chloroflexi/química , Chloroflexi/metabolismo , Carotenoides/metabolismo , Transferencia de Energía , Complejos de Proteína Captadores de Luz/química , Bacterioclorofilas/química , Proteínas Bacterianas/química , LuzRESUMEN
Heliobacteria are anoxygenic phototrophs that have a Type I homodimeric reaction center containing bacteriochlorophyll g (BChl g). Previous experimental studies have shown that in the presence of light and dioxygen, BChl g is converted into 81-OH-chlorophyll aF (hereafter Chl aF), with an accompanying loss of light-driven charge separation. These studies suggest that the reaction center only loses the ability to transfer electrons once both BChl g' molecules of the P800 special pair have been converted to Chl aF'. The present work confirms that the partially converted BChl g'/Chl aF' special pair remains functional in samples exposed to dioxygen by demonstrating its presence using hyperfine couplings obtained from Q-band 1H ENDOR, 2D 14N HYSCORE and DFT methods. The DFT calculations of the BChl g'/BChl g' homodimeric primary donor, which are based on the recently published X-ray crystal structure, predict that the unpaired electron spin is equally delocalized over both BChl g' molecules and provide an excellent match to the experimental hyperfine couplings of the anaerobic samples. Exposure to dioxygen leads to substantial changes in the hyperfine interactions, indicative of greater localization of the unpaired electron spin. The measured hyperfine couplings are reproduced in the DFT calculations by replacing one of the BChl g' molecules of the primary donor with a Chl aF' molecule. The calculations reveal that the spin density becomes localized on BChl g' in the heterodimeric primary donor. Time-dependent DFT calculations demonstrate that conversion of either or both of the accessory BChl g molecules and/or one of the BChl g' molecules of P800 to Chl aF' results in minor effects on the energy of the charge-separated states. In contrast, if both of the BChl g' molecules of P800 are converted a large increase in the energy of the charge-separated state occurs. This suggests that the reaction center remains functional when only one half of the dimer is converted, however, conversion of both halves of the P800 dimer leads to loss of function.
Asunto(s)
Bacterioclorofila A , Bacterioclorofilas , Clorofila A , Bacterioclorofilas/química , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
Photosynthesis is generally assumed to be initiated by a single photon1-3 from the Sun, which, as a weak light source, delivers at most a few tens of photons per nanometre squared per second within a chlorophyll absorption band1. Yet much experimental and theoretical work over the past 40 years has explored the events during photosynthesis subsequent to absorption of light from intense, ultrashort laser pulses2-15. Here, we use single photons to excite under ambient conditions the light-harvesting 2 (LH2) complex of the purple bacterium Rhodobacter sphaeroides, comprising B800 and B850 rings that contain 9 and 18 bacteriochlorophyll molecules, respectively. Excitation of the B800 ring leads to electronic energy transfer to the B850 ring in approximately 0.7 ps, followed by rapid B850-to-B850 energy transfer on an approximately 100-fs timescale and light emission at 850-875 nm (refs. 16-19). Using a heralded single-photon source20,21 along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. We also find that the probability distribution of the number of heralds per detected fluorescence photon supports the view that a single photon can upon absorption drive the subsequent energy transfer and fluorescence emission and hence, by extension, the primary charge separation of photosynthesis. An analytical stochastic model and a Monte Carlo numerical model capture the data, further confirming that absorption of single photons is correlated with emission of single photons in a natural light-harvesting complex.
