Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the H_A pocket of the RC. The presence of a 4-coordinate Zn^2 + ion in the H_A bacteriochlorin instead of BPhe results in a small decrease in the rates of the P* → P⁺H_A⁻ → P⁺Q_A⁻ electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn^2 + by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the H_A Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the H_B pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at H_A in both BChl- and Zn-BChl-containing RCs found in nature.     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q_a has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Q_a⁻ is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q_o of the cytochrome bc₁ complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q_a⁻ oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc₁ complex, which allows a fast transfer of quinone formed at the level of cyt bc₁ complex to the RCs. In agreement with this model, the fast phase of Q_a⁻ reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc₁. The PufX-deleted mutant displays only the slowest phase of Q_a⁻ oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc₁ and RCs.     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Sequential assembly of photosynthetic units in Rhodobacter sphaeroides as revealed by fast repetition rate analysis of variable bacteriochlorophyll a fluorescence

The development of functional photosynthetic units in Rhodobacter sphaeroides was followed by near infra-red fast repetition rate (IRFRR) fluorescence measurements that were correlated to absorption spectroscopy, electron microscopy and pigment analyses. To induce the formation of intracytoplasmic membranes (ICM) (greening), cells grown aerobically both in batch culture and in a carbon-limited chemostat were transferred to semiaerobic conditions. In both aerobic cultures, a low level of photosynthetic complexes was observed, which were composed of the reaction center and the LH1 core antenna. Interestingly, in the batch cultures the reaction centers were essentially inactive in forward electron transfer and exhibited low photochemical yields F_V/F_M, whereas the chemostat culture displayed functional reaction centers with a rather rapid (1-2 ms) electron transfer turnover, as well as a high F_V/F_M of ∼0.8. In both cases, the transfer to semiaerobiosis resulted in rapid induction of bacteriochlorophyll a synthesis that was reflected by both an increase in the number of LH1-reaction center and peripheral LH2 antenna complexes. These studies establish that photosynthetic units are assembled in a sequential manner, where the appearance of the LH1-reaction center cores is followed by the activation of functional electron transfer, and finally by the accumulation of the LH2 complexes.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides

The reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides has been studied with respect to the kinetics of charge recombination and to the phospholipid and ubiquinone (UQ) complements tightly associated with it. In the antenna-RC complexes, at 6.5 < pH < 9.0, P⁺Q_B⁻ recombines with a pH independent average rate constant <k> more than three times smaller than that measured in LH1-deprived RCs. At increasing pH values, for which <k> increases, the deceleration observed in RC-LH1 complexes is reduced, vanishing at pH > 11.0. In both systems kinetics are described by a continuous rate distribution, which broadens at pH > 9.5, revealing a strong kinetic heterogeneity, more pronounced in the RC-LH1 complex. In the presence of the antenna the Q_AQ_B⁻ state is stabilized by about 40 meV at 6.5 < pH < 9.0, while it is destabilized at pH > 11. The phospholipid/RC and UQ/RC ratios have been compared in chromatophore membranes, in RC-LH1 complexes and in the isolated peripheral antenna (LH2). The UQ concentration in the lipid phase of the RC-LH1 complexes is about one order of magnitude larger than the average concentration in chromatophores and in LH2 complexes. Following detergent washing RC-LH1 complexes retain 80-90 phospholipid and 10-15 ubiquinone molecules per monomer. The fractional composition of the lipid domain tightly bound to the RC-LH1 (determined by TLC and ³¹P-NMR) differs markedly from that of chromatophores and of the peripheral antenna. The content of cardiolipin, close to 10% weight in chromatophores and LH2 complexes, becomes dominant in the RC-LH1 complexes. We propose that the quinone and cardiolipin confinement observed in core complexes reflects the in vivo heterogeneous distributions of these components. Stabilization of the charge separated state in the RC-LH1 complexes is tentatively ascribed to local electrostatic perturbations due to cardiolipin.     

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: The qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700⁺ and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP⁺-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 μmol photons m⁻² s⁻¹ did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to ‘close’ all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q_A a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q_B-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.     

The chlorophyll a fluorescence induction pattern in chloroplasts upon repetitive single turnover excitations: Accumulation and function of QB-nonreducing centers

The increase of chlorophyll fluorescence yield in chloroplasts in a 12.5 Hz train of saturating single turnover flashes and the kinetics of fluorescence yield decay after the last flash have been analyzed. The approximate twofold increase in F_m relative to F_o, reached after 30-40 flashes, is associated with a proportional change in the slow (1-20 s) component of the multiphasic decay. This component reflects the accumulation of a sizeable fraction of Q_B-nonreducing centers. It is hypothesized that the generation of these centers occurs in association with proton transport across the thylakoid membrane. The data are quantitatively consistent with a model in which the fluorescence quenching of Q_B-nonreducing centers is reversibly released after second excitation and electron trapping on the acceptor side of Photosystem II.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Secondary metabolites from Eurotium species, Aspergillus calidoustus and A. insuetus common in Canadian homes with a review of their chemistry and biological activities

