Matthias Rögner

Detection of the water-binding sites of the oxygen-evolving complex of Photosystem II using W-band 17O electron-electron double resonance-detected NMR spectroscopy.

Water binding to the Mn(4)O(5)Ca cluster of the oxygen-evolving complex (OEC) of Photosystem II (PSII) poised in the S(2) state was studied via H(2)(17)O- and (2)H(2)O-labeling and high-field electron paramagnetic resonance (EPR) spectroscopy. Hyperfine couplings of coordinating (17)O (I = 5/2) nuclei were detected using W-band (94 GHz) electron-electron double resonance (ELDOR) detected NMR and Davies/Mims electron-nuclear double resonance (ENDOR) techniques. Universal (15)N (I = ½) labeling was employed to clearly discriminate the (17)O hyperfine couplings that overlap with (14)N (I = 1) signals from the D1-His332 ligand of the OEC (Stich Biochemistry 2011, 50 (34), 7390-7404). Three classes of (17)O nuclei were identified: (i) one μ-oxo bridge; (ii) a terminal Mn-OH/OH(2) ligand; and (iii) Mn/Ca-H(2)O ligand(s). These assignments are based on (17)O model complex data, on comparison to the recent 1.9 Å resolution PSII crystal structure (Umena Nature 2011, 473, 55-60), on NH(3) perturbation of the (17)O signal envelope and density functional theory calculations. The relative orientation of the putative (17)O μ-oxo bridge hyperfine tensor to the (14)N((15)N) hyperfine tensor of the D1-His332 ligand suggests that the exchangeable μ-oxo bridge links the outer Mn to the Mn(3)O(3)Ca open-cuboidal unit (O4 and O5 in the Umena et al. structure). Comparison to literature data favors the Ca-linked O5 oxygen over the alternative assignment to O4. All (17)O signals were seen even after very short (≤15 s) incubations in H(2)(17)O suggesting that all exchange sites identified could represent bound substrate in the S(1) state including the μ-oxo bridge. (1)H/(2)H (I = ½, 1) ENDOR data performed at Q- (34 GHz) and W-bands complement the above findings. The relatively small (1)H/(2)H couplings observed require that all the μ-oxo bridges of the Mn(4)O(5)Ca cluster are deprotonated in the S(2) state. Together, these results further limit the possible substrate water-binding sites and modes within the OEC. This information restricts the number of possible reaction pathways for O-O bond formation, supporting an oxo/oxyl coupling mechanism in S(4).

Toward the Complete Membrane Proteome High Coverage of Integral Membrane Proteins Through Transmembrane Peptide Detection

To attain a comprehensive membrane proteome of two strains of Corynebacterium glutamicum (l-lysine-producing and the characterized model strains), both sample pretreatment and analysis methods were optimized. Isolated bacterial membranes were digested with trypsin/cyanogen bromide or trypsin/chymotrypsin, and a complementary protein set was identified using the multidimensional protein identification technology (MudPIT). Besides a distinct number of cytosolic or membrane-associated proteins, the combined data analysis from both digests yielded 326 integral membrane proteins (∼50% of all predicted) covering membrane proteins both with small and large numbers of transmembrane helices. Also membrane proteins with a high GRAVY score (Kyte, J., and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132) were identified, and basic and acidic membrane proteins were evenly represented. A significant increase in hydrophobic peptides with distinctly higher sequence coverage of transmembrane regions was achieved by trypsin/chymotrypsin digestion in an organic solvent. The percentage of identified membrane proteins increased with protein size, yielding 80% of all membrane proteins above 60 kDa. Most prominently, almost all constituents of the respiratory chain and a high number of ATP-binding cassette transport systems were identified. This newly developed protocol is suitable for the quantitative comparison of membrane proteomes and will be especially useful for applications such as monitoring protein expression under different growth and fermentation conditions in bacteria such as C. glutamicum. Moreover with more than 50% coverage of all predicted membrane proteins (including the non-expressed species) this improved method has the potential for a close-to-complete coverage of membrane proteomes in general.

Psb27, a Cyanobacterial Lipoprotein, Is Involved in the Repair Cycle of Photosystem II

Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and 15N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.

