Rational redesign of the ferredoxin-NADP+-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production.

Utilization of electrons from the photosynthetic water splitting reaction for the generation of biofuels, commodities as well as application in biotransformations requires a partial rerouting of the photosynthetic electron transport chain. Due to its rather negative redox potential and its bifurcational function, ferredoxin at the acceptor side of Photosystem 1 is one of the focal points for such an engineering. With hydrogen production as model system, we show here the impact and potential of redox partner design involving ferredoxin (Fd), ferredoxin-oxido-reductase (FNR) and [FeFe]­hydrogenase HydA1 on electron transport in a future cyanobacterial design cell of Synechocystis PCC 6803. X-ray-structure-based rational design and the allocation of specific interaction residues by NMR-analysis led to the construction of Fd- and FNR-mutants, which in appropriate combination enabled an about 18-fold enhanced electron flow from Fd to HydA1 (in competition with equimolar amounts of FNR) in in vitro assays. The negative impact of these mutations on the Fd-FNR electron transport which indirectly facilitates H2 production (with a contribution of ≤42% by FNR variants and ≤23% by Fd-variants) and the direct positive impact on the Fd-HydA1 electron transport (≤23% by Fd-mutants) provide an excellent basis for the construction of a hydrogen-producing design cell and the study of photosynthetic efficiency-optimization with cyanobacteria.

Charge separation, stabilization, and protein relaxation in photosystem II core particles with closed reaction center.

The fluorescence kinetics of cyanobacterial photosystem II (PSII) core particles with closed reaction centers (RCs) were studied with picosecond resolution. The data are modeled in terms of electron transfer (ET) and associated protein conformational relaxation processes, resolving four different radical pair (RP) states. The target analyses reveal the importance of protein relaxation steps in the ET chain for the functioning of PSII. We also tested previously published data on cyanobacterial PSII with open RCs using models that involved protein relaxation steps as suggested by our data on closed RCs. The rationale for this reanalysis is that at least one short-lived component could not be described in the previous simpler models. This new analysis supports the involvement of a protein relaxation step for open RCs as well. In this model the rate of ET from reduced pheophytin to the primary quinone Q(A) is determined to be 4.1 ns(-1). The rate of initial charge separation is slowed down substantially from approximately 170 ns(-1) in PSII with open RCs to 56 ns(-1) upon reduction of Q(A). However, the free-energy drop of the first RP is not changed substantially between the two RC redox states. The currently assumed mechanistic model, assuming the same early RP intermediates in both states of RC, is inconsistent with the presented energetics of the RPs. Additionally, a comparison between PSII with closed RCs in isolated cores and in intact cells reveals slightly different relaxation kinetics, with a approximately 3.7 ns component present only in isolated cores.

How does photosystem 2 split water? The structural basis of efficient energy conversion.

Photosystem 2 (PS2) is the part of the photosynthetic apparatus that uses light energy to split water releasing oxygen, protons and electrons. Here, we present a model of the subunit organization of PS2 and the accompanying secondary antenna systems (phycobilisomes in cyanobacteria and the light-harvesting complexes in higher plants) and discuss possible physiological consequences of the proposed dimeric structure of PS2.

Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells.

Fluorescent DeltapH and DeltaPsi indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp. PCC 6803. Acridine yellow and Acridine orange proved to be the best DeltapH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically. Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane. In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies. Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus. Different from Synechocystis sp. PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis.

Novel approaches towards characterization of the high-affinity nucleotide binding sites on mitochondrial F1-ATPase by the fluorescence probes 3'-O-(1-naphthoyl)adenosine di- and triphosphate.

