Marc M. Nowaczyk

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).

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.

Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (μ-oxo) of the manganese tetramer

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn4O5Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522–3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55–60]}; (ii) ammonia displaces a water ligand to the outer manganese (MnA4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the MnA4-O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water.

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.

Rational wiring of photosystem II to hierarchical indium tin oxide electrodes using redox polymers

Photosystem II (PSII) is a multi-subunit enzyme responsible for solar-driven water oxidation to release O2 and highly reducing electrons during photosynthesis. The study of PSII in protein film photoelectrochemistry sheds light into its biological function and provides a blueprint for artificial water-splitting systems. However, the integration of macromolecules, such as PSII, into hybrid bio-electrodes is often plagued by poor electrical wiring between the protein guest and the material host. Here, we report a new benchmark PSII–electrode system that combines the efficient wiring afforded by redox-active polymers with the high loading provided by hierarchically-structured inverse opal indium tin oxide (IO-ITO) electrodes. Compared to flat electrodes, the hierarchical IO-ITO electrodes enabled up to an approximately 50-fold increase in the immobilisation of an Os complex-modified and a phenothiazine-modified polymer. When the Os complex-modified polymer is co-adsorbed with PSII on the hierarchical electrodes, photocurrent densities of up to ∼410 μA cm−2 at 0.5 V vs. SHE were observed in the absence of diffusional mediators, demonstrating a substantially improved wiring of PSII to the IO-ITO electrode with the redox polymer. The high photocurrent density allowed for the quantification of O2 evolution, and a Faradaic efficiency of 85 ± 9% was measured. As such, we have demonstrated a high performing and fully integrated host–guest system with excellent electronic wiring and loading capacity. This assembly strategy may form the basis of all-integrated electrode designs for a wide range of biological and synthetic catalysts.

Functional Characterization and Quantification of the Alternative PsbA Copies in Thermosynechococcus elongatus and Their Role in Photoprotection

The D1 protein (PsbA) of photosystem II (PSII) from Thermosynechococcus elongatus is encoded by a psbA gene family that is typical of cyanobacteria. Although the transcription of these three genes has been studied previously (Kós, P. B., Deák, Z., Cheregi, O., and Vass, I. (2008) Biochim. Biophys. Acta 1777, 74–83), the protein quantification had not been possible due to the high sequence identity between the three PsbA copies. The successful establishment of a method to quantify the PsbA proteins on the basis of reverse phase-LC-electrospray mass ionization-MS/MS (RP-LC-ESI-MS/MS) enables an accurate comparison of transcript and protein level for the first time ever. Upon high light incubation, about 70% PsbA3 could be detected, which closely corresponds to the transcript level. It was impossible to detect any PsbA2 under all tested conditions. The construction of knock-out mutants enabled for the first time a detailed characterization of both whole cells and also isolated PSII complexes. PSII complexes of the ΔpsbA1/psbA2 mutant contained only copy PsbA3, whereas only PsbA1 could be detected in PSII complexes from the ΔpsbA3 mutant. In whole cells as well as in isolated complexes, a shift of the free energy between the redox pairs in the PsbA3 complexes in comparison with PsbA1 could be detected by thermoluminescence and delayed fluorescence measurements. This change is assigned to a shift of the redox potential of pheophytin toward more positive values. Coincidentally, no differences in the QA-QB electron transfer could be observed in flash-induced fluorescence decay or prompt fluorescence measurements. In conclusion, PsbA3 complexes yield a better protection against photoinhibition due to a higher probability of the harmless dissipation of excess energy.

Structure and Function of Intact Photosystem 1 Monomers from the Cyanobacterium Thermosynechococcus elongatus

