Time-resolved spectroscopy of the primary photosynthetic processes of membrane-bound reaction centers from an antenna-deficient mutant of Rhodobacter capsulatus

The primary photosynthetic reactions in whole membranes of the antenna-deficient mutant strain U43 (pTXA6–10) of Rhodobacter capsulatus are studied by transient absorption and emission spectroscopy with subpicosecond time resolution. Extensive similarities between the transient absorption data on whole membranes and on isolated reaction centers support the idea that the primary processes in isolated reaction centers are not modified by the isolation procedure

Role of tyrosine M210 in the initial charge separation of reaction centers of Rhodobacter sphaeroides

Femtosecond spectroscopy was used in combination with site-directed mutagenesis to study the influence of tyrosine M210 (YM210) on the primary electron transfer in the reaction center of Rhodobacter sphaeroides. The exchange of YM210 to phenylalanine caused the time constant of primary electron transfer to increase from 3.5 f 0.4 ps to 16 f 6 ps while the exchange to leucine increased the time constant even more to 22 f 8 ps. The results suggest that tyrosine M210 is important for the fast rate of the primary electron transfer

Temperature dependence of the primary electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides

The primary electron transfer (ET) in reaction centers (RC) of Rhodobacter sphaeroides is investigated as a function of temperature with femtosecond time resolution. For temperatures from 300 to 25 K the ET to the bacteriopheophytin is characterized by a biphasic time dependence. The two time constants of τ1=3.5±0.4 ps and τ2=1.2±0.3 ps at T=300 K decrease continously with temperature to values of τ1=1.4±0.3 ps and τ2=0.3±0.15 ps at 25 K. The experimental results indicate that the ET is not thermally activated and that the same ET mechanisms are active at room and low temperatures. All observations are readily rationalized by a two-step ET model with the monomeric bacteriochlorophyll as a real electron carrier

Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre.

Photosynthesis uses a limited range of the solar spectrum, so enhancing spectral coverage could improve the efficiency of light capture. Here, we show that a hybrid reaction centre (RC)/yellow fluorescent protein (YFP) complex accelerates photosynthetic growth in the bacterium Rhodobacter sphaeroides. The structure of the RC/YFP-light-harvesting 1 (LH1) complex shows the position of YFP attachment to the RC-H subunit, on the cytoplasmic side of the RC complex. Fluorescence lifetime microscopy of whole cells and ultrafast transient absorption spectroscopy of purified RC/YFP complexes show that the YFP-RC intermolecular distance and spectral overlap between the emission of YFP and the visible-region (QX) absorption bands of the RC allow energy transfer via a Förster mechanism, with an efficiency of 40±10%. This proof-of-principle study demonstrates the feasibility of increasing spectral coverage for harvesting light using non-native genetically-encoded light-absorbers, thereby augmenting energy transfer and trapping in photosynthesis

Weak temperature dependence of P (+) H A (-) recombination in mutant Rhodobacter sphaeroides reaction centers

International audienceIn contrast with findings on the wild-type Rhodobacter sphaeroides reaction center, biexponential P (+) H A (-) → PH A charge recombination is shown to be weakly dependent on temperature between 78 and 298 K in three variants with single amino acids exchanged in the vicinity of primary electron acceptors. These mutated reaction centers have diverse overall kinetics of charge recombination, spanning an average lifetime from ~2 to ~20 ns. Despite these differences a protein relaxation model applied previously to wild-type reaction centers was successfully used to relate the observed kinetics to the temporal evolution of the free energy level of the state P (+) H A (-) relative to P (+) B A (-) . We conclude that the observed variety in the kinetics of charge recombination, together with their weak temperature dependence, is caused by a combination of factors that are each affected to a different extent by the point mutations in a particular mutant complex. These are as follows: (1) the initial free energy gap between the states P (+) B A (-) and P (+) H A (-) , (2) the intrinsic rate of P (+) B A (-) → PB A charge recombination, and (3) the rate of protein relaxation in response to the appearance of the charge separated states. In the case of a mutant which displays rapid P (+) H A (-) recombination (ELL), most of this recombination occurs in an unrelaxed protein in which P (+) B A (-) and P (+) H A (-) are almost isoenergetic. In contrast, in a mutant in which P (+) H A (-) recombination is relatively slow (GML), most of the recombination occurs in a relaxed protein in which P (+) H A (-) is much lower in energy than P (+) H A (-) . The weak temperature dependence in the ELL reaction center and a YLH mutant was modeled in two ways: (1) by assuming that the initial P (+) B A (-) and P (+) H A (-) states in an unrelaxed protein are isoenergetic, whereas the final free energy gap between these states following the protein relaxation is large (~250 meV or more), independent of temperature and (2) by assuming that the initial and final free energy gaps between P (+) B A (-) and P (+) H A (-) are moderate and temperature dependent. In the case of the GML mutant, it was concluded that the free energy gap between P (+) B A (-) and P (+) H A (-) is large at all times

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g. Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides,RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+QA− or P+QB−, respectively; (b) the kinetics of electron transfer from QA− to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (τ = 3.5 ps), followed by reduction of the bacteriopheophytin (τ = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (τ = 410 ± 30 and 47 ± 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type). EPR studies of the isolated RCs showed a characteristic g = 1.82 signal for the QA semiquinone coupled to the iron atom, which was indistinguishable from the wild type. It is concluded that GluM234 is not essential to the normal functioning of the acceptor quinone complex in bacterial RCs and that the role of bicarbonate in PS II is distinct from the role of this residue in bacterial RCs

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna.

The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl d, Chl f or bacteriochlorophyll (BChl) b to replace native BChl a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg-10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6 ps, energy transfer from Chl a to B850 BChl a remained highly efficient. We measured faster energy-transfer time constants for Chl d (3.5 ps) and Chl f (2.7 ps), which have red-shifted absorption maxima compared to Chl a. BChl b, red-shifted from the native BChl a, gave extremely rapid (≤0.1 ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans