Quinone-dependent delayed fluorescence from the reaction center of photosynthetic bacteria

Millisecond delayed fluorescence from the isolated reaction center of photosynthetic bacteria Rhodobacter sphaeroides was measured after single saturating flash excitation and was explained by thermal repopulation of the excited bacteriochlorophyll dimer from lower lying charge separated states. Three exponential components (fastest, fast, and slow) were found with lifetimes of 1.5, 102, and 865 ms and quantum yields of 6.4 x 10(-9), 2.2 x 10(-9), and 2.6 x 10(-9) (pH 8.0), respectively. While the two latter phases could be related to transient absorption changes, the fastest one could not. The fastest component, dominating when the primary quinone was prereduced, might be due to a small fraction of long-lived triplet states of the radical pair and/or the dimer. The fast phase observed in the absence of the secondary quinone, was sensitive to pH, temperature, and the chemical nature of the primary quinone. The standard free energy of the primary stable charge pair relative to that of the excited dimer was -910 +/- 20 meV at pH 8 and with native ubiquinone, and it showed characteristic changes upon pH and quinone replacement. The interaction energy ( approximately 50 meV) between the cluster of the protonatable groups around GluL212 and the primary semiquinone provides evidence for functional linkage between the two quinone binding pockets. An empirical relationship was found between the in situ free energy of the primary quinone and the rate of charge recombination, with practical importance in the estimation of the free energy levels from the easily available lifetime of the charge recombination. The ratio of the slow and fast components could be used to determine the pH dependence of the free energy level of the secondary stable charge pair relative to that of the excited dimer.     

HPLC purification of a biologically active membrane protein: the reaction center from photosynthetic bacteria

Reaction centers from Rhodopseudomonas sphaeroides R 26 have been isolated from a crude extract obtained by lauryldimethylamine oxide extraction of chromatophore membranes, by HPLC using a combination of surface-mediated and size exclusion chromatography. The eluted RCs exhibit a normal activity (t 1/2 of the back-reaction is 70 ms) and are recovered in good yield (over 50% based on the activity) and purity (based on the A 280 nm/A 800 nm = 1.30 +/- 0.05 ratio and the characteristic 3 polypeptides SDS-PAGE pattern). The elution time (5-10 mn) is about two orders of magnitude faster than for the classical purification techniques.     

Structure of the membrane-bound protein photosynthetic reaction center from Rhodobacter sphaeroides

The structure of the photosynthetic reaction center (RC) from Rhodobacter sphaeroides was determined at 3.1-A resolution by the molecular replacement method, using the Rhodopseudomonas viridis RC as the search structure. Atomic coordinates were refined with the difference Fourier method and restrained least-squares refinement techniques to a current R factor of 22%. The tertiary structure of the RC complex is stabilized by hydrophobic interactions between the L and M chains, by interactions of the pigments with each other and with the L and M chains, by residues from the L and M chains that coordinate to the Fe2+, by salt bridges that are formed between the L and M chains and the H chain, and possibly by electrostatic forces between the ends of helices. The conserved residues at the N-termini of the L and M chains were identified as recognition sites for the H chain.     

Bacteriopheophytin c in reaction center complexes of green photosynthetic bacteria

Two reaction center complexes prepared from cytoplasmic membranes of Chlorobium limicola f. thiosulfato-philum were compared by absorption and CD spectrophotometry. Bacteriopheophytin c (670 nm), which is optically active in one complex but not in the other, may serve as a secondary electron acceptor in the reaction center.     

Phylogeny of the PscB reaction center protein from green sulfur bacteria

The reaction center (RC) of green sulfur bacteria has iron—sulfur clusters as terminal acceptors and is related to the Type I RC found in Heliobacter sp. and in Photosystem I (PS I) of green plants and cyanobacteria. Degenerate primers were used to retrieve the genes coding for one of the RC proteins, PscB, from 11 strains of green sulfur bacteria. PCR using the same primers gave no product with a second group of strains and the protein from these strains did not crossreact with antibodies raised against purified PscB from the first group, suggesting the presence of a high degree of variability. The sequences shared a high degree of similarity in the region coding for the binding motif for the 4Fe–4S centers. However, the N-terminal portion of the deduced protein sequences was highly variable and contained a highly positively charged, low-complexity region with repeated tetrapeptides with two alanines flanked by proline or lysine. The PscB sequences obtained fell into two major groups, and the results suggested a lack of correlation between the pigmentation of the chlorosome antenna system and the reaction center protein. There is also a lack of correlation between the grouping of the pscB sequences and the phylogeny deduced from 16S rRNA.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effects of photosynthetic reaction center H protein domain mutations on photosynthetic properties and reaction center assembly in Rhodobacter sphaeroides

Purple bacterial photosynthetic reaction center (RC) H proteins comprise three cellular domains: an 11 amino acid N-terminal sequence on the periplasmic side of the inner membrane; a single transmembrane alpha-helix; and a large C-terminal, globular cytoplasmic domain. We studied the roles of these domains in Rhodobacter sphaeroides RC function and assembly, using a mutagenesis approach that included domain swapping with Blastochloris viridis RC H segments and a periplasmic domain deletion. All mutations that affected photosynthesis reduced the amount of the RC complex. The RC H periplasmic domain is shown to be involved in the accumulation of the RC H protein in the cell membrane, while the transmembrane domain has an additional role in RC complex assembly, perhaps through interactions with RC M. The RC H cytoplasmic domain also functions in RC complex assembly. There is a correlation between the amounts of membrane-associated RC H and RC L, whereas RC M is found in the cell membrane independently of RC H and RC L. Furthermore, substantial amounts of RC M and RC L are found in the soluble fraction of cells only when RC H is present in the membrane. We suggest that RC M provides a nucleus for RC complex assembly, and that a RC H/M/L assemblage results in a cytoplasmic pool of soluble RC M and RC L proteins to provide precursors for maximal production of the RC complex.     

Electron transfer and protein dynamics in the photosynthetic reaction center.

We have measured the kinetics of electron transfer (ET) from the primary quinone (Q(A)) to the special pair (P) of the reaction center (RC) complex from Rhodobacter sphaeroides as a function of temperature (5-300 K), illumination protocol (cooled in the dark and under illumination from 110, 160, 180, and 280 K), and warming rate (1.3 and 13 mK/s). The nonexponential kinetics are interpreted with a quantum-mechanical ET model (Fermi's golden rule and the spin-boson model), in which heterogeneity of the protein ensemble, relaxations, and fluctuations are cast into a single coordinate that relaxes monotonically and is sensitive to all types of relaxations caused by ET. Our analysis shows that the structural changes that occur in response to ET decrease the free energy gap between donor and acceptor states by 120 meV and decrease the electronic coupling between donor and acceptor states from 2.7 x 10(-4) cm(-1) to 1.8 x 10(-4) cm(-1). At cryogenic temperatures, conformational changes can be slowed or completely arrested, allowing us to monitor relaxations on the annealing time scale (approximately 10(3)-10(4) s) as well as the time scale of ET (approximately 100 ms). The relaxations occur within four broad tiers of conformational substates with average apparent Arrhenius activation enthalpies of 17, 50, 78, and 110 kJ/mol and preexponential factors of 10(13), 10(15), 10(21), and 10(25) s(-1), respectively. The parameterization provides a prediction of the time course of relaxations at all temperatures. At 300 K, relaxations are expected to occur from 1 ps to 1 ms, whereas at lower temperatures, even broader distributions of relaxation times are expected. The weak dependence of the ET rate on both temperature and protein conformation, together with the possibility of modeling heterogeneity and dynamics with a single conformational coordinate, make RC a useful model system for probing the dynamics of conformational changes in proteins.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

Electron transfer between the quinones in the photosynthetic reaction center and its coupling to conformational changes

The electron transfer between the two quinones Q(A) and Q(B) in the bacterial photosynthetic reaction center (bRC) is coupled to a conformational rearrangement. Recently, the X-ray structures of the dark-adapted and light-exposed bRC from Rhodobacter sphaeroides were solved, and the conformational changes were characterized structurally. We computed the reaction free energy for the electron transfer from to Q(B) in the X-ray structures of the dark-adapted and light-exposed bRC from Rb. sphaeroides. The computation was done by applying an electrostatic model using the Poisson-Boltzmann equation and Monte Carlo sampling. We accounted for possible protonation changes of titratable groups upon electron transfer. According to our calculations, the reaction energy of the electron transfer from to Q(B) is +157 meV for the dark-adapted and -56 meV for the light-exposed X-ray structure; i.e., the electron transfer is energetically uphill for the dark-adapted structure and downhill for the light-exposed structure. A common interpretation of experimental results is that the electron transfer between and Q(B) is either gated or at least influenced by a conformational rearrangement: A conformation in which the electron transfer from to Q(B) is inactive, identified with the dark-adapted X-ray structure, changes into an electron-transfer active conformation, identified with the light-exposed X-ray structure. This interpretation agrees with our computational results if one assumes that the positive reaction energy for the dark-adapted X-ray structure effectively prevents the electron transfer. We found that the strongly coupled pair of titratable groups Glu-L212 and Asp-L213 binds about one proton in the dark-adapted X-ray structure, where the electron is mainly localized at Q(A), and about two protons in the light-exposed structure, where the electron is mainly localized at Q(B). This finding agrees with recent experimental and theoretical studies. We compare the present results for the bRC from Rb. sphaeroides to our recent studies on the bRC from Rhodopseudomonas viridis. We discuss possible mechanisms for the gated electron transfer from to Q(B) and relate them to theoretical and experimental results.     