Time-Resolved Fluorescence Emission Measurements of Photosystem I Particles of Various Cyanobacteria: A Unified Compartmental Model

Photosystem I (PS-I) contains a small fraction of chlorophylls (Chls) that absorb at wavelengths longer than the primary electron donor P700. The total number of these long wavelength Chls and their spectral distribution are strongly species dependent. In this contribution we present room temperature time-resolved fluorescence data of five PS-I core complexes that contain different amounts of these long wavelength Chls, i.e., monomeric and trimeric photosystem I particles of the cyanobacteria Synechocystis sp. PCC 6803, Synechococcus elongatus, and Spirulina platensis, which were obtained using a synchroscan streak camera. Global analysis of the data reveals considerable differences between the equilibration components (3.4-15 ps) and trapping components (23-50 ps) of the various PS-I complexes. We show that a relatively simple compartmental model can be used to reproduce all of the observed kinetics and demonstrate that the large kinetic differences are purely the result of differences in the long wavelength Chl content. This procedure not only offers rate constants of energy transfer between and of trapping from the compartments, but also well-defined room temperature emission spectra of the individual Chl pools. A pool of red shifted Chls absorbing around 702 nm and emitting around 712 nm was found to be a common feature of all studied PS-I particles. These red shifted Chls were found to be located neither very close to P700 nor very remote from P700. In Synechococcus trimeric and Spirulina monomeric PS-I cores, a second pool of red Chls was present which absorbs around 708 nm, and emits around 721 nm. In Spirulina trimeric PS-I cores an even more red shifted second pool of red Chls was found, absorbing around 715 nm and emitting at 730 nm.

Light-Driven Water Splitting for (Bio-)Hydrogen Production: Photosystem 2 as the Central Part of a Bioelectrochemical Device

Abstract To establish a semiartificial device for (bio-)hydrogen production utilizing photosynthetic water oxidation, we report on the immobilization of a Photosystem 2 on electrode surfaces. For this purpose, an isolated Photosystem 2 with a genetically introduced His tag from the cyanobacterium Thermosynechococcus elongatus was attached onto gold electrodes modified with thiolates bearing terminal Ni(II)-nitrilotriacetic acid groups. Surface enhanced infrared absorption spectroscopy showed the binding kinetics of Photosystem 2, whereas surface plasmon resonance measurements allowed the amount of protein adsorbed to be quantified. On the basis of these data, the surface coverage was calculated to be 0.29 pmol protein cm−2, which is in agreement with the formation of a monomolecular film on the electrode surface. Upon illumination, the generation of a photocurrent was observed with current densities of up to 14 μA cm−2. This photocurrent is clearly dependent on light quality showing an action spectrum similar to an isolated Photosystem 2. The achieved current densities are equivalent to the highest reported oxygen evolution activities in solution under comparable conditions.

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.

Combination of A Photosystem 1-Based Photocathode and a Photosystem 2-Based Photoanode to a Z-Scheme Mimic for Biophotovoltaic Applications†