Asunto(s)
Complejos de Proteína Captadores de Luz , Fotones , Fotosíntesis , Rhodobacter sphaeroides , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Transferencia de Energía , Complejos de Proteína Captadores de Luz/química , Complejos de Proteína Captadores de Luz/metabolismo , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo , Fluorescencia , Procesos Estocásticos , Método de MontecarloRESUMEN
Process of photosynthesis in the green bacteria Chloroflexus (Cfx.) aurantiacus starts from absorption of light by chlorosomes, peripheral antennas consisting of thousands of bacteriochlorophyll c (BChl c) molecules combined into oligomeric structures. In this case, the excited states are formed in BChl c, energy of which migrates along the chlorosome towards the baseplate and further to the reaction center, where the primary charge separation occurs. Energy migration is accompanied by non-radiative electronic transitions between the numerous exciton states, that is, exciton relaxation. In this work, we studied dynamics of the exciton relaxation in Cfx. aurantiacus chlorosomes using differential femtosecond spectroscopy at cryogenic temperature (80 K). Chlorosomes were excited by 20-fs light pulses at wavelengths in the range from 660 to 750 nm, and differential (light-dark) absorption kinetics were measured at a wavelength of 755 nm. Mathematical analysis of the obtained data revealed kinetic components with characteristic times of 140, 220, and 320 fs, which are responsible for exciton relaxation. As the excitation wavelength decreased, the number and relative contribution of these components increased. Theoretical modelling of the obtained data was carried out based of the cylindrical model of BChl c. Nonradiative transitions between the groups of exciton bands were described by a system of kinetic equations. The model that takes into account energy and structural disorder of chlorosomes turned out to be the most adequate.
Asunto(s)
Chloroflexus , Chloroflexus/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Análisis Espectral , Bacterioclorofilas/química , FotosíntesisRESUMEN
Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-CâO or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.
Asunto(s)
Bacterioclorofilas , Clorofilidas , Hidrólisis , Dominio Catalítico , Descarboxilación , Bacterioclorofilas/química , Clorofila , Proteínas Bacterianas/metabolismoRESUMEN
The spectral features of energy donors and acceptors and the relationship between them in photosynthetic light-harvesting proteins are crucial for photofunctions of these proteins. Engineering energy donors and acceptors in light-harvesting proteins affords the means to increase our understanding of their photofunctional mechanisms. Herein, we demonstrate the conversion of energy-donating B800 bacteriochlorophyll (BChl) a to 3-acetyl chlorophyll (AcChl) a in light-harvesting complex 3 (LH3) from Rhodoblastus acidophilus by in situ oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. AcChl a in the B800 site exhibited a Qy band that was 111 nm blue-shifted with respect to BChl a in oxidized LH3. The structure of LH3 was barely influenced by the oxidation process, based on circular dichroism spectroscopy and size-exclusion chromatography evidence. In oxidized LH3, AcChl a transferred excitation energy to B820 BChl a, but the rate of excitation energy transfer (EET) was lower than in native LH3. The intracomplex EET in oxidized LH3 was slightly faster than in oxidized light-harvesting complex 2 (LH2). This difference is rationalized by an increase in overlap of the luminescence band of AcChl a with the long tail of the B820 absorption band in oxidized LH3 compared with that of the B850 band in oxidized LH2.
Asunto(s)
Bacterioclorofila A , Bacterioclorofilas , Bacterioclorofilas/química , Bacterioclorofila A/química , Clorofila A , Complejos de Proteína Captadores de Luz/química , Proteínas Bacterianas/químicaRESUMEN
The photosynthetic tetrapyrroles share a common structural feature comprised of a ß-ketoester motif embedded in an exocyclic ring (ring E). As part of a total synthesis program aimed at preparing native structures and analogues, 3-(3-methoxy-1,3-dioxopropyl)pyrrole was sought. The pyrrole is a precursor to analogues of ring C and the external framework of ring E. Four routes were developed. Routes 1-3 entail a Pd-mediated coupling process of a 3-iodopyrrole with potassium methyl malonate, whereas route 4 relies on electrophilic substitution of TIPS-pyrrole with methyl malonyl chloride. Together, the four routes afford considerable latitude. A long-term objective is to gain the capacity to create chlorophylls and bacteriochlorophylls and analogues thereof by facile de novo means for diverse studies across the photosynthetic sciences.