pH modulates the quinone position in the photosynthetic reaction center from Rhodobacter sphaeroides in the neutral and charge separated states

The structure of the photosynthetic reaction-center from Rhodobacter sphaeroides has been determined at four different pH values (6.5, 8.0, 9.0, 10.0) in the neutral and in charge separated states. At pH 8.0, in the neutral state, we obtain a resolution of 1.87 A, which is the best ever reported for the bacterial reaction center protein. Our crystallographic data confirm the existence of two different binding positions of the secondary quinone (QB). We observe a new orientation of QB in its distal position, which shows no ring-flip compared to the orientation in the proximal position. Datasets collected for the different pH values show a pH-dependence of the population of the proximal position. The new orientation of QB in the distal position and the pH-dependence could be confirmed by continuum electrostatics calculations. Our calculations are in agreement with the experimentally observed proton uptake upon charge separation. The high resolution of our crystallographic data allows us to identify new water molecules and external residues being involved in two previously described hydrogen bond proton channels. These extended proton-transfer pathways, ending at either of the two oxo-groups of QB in its proximal position, provide additional evidence that ring-flipping is not required for complete protonation of QB upon reduction.     

Characterization of protein matrix motions in the Rb. sphaeroides photosynthetic reaction center

We use Normal Mode Analysis to investigate motions in the photosynthetic reaction center (RC) protein. We identify the regions involved in concerted fluctuations of the protein matrix and analyze the normalized amplitudes and the directionality of the first few dominant modes. We also seek to quantify the coupling of normal modes to long-range electron transfer (ET). We find that a quasi-continuous spectrum of protein motions rather than one individual mode contributes to light-driven electron transfer. This is consistent with existing theoretical models (e.g. the spin-boson/dispersed polaron model) for the coupling of the protein and solvent "bath" to charge separation events. [Figure: see text].     

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-d-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Resolution and reconstitution of a bound Fe-S protein from the photosynthetic reaction center of Heliobacterium modesticaldum

The photosynthetic reaction center of Heliobacterium modesticaldum (HbRC) was isolated from membranes using n-dodecyl beta-D-maltopyranoside followed by sucrose density ultracentrifugation. The low-temperature EPR spectra of whole cells, isolated membranes, and HbRC complexes are similar, showing a single Fe-S cluster with g values of 2.067, 1.933, and 1.890 after illumination at 20 K, and a complex spectrum attributed to exchange interaction from two Fe-S clusters after illumination during freezing. The protein containing the Fe-S clusters was removed from the HbRC by washing it with 1.0 M NaCl and purified by ultrafiltration over a 30 kDa cutoff membrane. Analysis of the filtrate by SDS-PAGE showed a major band at approximately 8 kDa that was weakly stained with Coomassie Brilliant Blue and strongly stained with silver. The optical spectrum of the oxidized Fe-S protein shows a maximum at 410 nm, and the EPR spectrum of the reduced Fe-S protein shows a complex set of resonances similar to those found in 2[4Fe-4S] ferredoxins. The HbRC core was purified by DEAE ion-exchange chromatography and resolved by SDS-PAGE. The purified HbRC was composed of a band at ca. 40 kDa, which is identified as PshA, and several additional proteins. The isolated Fe-S protein rebinds spontaneously to purified HbRC cores, and the light-induced EPR signals of the Fe-S clusters are recovered. The flash-induced kinetics of the HbRC complex show two kinetic phases at room temperature, one with a lifetime of 75 ms and the other with a lifetime of 15 ms. The 75 ms component is lost when the Fe-S protein is removed from the HbRC complex, and it is regained when the Fe-S protein is rebound to HbRC cores. Thus, the 75 ms kinetic phase is derived from recombination of a terminal Fe-S cluster with P798(+), and the 15 ms kinetic phase is derived from recombination with an earlier acceptor, probably F(X). We suggest that the bound Fe-S protein present in the HbRC be designated PshB.