The fluorescence properties of 3'-O-(1-naphthoyl)adenosine di- and triphosphates (termed N-ADP and N-ATP, respectively) were investigated in detail. Of special importance for the use of these analogues as environmental probes is their high quantum yield (0.58 in water) and the polarity dependence of shape and wavelength position of the emission spectrum. Upon binding of N-ADP and N-ATP to mitochondrial F1-ATPase, the fluorescence intensity is markedly decreased, due to polarity changes and 'ground-state' quenching. Using this signal for equilibrium binding studies, three (at least a priori) equivalent nucleotide-binding sites were detected on the enzyme. The perspective intrinsic dissociation constants are as follows: N-ADP/Mg2+ 120 nM; N-ATP/Mg2+ 160 nM; N-ADP/EDTA 560 nM; N-ATP/EDTA 3500 nM. For bound ligand the environment was found to be rather unipolar; the rotational mobility of the fluorophore is restricted, its accessibility for iodide anions (as a quencher) is hindered. These facts show a location of the binding sites quite deeply embedded in the protein. The conformation of the binding domains is strongly dependent on the absence or presence of Mg2+, as can be seen from the relative efficiencies of the singlet-singlet energy transfer from tyrosine residues in the protein to bound naphthoyl moieties. Investigation of the binding kinetics revealed this process as biphasic (in presence of Mg2+). After the first fast step (kon greater than 1 X 10(6) M-1 X s-1), in which the analogue is bound to the enzyme, a slow local conformational rearrangement occurs.

Sequence of the two operons encoding the four core subunits of the cytochrome b(6)f complex from the thermophilic Cyanobacterium synechococcus elongatus.

The genes encoding cytochrome f (petA), cytochrome b(6) (petB), the Rieske FeS-protein (petC), and subunit IV (petD) of the cytochrome b(6)f complex from the thermophilic cyanobacterium Synechococcus elongatus were cloned and sequenced. Similar to other cyanobacteria, the structural genes are arranged in two short, single-copy operons, petC/petA and petB/petD, respectively. In addition, five open reading frames with homology to known orfs from the cyanobacterium Synechocystis PCC 6803 were identified in the immediate vicinity of these two operons.

Nucleotide sequence of the petB gene encoding cytochrome b6 from the mesophilic cyanobacterium Synechocystis PCC 6803: implications for evolution and function.

The gene encoding the cytochrome b6 subunit (petB) of the cytochrome b6f complex has been isolated, cloned and sequenced by nonradioactive methods from genomic DNA of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 666 nucleotides, coding for a polypeptide with a molecular mass of 25.02 kDa. In contrast to higher plant petB sequences an aminoterminal extension of seven amino acids occurs. Aminoterminal sequencing of the isolated protein excludes--different from higher plants--the existence of an intron after the first amino acids but indicate the posttranslational removal of three amino acids from the amino terminus. The aminoterminal extension--found only in non-nitrogen-fixing, unicellular cyanobacteria--shows a high degree of homology between different species.

Isolation of membrane protein subunits in their native state: evidence for selective binding of chlorophyll and carotenoid to the b(6) subunit of the cytochrome b(6)f complex.

Cytochrome (cyt) b-c complexes play a central role in electron transfer chains and are almost ubiquitous in nature. Although similar in their basic structure and function, the cyt b(6)f complex of photosynthetic membranes and its counterpart, the mitochondrial cyt bc(1) complex, show some characteristic differences which cannot be explained by the high resolution structure of the cyt bc(1) complex alone. Especially the presence of a chlorophyll molecule is a striking feature of all cyt b(6)f complex preparations described so far, imposing questions as to its structural and functional role. To allow a more detailed characterization, we here report the preparation of native subunits cyt b(6) and IV starting from a monomeric cyanobacterial cyt b(6)f complex. Spectroscopical and reversed-phase HPLC analyses of the purified cyt b(6) subunit showed that it contained not only two b-type hemes, but also one chlorophyll a molecule and a cyanobacterial carotenoid, echinenone. Evidence for selective binding of both pigments to this subunit is presented and their putative function is discussed.

The photosystem I trimer of cyanobacteria: molecular organization, excitation dynamics and physiological significance.

The photosystem I complex organized in cyanobacterial membranes preferentially in trimeric form participates in electron transport and is also involved in dissipation of excess energy thus protecting the complex against photodamage. A small number of longwave chlorophylls in the core antenna of photosystem I are not located in the close vicinity of P700, but at the periphery, and increase the absorption cross-section substantially. The picosecond fluorescence kinetics of trimers resolved the fastest energy transfer components reflecting the equilibration processes in the core antenna at different redox states of P700. Excitation kinetics in the photosystem I bulk antenna is nearly trap-limited, whereas excitation trapping from longwave chlorophyll pools is diffusion-limited and occurs via the bulk antenna. Charge separation in the photosystem I reaction center is the fastest of all known reaction centers.

Photosynthetic and respiratory electron transport in the alkaliphilic cyanobacterium Arthrospira (Spirulina) platensis.

