Rhodobacter Sphaeroides Research Articles

An ancient metabolite damage-repair system sustains photosynthesis in plants

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major catalyst in the conversion of carbon dioxide into organic compounds in photosynthetic organisms. However, its activity is impaired by binding of inhibitory sugars such as xylulose-1,5-bisphosphate (XuBP), which must be detached from the active sites by Rubisco activase. Here, we show that loss of two phosphatases in Arabidopsis thaliana has detrimental effects on plant growth and photosynthesis and that this effect could be reversed by introducing the XuBP phosphatase from Rhodobacter sphaeroides. Biochemical analyses revealed that the plant enzymes specifically dephosphorylate XuBP, thus allowing xylulose-5-phosphate to enter the Calvin-Benson-Bassham cycle. Our findings demonstrate the physiological importance of an ancient metabolite damage-repair system in degradation of by-products of Rubisco, and will impact efforts to optimize carbon fixation in photosynthetic organisms.

Delocalized electronic excitations and their role in directional charge transfer in the reaction center of Rhodobacter sphaeroides.

In purple bacteria, the fundamental charge-separation step that drives the conversion of radiation energy into chemical energy proceeds along one branch-the A branch-of a heterodimeric pigment-protein complex, the reaction center. Here, we use first principles time-dependent density functional theory (TDDFT) with an optimally-tuned range-separated hybrid functional to investigate the electronic and excited-state structure of the six primary pigments in the reaction center of Rhodobacter sphaeroides. By explicitly including amino-acid residues surrounding these six pigments in our TDDFT calculations, we systematically study the effect of the protein environment on energy and charge-transfer excitations. Our calculations show that a forward charge transfer into the A branch is significantly lower in energy than the first charge transfer into the B branch, in agreement with the unidirectional charge transfer observed experimentally. We further show that the inclusion of the protein environment redshifts this excitation significantly, allowing for energy transfer from the coupled Qx excitations. Through analysis of transition and difference densities, we demonstrate that most of the Q-band excitations are strongly delocalized over several pigments and that both their spatial delocalization and charge-transfer character determine how strongly affected they are by thermally-activated molecular vibrations. Our results suggest a mechanism for charge-transfer in this bacterial reaction center and pave the way for further first-principles investigations of the interplay between delocalized excited states, vibronic coupling, and the role of the protein environment in this and other complex light-harvesting systems.

Mechanism of Asparagine-Mediated Proton Transfer in Photosynthetic Reaction Centers.

In photosynthetic reaction centers from purple bacteria (PbRCs), light-induced charge separation leads to the reduction of the terminal electron acceptor quinone, QB. The reduction of QB to QB•- is followed by protonation via Asp-L213 and Ser-L223 in PbRC from Rhodobacter sphaeroides. However, Asp-L213 is replaced with nontitratable Asn-L222 and Asn-L213 in PbRCs from Thermochromatium tepidum and Blastochloris viridis, respectively. Here, we investigated the energetics of proton transfer along the asparagine-involved H-bond network using a quantum mechanical/molecular mechanical approach. The potential energy profile for the H-bond between H3O+ and the carbonyl O site of Asn-L222 shows that the proton is predominantly localized at the Asn-L222 moiety in the T. tepidum PbRC protein environment, easily forming the enol species. The release of the proton from the amide -NH2 site toward Ser-L232 via tautomerization suffers from the energy barrier. Upon reorientation of Asn-L222, the enol -OH site forms a short low-barrier H-bond with Ser-L232, facilitating protonation of QB•- in a Grotthuss-like mechanism. This is a basis of how asparagine or glutamine side chains function as acceptors/donors in proton transfer pathways.

Chlorophyll a Synthesis in Rhodobacter sphaeroides by Chlorophyll Synthase of Nicotiana tabacum.