Until now, the functional and structural characterization of monomeric photosystem 1 (PS1) complexes from Thermosynechococcus elongatus has been hampered by the lack of a fully intact PS1 preparation; for this reason, the three-dimensional crystal structure at 2.5 A resolution was determined with the trimeric PS1 complex [Jordan, P., et al. (2001) Nature 411 (6840), 909-917]. Here we show the possibility of isolating from this cyanobacterium the intact monomeric PS1 complex which preserves all subunits and the photochemical activity of the isolated trimeric complex. Moreover, the equilibrium between these complexes in the thylakoid membrane can be shifted by a high-salt treatment in favor of monomeric PS1 which can be quantitatively extracted below the phase transition temperature. Both monomers and trimers exhibit identical posttranslational modifications of their subunits and the same reaction centers but differ in the long-wavelength antenna chlorophylls. Their chlorophyll/P700 ratio (108 for the monomer and 112 for the trimer) is slightly higher than in the crystal structure, confirming mild preparation conditions. Interaction of antenna chlorophylls of the monomers within the trimer leads to a larger amount of long-wavelength chlorophylls, resulting in a higher photochemical activity of the trimers under red or far-red illumination. The dynamic equilibrium between monomers and trimers in the thylakoid membrane may indicate a transient monomer population in the course of biogenesis and could also be the basis for short-term adaptation of the cell to changing environmental conditions.

Structure of Psb27 in solution: implications for transient binding to photosystem II during biogenesis and repair.

Psb27 is a membrane-extrinsic subunit of photosystem II (PSII) where it is involved in the assembly and maintenance of this large membrane protein complex that catalyzes one of the key reactions in the biosphere, the light-induced oxidation of water. Here, we report for the first time the structure of Psb27 that was not observed in the previous crystal structures of PSII due to its transient binding mode. The Psb27 structure shows that the core of the protein is a right-handed four-helix bundle with an up-down-up-down topology. The electrostatic potential of the surface generated by the amphipathic helices shows a dipolar distribution which fits perfectly to the major PsbO binding site on the PSII complex. Moreover, the presented docking model could explain the function of Psb27, which prevents the binding of PsbO to facilitate the assembly of the Mn(4)Ca cluster.

Structure, ligands and substrate coordination of the oxygen-evolving complex of photosystem II in the S2 state: a combined EPR and DFT study

The S2 state of the oxygen-evolving complex of photosystem II, which consists of a Mn4O5Ca cofactor, is EPR-active, typically displaying a multiline signal, which arises from a ground spin state of total spin ST = 1/2. The precise appearance of the signal varies amongst different photosynthetic species, preparation and solvent conditions/compositions. Over the past five years, using the model species Thermosynechococcus elongatus, we have examined modifications that induce changes in the multiline signal, i.e. Ca(2+)/Sr(2+)-substitution and the binding of ammonia, to ascertain how structural perturbations of the cluster are reflected in its magnetic/electronic properties. This refined analysis, which now includes high-field (W-band) data, demonstrates that the electronic structure of the S2 state is essentially invariant to these modifications. This assessment is based on spectroscopies that examine the metal centres themselves (EPR, (55)Mn-ENDOR) and their first coordination sphere ligands ((14)N/(15)N- and (17)O-ESEEM, -HYSCORE and -EDNMR). In addition, extended quantum mechanical models from broken-symmetry DFT now reproduce all EPR, (55)Mn and (14)N experimental magnetic observables, with the inclusion of second coordination sphere ligands being crucial for accurately describing the interaction of NH3 with the Mn tetramer. These results support a mechanism of multiline heterogeneity reported for species differences and the effect of methanol [Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 829], involving small changes in the magnetic connectivity of the solvent accessible outer MnA4 to the cuboidal unit Mn3O3Ca, resulting in predictable changes of the measured effective (55)Mn hyperfine tensors. Sr(2+) and NH3 replacement both affect the observed (17)O-EDNMR signal envelope supporting the assignment of O5 as the exchangeable μ-oxo bridge and it acting as the first site of substrate inclusion.

NdhP and NdhQ: Two Novel Small Subunits of the Cyanobacterial NDH-1 Complex

The subunit composition of the NAD(P)H dehydrogenase complex of Thermosynechococcus elongatus was analyzed by different types of mass spectrometry. All 15 known subunits (NdhA-NdhO) were identified in the purified NDH-1L complex. Moreover, two additional intact mass tags of 4902.7 and 4710.5 Da could be assigned after reannotation of the T. elongatus genome. NdhP and NdhQ are predicted to contain a single transmembrane helix each, and homologues are apparent in other cyanobacteria. Additionally, ndhP is present in some cyanophages in a cluster of PSI genes and exhibits partial similarity to NDF6, a subunit of the plant NDH-1 complex.