Photosynthetic and respiratory electron transport and their interplay with ion transport have been studied in Arthrospira platensis, a filamentous alkaliphilic cyanobacterium living in hypersaline lakes. As typical for alkaliphiles, A. platensis apparently does not maintain an outward positive pH gradient at its plasma membrane. Accordingly, sodium extrusion occurs via an ATP-dependent primary sodium pump, in contrast to the Na(+)/H(+) antiport in most cyanobacteria. A. platensis is strongly dependent on sodium/bicarbonate symport for the uptake of inorganic carbon. Sodium extrusion in the presence of the Photosystem II inhibitor diuron indicates that a significant amount of ATP is supplied by cyclic electron transport around Photosystem I, the content of which in A. platensis is exceptionally high. Plastoquinol is oxidized by two parallel pathways, via the cytochrome b (6) f complex and a putative cytochrome bd complex, both of which are active in the light and in the dark.

Photosystem I from the unusual cyanobacterium Gloeobacter violaceus.

Photosystem I (PS I) from the primitive cyanobacterium Gloeobacter violaceus has been purified and characterised. Despite the fact that the isolated complexes have the same subunit composition as complexes from other cyanobacteria, the amplitude of flash-induced absorption difference spectra indicates a much bigger antenna size with about 150 chlorophylls per P700 as opposed to the usual 90. Image analysis of the PS I preparation from Gloeobacter reveals that the PS I particles exist both in a trimeric and in a monomeric form and that their size and shape closely resembles other cyanobacterial PS I particles. However, the complexes exhibit a higher molecular weight as could be shown by gel filtration. The preparation contains novel polypeptides not related to known Photosystem I subunits. The N-terminal sequence of one of those polypeptides has been determined and reveals no homology to known or hypothetical proteins. Immunoblotting shows a cross-reaction of three of the polypeptide bands with an antibody raised against the major LHC from the diatom Cyclotella cryptica. Electron microscopy reveals a novel T-shaped complex which has never been observed in any other cyanobacterial PS I preparation. 77 K spectra of purified PS I show an extreme blue-shift of the fluorescence emission, indicating an unusual organisation of the PS I antenna system in Gloeobacter.

Spectroscopic characterization of the ATPase of the thermophilic bacterium PS3 and its isolated subunits.

The ATPase of the thermophilic bacterium PS3, TF0F1, and its subunits has been isolated and their absorption and fluorescence spectra have been measured. The following results were obtained: The tryptophan content of the subunits was determined spectroscopically. Although tryptophan (Trp) and tyrosine (Tyr) are found in TF1, the fluorescence spectrum of native TF1 and its subunits is dominated by Tyr fluorescence; this is in contrast to other proteins. Among (native) TF1 and its subunits only TF1 and the alpha-subunit show a weak fluorescence of Trp, which is blue-shifted, indicating a location in a strongly hydrophobic environment. TF0 fluorescence is dominated by the strong Trp fluorescence. TF0F1 fluorescence is also dominated by the Trp residues. Additionally, its fluorescence is higher than the sum of the isolated TF0 and TF1, indicating marked changes in the microenvironment of the fluorescing aminoacids upon binding of TF1 to TF0.

[In vitro and in vivo studies of diclofenac suppositories in humans]. / Zur Beziehung von in-vitro- und in-vivo-Untersuchungen beim Menschen am Beispiel von Diclofenac-Suppositorien.

Five different preparations of diclofenac-suppositories (A, B, C, D, E) are investigated for in vitro liberation (modified paddle method) and for in vivo bioavailability in man (8-12 subjects) in a crossover design. In vitro, the time at which 63.2% of the drug are liberated (tL) is 14, 20, 25, 14 and 3.5 min for the preparations A, B, C, D and E, respectively. The steady state concentrations from the preparations B and C are 51 and 11%, respectively, and lower than for the others. In vivo, the time of the concentration maximum (tmax) in min is (mean +/- S mean): A = 68 +/- 18, B = 72 +/- 9, C = 120 +/- 22, D = 42 +/- 3, E = 24 +/- 0.5. The concentration maximum (cmax) in mumol . l-1 at tmax is (mean +/- S mean): A = 5.9 +/- 0.8, B = 3.9 +/- 0.3, C = 3.1 +/- 0.4, D = 5.7 +/- 0.6, and E = 5.5 +/- 0.8. The area under the curve (AUC) for all the preparations has found to be between 8.7 and 10,6 mumol . h . l-1. There is a significant correlation between the in vitro parameter tL and the in vivo data of tmax and cmax, respectively. In consequence, the invasion behaviour in vivo can be derived from in vitro data for drugs with similar good physicochemical properties as diclofenac-Na.