The production of phytylated chlorophyll a (Chl aP) in Rhodobacter sphaeroides, which uses phytylated bacteriochlorophyll a (BChl aP), is the first step in expanding the light absorption spectra. Unlike the chlorophyll synthase (ChlG) of the Synechocystis sp. PCC6803, ChlGs of angiosperms, including Arabidopsis thaliana, Nicotiana tabacum, Avena sativa, and Oryza sativa, showed bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorophyllide a (BChlide a), geranylgeranylated BChl a (BChl aGG), and BChl aP, collectively called bacteriochlorins. Among the angiosperm ChlGs, N. tabacum ChlG had the highest bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorins. Expression of N. tabacum chlG in R. sphaeroides resulted in the formation of free Chl aP in the presence of BChl aP during photoheterotrophic growth, even though reactive oxygen species were generated.

PEG effects on excitonic properties of LH2 from Rhodobacter sphaeroides 2.4.1 in different environments

The effects of Polyethylene glycol (PEG) on B800 and B850 excitonic properties of the Light-Harvesting complex 2 (LH2) from Rhodobacter (Rba.) sphaeroides 2.4.1 in micellar solutions of detergents and phospholipid bilayers were investigated by a series of spectroscopies. Compared to the micellar solution, in the lipid bilayer, the lateral force applied on LH2 changed the excitonic structure of B850 and weakened the excitonic interaction between B800 molecules. In micelles, PEG addition causes LH2 aggregation, resulting in the enhancement of B850 excitonic coupling inter-complexes and conformational change of the bound carotenoid (Car). In liposomes, PEG addition causes ∼37 % fluorescence quenching of B850 excitons in the embedded LH2s by changing the surrounding hydration state.

Ribonuclease E strongly impacts bacterial adaptation to different growth conditions

ABSTRACT Adaptation of bacteria to changes in their environment is often accomplished by changes of the transcriptome. While we learned a lot on the impact of transcriptional regulation in bacterial adaptation over the last decades, much less is known on the role of ribonucleases. This study demonstrates an important function of the endoribonuclease RNase E in the adaptation to different growth conditions. It was shown previously that RNase E activity does not influence the doubling time of the facultative phototroph Rhodobacter sphaeroides during chemotrophic growth, however, it has a strong impact on phototrophic growth. To better understand the impact of RNase E on phototrophic growth, we now quantified gene expression by RNA-seq and mapped 5’ ends during chemotrophic growth under high oxygen or low oxygen levels and during phototrophic growth in the wild type and a mutant expressing a thermosensitive RNase E. Based on the RNase E-dependent expression pattern, the RNAs could be grouped into different classes. A strong effect of RNase E on levels of RNAs for photosynthesis genes was observed, in agreement with poor growth under photosynthetic conditions. RNase E cleavage sites and 5’ ends enriched in the rne ts mutant were differently distributed among the gene classes. Furthermore, RNase E affects the level of RNAs for important transcription factors thus indirectly affecting the expression of their regulons. As a consequence, RNase E has an important role in the adaptation of R. sphaeroides to different growth conditions.

Conversion of light into electricity in a semi-synthetic system based on photosynthetic bacterial chromatophores

Chromatophores (Chr) from photosynthetic nonsulfur purple bacterium Rhodobacter sphaeroides immobilized onto a Millipore membrane filter (MF) and sandwiched between two semiconductor indium tin oxide (ITO) electrodes (termed ITO|Chr – MF|ITO) have been used to measure voltage (ΔV) induced by continuous illumination. The maximum ΔV was detected in the presence of ascorbate / N,N,N’N'-tetramethyl-p-phenylenediamine couple, coenzyme UQ0, disaccaride trehalose and antimycin A, an inhibitor of cytochrome bc1 complex. In doing so, the light-induced electron transfer in the reaction centers was the major source of photovoltages. The stability of the voltage signal upon prolonged irradiation (>1 h) may be due to the maintenance of a conformation that is optimal for the functioning of integral protein complexes and stabilization of lipid bilayer membranes in the presence of trehalose. Retaining ∼70 % of the original photovoltage performance on the 30th day of storage at 23 °C in the dark under air was achieved after re-injection of fresh buffer (∼40 μL) containing redox mediators into the ITO|Chr – MF|ITO system. The approach we use is easy and can be extended to other biological intact systems (cells, thylakoid membranes) capable of converting energy of light.