Asunto(s)
Pirroles , Tetrapirroles , Pirroles/química , Clorofila/química , Bacterioclorofilas/química , FotosíntesisRESUMEN
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
In green photosynthetic bacteria, light is absorbed by bacteriochlorophyll (BChl) c/d/e oligomers, which are located in chlorosomes - unique structures created by Nature to collect the energy of very weak light fluxes. Using coherent femtosecond spectroscopy at cryogenic temperature, we detected and studied low-frequency vibrational motions of BChl c oligomers in chlorosomes of the green bacteria Chloroflexus (Cfx.) aurantiacus. The objects of the study were chlorosomes isolated from the bacterial cultures grown under different light intensity. It was found that the Fourier spectrum of low-frequency coherent oscillations in the Qy band of BChl c oligomers depends on the light intensity used for the growth of bacteria. It turned out that the number of low-frequency vibrational modes of chlorosomes increases as illumination under which they were cultivated decreases. Also, the frequency range within which these modes are observed expands, and frequencies of the most modes change. Theoretical modeling of the obtained data and analysis of the literature led to conclusion that the structural basis of Cfx. aurantiacus chlorosomes are short linear chains of BChl c combined into more complex structures. Increase in the length of these chains in chlorosomes grown under weaker light leads to the observed changes in the spectrum of vibrations of BChl c oligomers. This increase is an effective mechanism for bacteria adaptation to changing external conditions.
Asunto(s)
Bacterioclorofilas , Chloroflexus , Bacterioclorofilas/química , Proteínas Bacterianas/química , Análisis Espectral , Bacterias , LuzRESUMEN
Photoheterotrophic bacteria harvest light energy using either proton-pumping rhodopsins or bacteriochlorophyll (BChl)-based photosystems. The bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for both systems. Here, we show that BChl is expressed between 4°C and 22°C in the dark, whereas xanthorhodopsin is expressed only at temperatures below 16°C and in the presence of light. Thus, cells grown at low temperatures under a natural light-dark cycle contain both BChl-based photosystems and xanthorhodopsins with a nostoxanthin antenna. Flash photolysis measurements proved that both systems are photochemically active. The captured light energy is used for ATP synthesis and stimulates growth. Thus, S. glacialis AAP5 represents a chlorophototrophic and a retinalophototrophic organism. Our analyses suggest that simple xanthorhodopsin may be preferred by the cells under higher light and low temperatures, whereas larger BChl-based photosystems may perform better at lower light intensities. This indicates that the use of two systems for light harvesting may represent an evolutionary adaptation to the specific environmental conditions found in alpine lakes and other analogous ecosystems, allowing bacteria to alternate their light-harvesting machinery in response to large seasonal changes of irradiance and temperature.
Asunto(s)
Bacterioclorofilas , Lagos , Bacterioclorofilas/química , Lagos/análisis , Protones , Bombas de Protones , Ecosistema , Proteínas Bacterianas/metabolismo , Bacterias/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , FotosíntesisRESUMEN
The mechanism of bacteriochlorophyll photooxidation in light-harvesting complexes of a number of purple photosynthetic bacteria when the complexes are excited into the carotenoid absorption bands remains unclear for many years. Here, using narrow-band laser illumination we measured action spectrum of this process for the spectral ranges of carotenoid and bacteriochlorophyll. It is shown that bacteriochlorophyll excitation results in almost no photooxidation of these molecules, while carotenoid excitation leads to oxidation with quantum yield of about 0,0003. Low value of the yield enabled an assumption that the studied process is initiated by the triplet states of the main carotenoids of the complexes with the number of conjugated double-bond chain length of N = 11. Interaction of these states with oxygen facilitates formation, though with low efficiency, of the excited singlet oxygen, which oxidizes bacteriochlorophylls. The carotenoid triplet states are formed in the process of the earlier studied singlet-triplet fission. The obtained results point at the necessity of reconsidering the functions of carotenoids in the light-harvesting complexes of purple bacteria.