Overexpression and reconstitution of a Rieske iron-sulfur protein from the cyanobacterium Synechocystis PCC 6803.

Chloroplast cyt b6f complexes as well as mitochondrial and bacterial cyt bc1 complexes contain a high potential Rieske iron-sulfur protein which is essential for their function. To characterise the isolated Rieske protein from the mesophilic cyanobacterium Synechocystis PCC6803 we cloned the encoding gene into an expression vector and overexpressed the protein in E. coli. In cells overexpressing the protein no typical Rieske type EPR signal was detected neither in membranes nor in inclusion bodies where the majority of the protein was deposited. The inclusion bodies were isolated from the E. coli cells and denaturated with 8 M urea. With a single anion exchange chromatographic step a pure protein could be obtained which was used for further experiments. The NifS like protein IscS was recently reported to mediate the incorporation of iron-sulfur clusters into ferredoxin in vitro. We used the recombinant IscS protein for the incorporation of the cluster into the folded Rieske apoprotein. Spectroscopic characterisation of the resultant protein by CD and EPR spectroscopy showed the presence of a typical Rieske iron-sulfur centre.

Charge separation kinetics in intact photosystem II core particles is trap-limited. A picosecond fluorescence study.

The fluorescence kinetics in intact photosystem II core particles from the cyanobacterium Thermosynechococcus elongatus have been measured with picosecond resolution at room temperature in open reaction centers. At least two new lifetime components of approximately 2 and 9 ps have been resolved in the kinetics by global analysis in addition to several known longer-lived components (from 42 ps to approximately 2 ns). Kinetic compartment modeling yields a kinetic description in full agreement with the one found recently by femtosecond transient absorption spectroscopy [Holzwarth et al. (2005) submitted to Proc. Natl. Acad. Sci. U.S.A.]. We have for the first time resolved directly the fluorescence spectrum and the kinetics of the equilibrated excited reaction center in intact photosystem II and have found two early radical pairs before the electron is transferred to the quinone Q(A). The apparent lifetime for primary charge separation is 7 ps, that is, by a factor of 8-12 faster than assumed on the basis of earlier analyses. The main component of excited-state decay is 42 ps. The effective primary charge separation rate constant is 170 ns(-)(1), and the secondary electron-transfer rate constant is 112 ns(-)(1). Both electron-transfer steps are reversible. Electron transfer from pheophytin to Q(A) occurs with an apparent overall lifetime of 350 ps. The energy equilibration between the CP43/CP47 antenna and the reaction center occurs with a main apparent lifetime of approximately 1.5 ps and a minor 10 ps lifetime component. Analysis of the overall trapping kinetics based on the theory of energy migration and trapping on lattices shows that the charge separation kinetics in photosystem II is extremely trap-limited and not diffusion-to-the-trap-limited as claimed in several recent papers. These findings support the validity of the assumptions made in deriving the earlier exciton radical pair equilibrium model [Schatz, G. H., Brock, H., and Holzwarth, A. R. (1988) Biophys. J. 54, 397-405].

Kinetics and mechanism of electron transfer in intact photosystem II and in the isolated reaction center: pheophytin is the primary electron acceptor.

The mechanism and kinetics of electron transfer in isolated D1/D2-cyt(b559) photosystem (PS) II reaction centers (RCs) and in intact PSII cores have been studied by femtosecond transient absorption and kinetic compartment modeling. For intact PSII, a component of approximately 1.5 ps reflects the dominant energy-trapping kinetics from the antenna by the RC. A 5.5-ps component reflects the apparent lifetime of primary charge separation, which is faster by a factor of 8-12 than assumed so far. The 35-ps component represents the apparent lifetime of formation of a secondary radical pair, and the approximately 200-ps component represents the electron transfer to the Q(A) acceptor. In isolated RCs, the apparent lifetimes of primary and secondary charge separation are approximately 3 and 11 ps, respectively. It is shown (i) that pheophytin is reduced in the first step, and (ii) that the rate constants of electron transfer in the RC are identical for PSII cores and for isolated RCs. We interpret the first electron transfer step as electron donation from the primary electron donor Chl(acc D1). Thus, this mechanism, suggested earlier for isolated RCs at cryogenic temperatures, is also operative in intact PSII cores and in isolated RCs at ambient temperature. The effective rate constant of primary electron transfer from the equilibrated RC* excited state is 170-180 ns(-1), and the rate constant of secondary electron transfer is 120-130 ns(-1).