In photosynthesis, conversion of solar energy into chemical energy follows a Z-scheme, which involves two sequential photoinduced charge separation steps (Figure 1 A). First, upon water splitting at photosystem 2 (PS2), the excited electrons are transferred through an electron transport chain that generates a chemiosmotic potential, which provides the energy for ATP synthesis. Then, at photosystem 1 (PS1), upon light absorption and charge separation, the electrons are transferred via ferredoxin to ferredoxin–NADP+ oxidoreductase for the production of NADPH. Figure 1 Electron-transfer pathways in the Z-scheme of natural photosynthesis (A) and in the proposed coupled PS2/PS1 system (B): All of the potentials are given versus the standard hydrogen electrode (SHE) in volts (data for the natural Z-scheme according to ... The charge separation processes in the Z-scheme inspired the design of photosynthesis-like systems based on organic and inorganic photosensitizers to convert solar energy into chemical energy.1 Exploiting the yield in light collection of photosynthetic proteins may further increase the efficiency of solar to chemical energy conversion in semi-artificial devices. Various photobioelectrochemical half-cells based either on PS12–4 or PS25–7 were suggested. However, until now autonomous solar to chemical energy conversion could not be demonstrated. The electrons provided by PS2 are insufficiently energetic, and PS1-based systems require sacrificial electron donors4 or an externally applied potential2 to sustain solar to chemical energy conversion. These limitations may be overcome by the serial coupling of both light excitation steps of PS1 and PS2 in a semi-artificial photosynthesis device (Figure 1 B). In analogy to the natural Z-scheme, PS2 would extract electrons from water, which are then transferred to PS1. The charge separation at PS1 would provide electrons which are energetic enough for H2 evolution if a suitable catalyst such as a hydrogenase was efficiently coupled to the PS1 reaction,8 as has been proposed previously5, 9, 10 to mimic the last step in the Z-scheme (that is, NADPH synthesis). To achieve maximum yields in solar energy conversion, the energy from the charge separation at PS2, that is, the first step in the Z-scheme, needs to be recovered as well. Herein, we present the first experimental set-up to serially couple PS2 and PS1. We focus on the generation of electrical energy from the difference in potential between the acceptor side of PS2 and the donor side of PS1 (Figure 1 B), that is, that part which contributes to ATP synthesis in the Z-scheme of natural photosynthesis. Our cell is designed to enable the extension of the principle to simultaneous electrical and chemical energy generation (full Z-scheme mimic). We have previously shown that electrochemical half-cells based on either PS2 or PS1 can be constructed separately: The reducing site of PS2 was contacted to an electrode via an Os-complex modified hydrogel, resulting in high photocurrent densities with unprecedented stability.6 Similarly, the oxidizing site of PS1 was contacted (again by means of an Os-complex modified redox hydrogel) with an electrode, which resulted in the generation of high cathodic photocurrents.2 In both cases the same redox polymer was used. The combination of a PS1-based photocathode and a PS2-based photoanode (Figure 2) results in a photovoltaic cell that operates as a closed system without any sacrificial electron donors or acceptors: Under illumination, water is oxidized to oxygen in the anodic compartment by PS2, and oxygen is reduced in the cathodic compartment by PS1 via methyl viologen (MV); the latter is reduced by PS1 and then regenerated by oxygen reduction, leading finally to water.12 Figure 2 Representation of the proposed biophotovoltaic cell combining a PS2-based photoanode and a PS1-based photocathode. Upon absorption of photons, water molecules are split into electrons and protons. The electrons are transferred to the cathodic half-cell ... Notably, the harvest of electrical energy from two coupled light reactions analogous to the Z-scheme in nature (Figure 1) requires different redox potentials of the respective redox hydrogels that wire PS1 and PS2 to their electrodes. The electrical power output of such a photobiovoltaic cell will be determined by 1) the difference in the formal potential of the two redox polymers and 2) the photocurrent density. The electron-transfer communication between the photoanode and PS2 was achieved with an imidazole-coordinated bispyridyl osmium complex-based redox hydrogel (polymer Os1, formal potential: 395 mV vs SHE). At an applied potential of 500 mV vs. SHE, photocurrent densities of up to 45 μA cm−2 are obtained.6 At the photocathode, PS1 was immobilized via a pyridine-coordinated bispyridyl osmium complex-based redox hydrogel13 (polymer Os2) with a more positive formal redox potential of 505 mV vs SHE (for structures of polymer Os1 and Os2, see the Supporting Information). At an applied potential of 200 mV vs. SHE, cathodic photocurrent densities up to 3 μA cm−2 are obtained (see the Supporting Information). The two-compartment cell with the PS2/Os1-based photoanode and the PS1/Os2-based photocathode allows separate electrolyte and buffer optimization for each protein complex. As the potential of the two redox polymers is almost pH-independent, the difference in pH values between the two compartments should not significantly affect the open-circuit voltage. Electrical connection and illumination of both half-cells generate a steady-state photocurrent of about 1 μA cm−2, which disappears upon switching off the light (Figure 3, left). Switching off illumination exclusively on the anode side (PS2) results in a decrease of the photocurrent, which could be restored by switching the light on again (Figure 3, right). Figure 3 Photocurrent density of the proposed biophotovoltaic cell combining the PS2/Os1-based photoanode and the PS1/Os2-based photocathode as shown in Figure 2. The light status of the respective photoelectrode is indicated by O=light on and C=light ... To further confirm the contribution of the PS2/Os1-based photoanode to the overall photocurrent, dinoterb (2,4-dinitro-6-tert-butylphenol), a herbicide that blocks the QB site of the D1 subunit of PS2, was added to the photoanode compartment to deactivate PS2 while both half-cells were continuously illuminated. As shown in the Supporting Information, Figure S3, the photocurrent density substantially decreases upon addition of dinoterb. To determine the short-circuit current density (ISC), the open-circuit voltage (VOC), and the maximal cell power output (Pcell), both photoanode and photocathode were externally connected by a variable resistor. ISC is given by the intersection point of the linear fit of the current density over cell voltage plot and the y axis, and VOC is determined by the intersection point with the x axis (Figure 4). The following values were obtained: ISC=(2.0±0.7) μA cm−2, VOC=(90±20) mV (Figure 4, left), Pcell=(23±10) nW cm−2 (Figure 4, right). The fill factor (ff) is 0.128. The conversion efficiency η for the system, that is, the ratio of power output to power input, is 3.6×10−7, with the maximal power input (349 W m−2) resulting from the LEDs used. Figure 4 Determination of the short-circuit current density (ISC), the open-circuit voltage (VOC), and the maximal cell power density output (Pcell) for the biophotovoltaic cell, combining PS2/Os1-based photoanode and PS1/Os2-based photocathode via an external ... The determined VOC correlates with the difference in the formal potentials of the two redox hydrogels Os1 and Os2, while the maximum current density of the complete photovoltaic cell is limited by the PS1/Os2-based photocathode. Thus, the photocurrent density of the PS1/Os2 half-cell in combination with the relatively low potential difference between the two redox hydrogels limits at this stage the performance of the biophotovoltaic cell. However, the main goal was to proof the feasibility of connecting a PS2-based photoanode and a PS1-based photocathode in a Z-scheme-analogue setup. In future, polymer design for a better electron transfer to PS1 may enable an increased current density by up to one or two orders of magnitude.2 Additionally, an enhancement in power density can be achieved by higher PS1 and/or PS2 loadings using for example, nanostructured electrode surfaces.14, 15 Furthermore, the potential difference between the polymer-tethered redox species may be tuned and optimally adapted to PS1 or PS2. While the potential of the redox polymer Os2 at the photocathode matches well with PS1, the redox polymer used with PS2 could be about 400 mV more negative to match the potential of the acceptor site of PS2 (Figure 1). This would enable a significant increase in cell voltage and power density. In conclusion, we have shown the serial coupling of two independent processes of light capturing by PS2 and PS1 yielding a fully closed and autonomous biophotovoltaic cell. This is fundamentally different from previously reported biophotovoltaic devices,14, 16 as it provides the basis for the future use of this “biobattery” in combination with various catalysts: The very reactive electrons may be used for chemical energy conversion instead of reducing oxygen by methyl viologen. Notably, the separation of the oxygen evolving PS2 photoanode from the PS1 photocathode opens the possibility to couple PS1 with oxygen-sensitive biocatalysts such as nitrogenases, CO2 reducing enzymes of the Calvin cycle, or hydrogenases for the production of biohydrogen.10 In future, this principle will allow the full energetic exploitation of the photosynthetic Z-scheme in a single set-up.