The Photoactive Photosynthetic Reaction Center of a Rhodobacter sphaeroides Mutant Lacking 3-Vinyl (Bacterio)Chlorophyllide a Hydratase Contains 3-Vinyl Bacteriochlorophyll a.

Rhodobacter sphaeroides mutant BF-lacking 3-vinyl (bacterio)chlorophyllide a hydratase (BchF)-accumulates chlorophyllide a (Chlide a) and 3-vinyl bacteriochlorophyllide a (3V-Bchlide a). BF synthesizes 3-vinyl bacteriochlorophyll a (3V-Bchl a) through prenylation of 3V-Bchlide a and assembles a novel reaction center (V-RC) using 3V-Bchl a and Mg-free 3-vinyl bacteriopheophytin a (3V-Bpheo a) at a molar ratio of 2:1. We aimed to verify whether a bchF-deleted R. sphaeroides mutant produces a photochemically active RC that facilitates photoheterotrophic growth. The mutant grew photoheterotrophically-implying a functional V-RC-as confirmed by the emergence of growth-competent suppressors of bchC-deleted mutant (BC) under irradiation. Suppressor mutations in BC were localized to bchF, which diminished BchF activity and caused 3V-Bchlide a accumulation. bchF expression carrying the suppressor mutations in trans resulted in the coproduction of V-RC and wild-type RC (WT-RC) in BF. The V-RC had a time constant (τ) for electron transfer from the primary electron donor P (a dimer of 3V-Bchl a) to the A-side containing 3V-Bpheo a (HA) similar to that of the WT-RC and a 60% higher τ for electron transfer from HA to quinone A (QA). Thus, the electron transfer from HA to QA in the V-RC should be slower than that in the WT-RC. Furthermore, the midpoint redox potential of P/P+ of the V-RC was 33 mV more positive than that of the WT-RC. R. sphaeroides, thus, synthesizes the V-RC when 3V-Bchlide a accumulates. The V-RC can support photoheterotrophic growth; however, its photochemical activity is inferior to that of the WT-RC. IMPORTANCE 3V-Bchlide a is an intermediate in the bacteriochlorophyll a (Bchl a)-specific biosynthetic branch and prenylated by bacteriochlorophyll synthase. R. sphaeroides synthesizes V-RC that absorbs light at short wavelengths. The V-RC was not previously discovered because 3V-Bchlide a does not accumulate during the growth of WT cells synthesizing Bchl a. The levels of reactive oxygen species increased with the onset of photoheterotrophic growth in BF, resulting in a long lag period. Although the inhibitor of BchF is unknown, the V-RC may act as a substitute for the WT-RC when BchF is completely inhibited. Alternatively, it may act synergistically with WT-RC at low levels of BchF activity. The V-RC may broaden the absorption spectra of R. sphaeroides and supplement its photosynthetic ability at various wavelengths of visible light to a greater extent than that by the WT-RC alone.

From primordial clocks to circadian oscillators

Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator1. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock2. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood3–6, but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism7–9. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides, which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.

New TSPO Crystal Structures of Mutant and Heme-Bound Forms with Altered Flexibility, Ligand Binding, and Porphyrin Degradation Activity.