As part of studies of metabolites from fungi common in the built environment in Canadian homes, we investigated metabolites from strains of three Eurotium species, namely E. herbariorum, E. amstelodami, and E. rubrum as well as a number of isolates provisionally identified as Aspergillus ustus. The latter have been recently assigned as the new species A. insuetus and A. calidoustus. E. amstelodami produced neoechinulin A and neoechinulin B, epiheveadride, flavoglaucin, auroglaucin, and isotetrahydroauroglaucin as major metabolites. Minor metabolites included echinulin, preechinulin and neoechinulin E. E. rubrum produced all of these metabolites, but epiheveadride was detected as a minor metabolite. E. herbariorum produced cladosporin as a major metabolite, in addition to those found in E. amstelodami. This species also produced questin and neoechinulin E as minor metabolites. This is the first report of epiheveadride occurring as a natural product, and the first nonadride isolated from Eurotium species. Unlike strains from mainly infection-related samples, largely from Europe, neither ophiobolins G and H nor austins were detected in the Canadian strains of A. insuetus and A. calidoustus tested, all of which had been reported from the latter species. TMC-120 A, B, C and a sesquiterpene drimane are reported with certainty for the first time from indoor isolates, as well as two novel related methyl isoquinoline alkaloids.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Egg production of the copepod Acartia bifilosa in two contrasting European estuaries in relation to seston composition

The egg production of the copepod Acartia bifilosa was measured and related to environmental variables and food availability in two estuaries located in the same biogeographic region (Bay of Biscay) but showing very strong differences in abiotic and biotic features: the Gironde estuary (France) and the estuary of Mundaka (Spain). The study was conducted during the spring-summer-autumn period of 1994. Food availability was evaluated by analysing the chlorophyll a (Chl a), the particulate organic carbon (POC) and the easily extractable macromolecular compounds such as proteins, carbohydrates and lipids of the seston. The egg production of copepods was estimated from field incubations with natural water, and phytoplankton feeding of adult females was estimated by means of the gut fluorescence method. The nutritional environment of the Gironde was characterised by high amounts of suspended particulate matter (SPM) with low food value, emphasising the mainly detrital origin of the organic matter (OM). In Mundaka, the higher contribution of phytoplankton to the seston led to marked increases in particulate food value accounting for up to 35% of organic matter. The weight-specific egg production was found to be sharply higher in Mundaka (ranging from 0.2 to 0.63×10⁻³ day⁻¹) than in the Gironde (ranging from 0 to 0.13×10⁻³ day⁻¹), but the seasonal trend of variations was similar, the highest weight-specific egg production rates occurring in early summer and the lowest in autumn in both estuaries. Egg production was not correlated linearly with temperature since maximal egg production occurred at intermediate temperatures. In Mundaka, the egg production showed a significant positive correlation with the chlorophyll and the Chl/SPM and the POC/SPM ratios. This coupled with higher values of algal food availability (Chl a/SPM: 10 to 1870 μg g⁻¹) and gut fluorescence (between 0.12 and 0.38 ng Chl a Eq ind⁻¹) indicate that a herbivorous diet could cover the energy requirements of A. bifilosa and support egg production. In the Gironde, the algal food availability and the gut fluorescence were lower (Chl a/SPM: 10 to 80 μg g⁻¹; GF: 0.09 and 0.25 ng Chl a Eq ind⁻¹), and the egg production showed significant positive correlation with the particulate food value, suggesting that other sources of carbon rather than phytoplankton were responsible for the observed changes in egg production. Results indicate that the particular seston properties of each system may be responsible for the noticeable differences in A. bifilosa fertility among estuaries.     

kLa of stirred tank bioreactors revisited

By means of improved feedback control k_La measurements become possible at a precision and reproducibility that now allow a closer look at the influences of power input and aeration rate on the oxygen mass transfer. These measurements are performed online during running fermentations without a notable impact on the biochemical conversion processes. A closer inspection of the mass transfer during cultivations showed that at least the number of impellers influences mass transfer and mixing: On the laboratory scale, two hollow blade impellers clearly showed a larger k_La than the usually employed three impeller versions when operated at the same agitation power and aeration rate. Hollow blade impellers are preferable under most operational conditions because of their perfect gas handling capacity. Mixing time studies showed that these two impeller systems are also preferable with respect to mixing. Furthermore the widths of the baffle bars depict a significant influence on the k_La. All this clearly supports the fact that it is not only the integral power density that finally determines k_La.     

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA1 or psbA3 gene

The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA₃ gene compared to that deduced from the psbA₁ gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA₁ and psbA₂ genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of Pheo_D1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S₂Q_A^−• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the Q_B pocket. The temperature dependences of the S₂Q_A^−• charge recombination show that the strength of the H-bond to Pheo_D1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of Q_A (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P₆₈₀^+• and Y_Z is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P₆₈₀/P₆₈₀^+• couple (and hence that of ¹P₆₈₀^*/P₆₈₀^+•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.     

Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis

Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O₂ (mg Chl a)^− 1 h^− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl₂. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.