Towards structural determination of the water-splitting enzyme

A photosystem II preparation from the thermophilic cyanobacterium Synechococcus elongatus, which is especially suitable for three-dimensional crystallization in a fully active form was developed. The efficient purification method applied here yielded 10 mg of protein of a homogenous dimeric complex of about 500 kDa within 2 days. Detailed characterization of the preparation demonstrated a fully active electron transport chain from the manganese cluster to plastoquinone in the QB binding site. The oxygen-evolving activity, 5000–6000 μmol of O2/(h·mg of chlorophyll), was the highest so far reported and is maintained even at temperatures as high as 50 °C. The crystals obtained by the vapor diffusion method diffracted to a resolution of 4.3 Å. The space group was determined to be P212121 with four photosystem II dimers per unit cell. Analysis of the redissolved crystals revealed that activity, supramolecular organization, and subunit composition were maintained during crystallization.

Wiring photosynthetic enzymes to electrodes

The efficient electron transfer between redox enzymes and electrode surfaces can be obtained by wiring redox enzymes using, for instance, polymer-bound redox relays as has been demonstrated as a basis for the design of amperometric biosensors, logic gates or sensor arrays and more general as a central aspect of “bioelectrochemistry”. Related devices allow exploiting the unique catalytic properties of enzymes, among which photosynthetic enzymes are especially attractive due to the possibility to trigger the redox reactions upon irradiation with light. Photocatalytic properties such as the light-driven water splitting by photosystem 2 make them unique candidates for the development of semiartificial devices which convert light energy into stable chemical products, like hydrogen. This review summarizes recent concepts for the integration of photosystem 1 and photosystem 2 into bioelectrochemical devices with special focus on strategies for the design of electron transfer pathways between redox enzymes and conductive supports.

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.