The ancient protein TSPO (translocator protein 18kD) is found in all kingdoms and was originally identified as a binding site of benzodiazepine drugs. Its physiological function remains unclear, although porphyrins are conserved ligands. Several crystal structures of bacterial TSPO and nuclear magnetic resonance structures of a mouse form have revealed monomer and dimer configurations, but there have been no reports of structures with a physiological ligand. Here, we present the first X-ray structures of Rhodobacter sphaeroides TSPO with a physiological ligand bound. Two different variants (substituting threonine for alanine at position 139 (A139T) and phenylalanine for alanine at position 138 (A138F)) yielded well-diffracting crystals giving structures of both apo- and heme-containing forms. Both variants have wild-type micromolar affinity for heme and protoporphyrin IX, but A139T has very low ability to accelerate the breakdown of porphyrin in the presence of light and oxygen. The binding of heme to one protomer of the dimer of either mutant induces a more rigid structure, both in the heme-binding protomer and the protomer without heme bound, demonstrating an allosteric response. Ensemble refinement of the X-ray data reveals distinct regions of altered flexibility in response to single heme binding to the dimer. The A139T variant shows a more rigid structure overall, which may relate to extra hydrogen bonding of waters captured in the heme crevice. As TSPO has been suggested to have a role in heme delivery from mitochondria to the cytoplasm, the new structures provide potential clues regarding the structural basis of such activity.

Cryo-EM structure of the four-subunit Rhodobacter sphaeroides cytochrome bc1 complex in styrene maleic acid nanodiscs

Cytochrome bc1 complexes are ubiquinol:cytochrome c oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome b, cytochrome c1, and the Rieske iron-sulfur subunit, but the function of mitochondrial cytochrome bc1 complexes is modified by up to eight supernumerary subunits. The cytochrome bc1 complex from the purple phototrophic bacterium Rhodobactersphaeroides has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene-maleic acid copolymer to purify the R. sphaeroides cytochrome bc1 complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome bc1 complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome c1 subunits. We observe a quinone at the Qo quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome b subunits, with some spanning both of the two monomers that make up the dimeric complex.

Supramolecular Biohybrid Construct for Photoconversion Based on a Bacterial Reaction Center Covalently Bound to Cytochrome c by an Organic Light Harvesting Bridge.

A supramolecular construct for solar energy conversion is developed by covalently bridging the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides and cytochrome c (Cyt c) proteins with a tailored organic light harvesting antenna (hCy2). The RC-hCy2-Cyt c biohybrid mimics the working mechanism of biological assemblies located in the bacterial cell membrane to convert sunlight into metabolic energy. hCy2 collects visible light and transfers energy to the RC, increasing the rate of photocycle between a RC and Cyt c that are linked in such a way that enhances proximity without preventing protein mobility. The biohybrid obtained with average 1 RC/10 hCy2/1.5 Cyt c molar ratio features an almost doubled photoactivity versus the pristine RC upon illumination at 660 nm, and ∼10 times higher photocurrent versus an equimolar mixture of the unbound proteins. Our results represent an interesting insight into photoenzyme chemical manipulation, opening the way to new eco-sustainable systems for biophotovoltaics.

Intracytoplasmic-membrane development in alphaproteobacteria involves the homolog of the mitochondrial crista-developing protein Mic60

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R.sphaeroides. We show that the three α helices of mitochondrial Mic60's mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs and have therefore improved our understanding of the nature of the mitochondrial ancestor.

Calculated vibrational properties of pigments in protein binding sites 2: Semiquinones in photosynthetic proteins

[QA– – QA] Fourier transform infrared difference spectra have previously been obtained using purple bacterial reaction centers from Rhodobacter sphaeroides with unlabeled, 18O and 13C isotope labeled phylloquinone (PhQ, also known as vitamin K1) incorporated into the QA protein binding site (Breton, (1997), Proc. Natl. Acad. Sci. USA94 11318–11323). The nature of the bands in these spectra and the isotope induced band shifts are poorly understood, especially for the phyllosemiquinone anion (PhQ–) state. To aid in the interpretation of the bands in these experimental spectra, ONIOM type QM/MM vibrational frequency calculations were undertaken. Calculations were also undertaken for PhQ– in solution. Surprisingly, both sets of calculated spectra are similar and agree well with the experimental spectra. This similarity suggests pigment-protein interactions do not perturb the electronic structure of the semiquinone in the QA binding site. This is not found to be the case for the neutral PhQ species in the same protein binding site. PhQ also occupies the A1 protein binding site in photosystem I, and the vibrational properties of PhQ– in the QA and A1 binding sites are compared and shown to exhibit considerable differences. These differences probably arise because of changes in the degree of asymmetry of hydrogen bonding of PhQ– in the A1 and QA binding sites.