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase.

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.

Supramolecular architecture of cyanobacterial thylakoid membranes: How is the phycobilisome connected with the photosystems?

Cyanobacteria, as the most simple organisms to perform oxygenic photosynthesis differ from higher plants especially with respect to the thylakoid membrane structure and the antenna system used to capture light energy. Cyanobacterial antenna systems, the phycobilisomes (PBS), have been shown to be associated with Photosystem 2 (PS 2) at the cytoplasmic side, forming a PS 2-PBS-supercomplex, the structure of which is not well understood. Based on structural data of PBS and PS 2, a model for such a supercomplex is presented. Its key features are the PS 2 dimer as prerequisite for formation of the supercomplex and the antiparallel orientation of PBS-cores and the two PS 2 monomers which form the 'contact area' within the supercomplex. Possible consequences for the formation of 'superstructures' (PS 2-PBS rows) within the thylakoid membrane under so-called 'state 1' conditions are discussed. As there are also indications for specific functional connections of PBS with Photosystem 1 (PS 1) under so-called 'state 2' conditions, we show a model which reconciles the need for a structural interaction between PBS and PS 1 with the difference in structural symmetry (2-fold rotational symmetry of PBS-cores, 3-fold rotational symmetry of trimeric PS 1). Finally, the process of dynamic coupling and uncoupling of PBS to PS 1 and PS 2, based on the presented models, shows analogies to mechanisms for the regulation of photosynthetic electron flow in higher plants-despite the very different organization of their thylakoid membranes in comparison to cyanobacteria.

[Pseudohypoparathyroidism--a case report]. / Pseudohypoparathyreoidismus--eine Kasuistik.

The clinical picture of pseudohypoparathyroidism described by Albright and co-workers represents a morphological, functional and laboratory-chemical combination of symptoms which in the classical case is characterized by proportionate nanism, round face, oligophrenia and neuromuscular overexcitability, radiological changes as well as hypocalcaemia and hyperphosphataemia in increased parathormone. On the basis of casuistics typical findings are demonstrated and discussed with the data in literature.

Expression of a higher plant psbA gene in Synechocystis 6803 yields a functional hybrid photosystem II reaction center complex.

The psbA gene codes for the D1 polypeptide of the photosystem II reaction center complex and is found in all photosynthetic organisms that carry out oxygenic photosynthesis. Here we describe the construction and characterization of a strain of the cyanobacterium Synechocystis sp PCC 6803 in which the three endogenous psbA genes are replaced by a single psbA gene from the chloroplast genome of the higher plant Poa annua. The resulting chimeric strain, KWPAS, grows photoautotrophically with a doubling time of 26 hours compared with 20 hours for wild-type Synechocystis 6803. The mutant oxidizes water to oxygen at light-saturated rates comparable with wild type, despite differences in 15% of the primary structure of D1 between these species. RNA gel blot analysis indicates the presence in KWPAS of a psbA transcript of approximately 1.25 kilobases, consistent with the chloroplast promoter also acting as a promoter in Synechocystis. By using antibodies specific for the carboxyl-terminal extension of the D1 polypeptide of higher plants, we showed that the D1 polypeptide synthesized by KWPAS is post-translationally modified at the carboxyl terminus, probably through processing. A detailed biophysical analysis of the chimeric photosystem II complex indicated that the rates of forward electron transfer are similar to wild type. The rates of charge recombination between the donor and acceptor sides of the reaction center are, however, accelerated by as much as a factor of nine (QA- to S2) and are the most likely explanation for the lower rate of photoautotrophic growth in the mutant. We conclude that the psbA gene from a higher plant can be expressed in cyanobacteria and its product processed and assembled into a functional chimeric photosystem II reaction center.