Asunto(s)
Bacterioclorofilas , Carotenoides , Bacterioclorofilas/química , Carotenoides/química , Complejos de Proteína Captadores de Luz , Oxígeno Singlete , OxígenoRESUMEN
In the structure of photosynthetic reaction center (RC) of the purple bacterium Cereibacter sphaeroides the highly conserved amino acid residue Ile-M206 is located near the bacteriochlorophyll dimer P, which is the primary electron donor, and the monomeric bacteriochlorophyll BA, which is the nearest electron acceptor. Since Ile-M206 is close to the C2-acetyl group of bacteriochlorophyll PB, the hydroxyl group of Tyr-M210, and to the C9-keto group of bacteriochlorophyll BA, as well as to the water molecule near the latter group, this site can be used for introducing mutations in order to study mechanisms of primary photochemical processes in the RC. Previously it was shown that the IleâGlu substitution at the M204 position (analog of M206 in the RC of C. sphaeroides) in the RC of the closely related purple non-sulfur bacterium Rhodobacter capsulatus significantly affected kinetics of the P+HA- state formation, whereas the M204 IleâGln substitution led to the loss of BChl BA molecule from the complex structure. In the present work, it is shown that the single I(M206)Q or double I(M206)Q + F(M208)A amino acid substitutions in the RC of C. sphaeroides do not change the pigment composition and do not markedly influence redox potential of the primary electron donor. However, substitution of Ile M206 by Gln affected positions and amplitudes of the absorption bands of bacteriochlorophylls, increased lifetime of the primary electron donor P* excited state from 3.1 ps to 22 ps, and decreased quantum yield of the P+QA- state formation to 60%. These data suggest significant changes in the pigment-protein interactions in the vicinity of the primary electron donor P and the nearest electron acceptor BA. A considerable decrease was also noticed in the resistance of the mutant RC to thermal denaturation, which was more pronounced in the RC with the double substitution I(M206)Q + F(M208)A. This was likely associated with the disruption of the dense packing of the protein near bacteriochlorophylls PB and BA. Possible reasons for different effects of identical mutations on the properties of two highly homologous RCs from closely related purple non-sulfur bacteria are discussed.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética , Rhodobacter sphaeroides , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Bacterioclorofilas/química , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Cinética , Aminoácidos/metabolismo , Agua/metabolismo , Transporte de ElectrónRESUMEN
Chlorosomes of green bacteria can be considered as a prototype of future artificial light-harvesting devices due to their unique property of self-assembly of a large number of bacteriochlorophyll (BChl) c/d/e molecules into compact aggregates. The presence of carotenoids (Cars) in chlorosomes is very important for photoprotection, light harvesting and structure stabilization. In this work, we studied for the first time the electrochromic band shift (Stark effect) in Cars of the phototrophic filamentous green bacterium Chloroflexus (Cfx.) aurantiacus induced by fs light excitation of the main pigment, BChl c. The high accuracy of the spectral measurements permitted us to extract a small wavy spectral feature, which, obviously, can be associated with the dynamic shift of the Car absorption band. A global analysis of spectroscopy data and theoretical modeling of absorption spectra showed that near 60% of Cars exhibited a red Stark shift of ~ 25 cm-1 and the remaining 40% exhibited a blue shift. We interpreted this finding as evidence of various orientations of Car in chlorosomes. We estimated the average value of the light-induced electric field strength in the place of Car molecules as ~ 106 V/cm and the average distance between Car and the neighboring BChl c as ~ 10 Å. We concluded that the dynamics of the Car electrochromic band shift mainly reflected the dynamics of exciton migration through the chlorosome toward the baseplate within ~ 1 ps. Our work has unambiguously shown that Cars are sensitive indicators of light-induced internal electric fields in chlorosomes.