The exploration of hydrogen production promoting mechanism of Rhodobacter sphaeroides by expressing tetA under tetracycline stress

Photo-fermentative hydrogen production rate improving has been a key limiting link in the field of biological hydrogen production by photosynthetic bacterium (PSB) in recent years. In our previous study, apparent hydrogen production enhancement was observed in R. sphaeroides HY01 with tetA expressed under tetracycline (Tc), which attracted our interests. It was found that Tc-resistant strains always presented stable hydrogen production promotion on the maximum hydrogen production rate (Rm), which was approximately 30% with Tc addition. Figuring out the underlying connections between Tc resistance and hydrogen production of R. sphaeroides may provide new methods for obtaining engineered PSB with high hydrogen production performance. In this study, basing on experimental tests, hydrogen promotion of resistant strain was found to be Tc dose-dependent. Some relevant gene expression differences on hydrogen production of Rodobacter sphaeroides induced by the expression of TetA and the addition of Tc were also tested and discussed.

The Role of Electrostatic Binding Interfaces in the Performance of Bacterial Reaction Center Biophotoelectrodes.

Photosynthetic reaction centers (RCs) efficiently capture and convert solar radiation into electrochemical energy. Accordingly, RCs have the potential as components in biophotovoltaics, biofuel cells, and biosensors. Recent biophotoelectrodes containing the RC from the bacterium Rhodobacter sphaeroides utilize a natural electron donor, horse heart cytochrome c (cyt c), as an electron transfer mediator with the electrode. In this system, electrostatic interfaces largely control the protein-electrode and protein-protein interactions necessary for electron transfer. However, recent studies have revealed kinetic bottlenecks in cyt-mediated electron transfer that limit biohybrid photoelectrode efficiency. Here, we seek to understand how changing protein-protein and protein-electrode interactions influence RC turnover and biophotoelectrode efficiency. The RC-cyt c binding interaction was modified by substituting interfacial RC amino acids. Substitutions Asn-M188 to Asp and Gln-L264 to Glu, which are known to produce a higher cyt-binding affinity, led to a decrease in the RC turnover frequency (TOF) at the electrode, suggesting that a decrease in cyt c dissociation was rate-limiting in these RC variants. Conversely, an Asp-M88 to Lys substitution producing a lower binding affinity had little effect on the RC TOF, suggesting that a decrease in the cyt c association rate was not a rate-limiting factor. Modulating the electrode surface with a self-assembled monolayer that oriented the cyt c to face the electrode did not affect the RC TOF, suggesting that the orientation of cyt c was also not a rate-limiting factor. Changing the ionic strength of the electrolyte solution had the most potent impact on the RC TOF, indicating that cyt c mobility was important for effective electron donation to the photo-oxidized RC. An ultimate limitation for the RC TOF was that cyt c desorbed from the electrode at ionic strengths above 120 mM, diluting its local concentration near the electrode-adsorbed RCs and resulting in poor biophotoelectrode performance. These findings will guide further tuning of these interfaces for improved performance.

Mutual supply of carbon and nitrogen sources in the co-culture of aerial microalgae and nitrogen-fixing bacteria

This study investigated co-culture for supply of carbon (C) and nitrogen (N) sources by aerial microalgae and N-fixing bacteria. The aerial microalga Coelastrella sp. KGU-HN001 was isolated from the surface of steel signs in Japan. After examining monocultures of the microalga and N-fixing bacterium Rhodobacter sphaeroides, mutual nutrient supply was evaluated in co-culture using a medium without C and N sources. In co-culture under aerobic conditions, the cell number, biomass, total organic C in cells and the total N of cell suspensions increased. Carbohydrate was secreted from the microalga and nitrogenase activity was expressed by the bacterium. Addition of an N source enhanced N fixation in the co-culture. These results indicated that C and N fixation by the microalga and the bacterium occurred under aerobic conditions, and the microorganisms were able to grow by self-supplying nutrients even under nutrient deficiencies.

Rhodobacter sphaeroides as a model to study the ecotoxicity of 1-alkyl-3-methylimidazolium bromide.

The purple non-sulfur bacterium Rhodobacter sphaeroides was selected as a biological model to investigate its response to the toxicity of 1-alkyl-3-methylimidazolium bromide ([Cnmim]Br), a type of ionic liquid (IL), with different alkyl chain lengths (n describes the number of carbon atoms in the alkyl chain). The inhibition of bacterial growth by [Cnmim]Br was positively correlated with n. Morphological characterization revealed that [Cnmim]Br caused cell membrane perforation. The signal amplitude of the electrochromic absorption band shift of endogenous carotenoids showed a negatively linear correlation with n, and the amplitude of the blue-shift of the B850 band in light-harvesting complex 2 showed a positively linear correlation with n. Furthermore, an increase in blocked ATP synthesis and increase in antioxidant enzyme activity were observed in chromatophores treated with ILs containing longer alkyl chains. In summary, the purple bacterium can be developed as a model to monitor ecotoxicity and examine the mechanism of IL toxicity.

Photoinduced electron transfer in cytochrome bc1: Dynamics of rotation of the Iron-sulfur protein during bifurcated electron transfer from ubiquinol to cytochrome c1 and cytochrome bL

The electron transfer reactions within wild-type Rhodobacter sphaeroides cytochrome bc1 (cyt bc1) were studied using a binuclear ruthenium complex to rapidly photooxidize cyt c1. When cyt c1, the iron‑sulfur center Fe2S2, and cyt bH were reduced before the reaction, photooxidation of cyt c1 led to electron transfer from Fe2S2 to cyt c1 with a rate constant of ka = 80,000 s−1, followed by bifurcated reduction of both Fe2S2 and cyt bL by QH2 in the Qo site with a rate constant of k2 = 3000 s−1. The resulting Q then traveled from the Qo site to the Qi site and oxidized one equivalent each of cyt bL and cyt bH with a rate constant of k3 = 340 s−1. The rate constant ka was decreased in a nonlinear fashion by a factor of 53 as the viscosity was increased to 13.7. A mechanism that is consistent with the effect of viscosity involves rotational diffusion of the iron‑sulfur protein from the b state with reduced Fe2S2 close to cyt bL to one or more intermediate states, followed by rotation to the final c1 state with Fe2S2 close to cyt c1, and rapid electron transfer to cyt c1.

Reconstituted LH2 in multilayer membranes induced by poly-l-lysine: Structure of supramolecular and electronic states

To explore the effect of cell membrane stacking on the function of light-harvesting complex 2 (LH2) in purple nonsulfur photosynthetic bacteria, LH2 from Rhodobacter sphaeroides 2.4.1 (R. sph 2.4.1) was reconstituted into lipid bilayer vesicles (LH2@liposome) and further formed multi-layer structure by electrostatic interaction with poly-l-lysine (LH2@liposome/PLL), which was characterized by cryo-electron microscopy (cryo-EM) and TEM. When embedded in liposomes and additionally in multi-layer liposomes, the absorption band, zero-crossing point of CD signals and fluorescence emission of LH2 B850 excitons were observed to uniformly have 1–2 nm red-shifting. Combining with the corresponding fluorescence quench and the generation of shorter-living fluorescence species, a new excitonic species generated through B850 structural splitting was proposed. By FT-Raman and triplet carotenoid dynamics, the structural mechanism was deduced and discussed. Briefly, all environmental changes, including LH2 aggregating and multi-layer membrane stacking, eventually applied forces on B850 exciton molecules mainly through the hydrogen bonding between the C3-acetyl carbonyl groups of B850 BChls and Tyr44 and 45 residues at C-terminus of α-polypeptides. The effect of multi-layer structure on LH2 could be assigned as a kind of photoprotection.