Photosystem 1 (PS1) triggers the most energetic light‐induced charge‐separation step in nature and the in vivo electron‐transfer rates approach 50 e− s−1 PS1−1. Photoelectrochemical devices based on ...this building block have to date underperformed with respect to their semiconductor counterparts or to natural photosynthesis in terms of electron‐transfer rates. We present a rational design of a redox hydrogel film to contact PS1 to an electrode for photocurrent generation. We exploit the pH‐dependent properties of a poly(vinyl)imidazole Os(bispyridine)2Cl polymer to tune the redox hydrogel film for maximum electron‐transfer rates under optimal conditions for PS1 activity. The PS1‐containing redox hydrogel film displays electron‐transfer rates of up to 335±14 e− s−1 PS1−1, which considerably exceeds the rates observed in natural photosynthesis or in other semiartificial systems. Under O2 supersaturation, photocurrents of 322±19 μA cm−2 were achieved. The photocurrents are only limited by mass transport of the terminal electron acceptor (O2). This implies that even higher electron‐transfer rates may be achieved with PS1‐based systems in general.
The hybrid leaf: Fine‐tuning the properties of stimuli‐responsive redox hydrogels by means of pH‐induced film collapse, cross‐linking, and resolvation generates the highest photocurrents to date for photosystem 1 (PS1) contacted to conductive electrodes (see figure; MV=methyl viologen).
Summary
In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light‐dependent reduction of O2 to H2O. This alleviates electron pressure on the ...photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero‐oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase‐like complex (NDH‐1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH‐1 types have been characterized in cyanobacteria: NDH‐11 and NDH‐12, which function in respiration; and NDH‐13 and NDH‐14, which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (∆flv1 and Δflv3) and the double NDH‐1 mutants (∆d1d2, which is deficient in NDH‐11,2 and ∆d3d4, which is deficient in NDH‐13,4), we studied triple mutants lacking one of Flv1 or Flv3, and NDH‐11,2 or NDH‐13,4. We show that the presence of either Flv1/3 or NDH‐11,2, but not NDH‐13,4, is indispensable for survival during changes in growth conditions from high CO2/moderate light to low CO2/high light. Our results show functional redundancy between FDPs and NDH‐11,2 under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH‐11,2, allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO2 and light availability.
Significance Statement
Flavodiiron proteins and NDH‐1 complex ensure survival of cyanobacterial cells by cooperatively safeguarding the photosynthetic apparatus against excessive reduction. Ferredoxin probably functions as an electron donor to both flavodiiron proteins and NDH‐1 allowing their functions to be dynamically coordinated for photoprotection of photosynthesis under variable CO2 and light availability.
Requirements concerning the construction of a minimal photosynthetic design cell with direct coupling of water-splitting photosynthesis and H2 production are discussed in the present paper. Starting ...from a cyanobacterial model cell, Synechocystis PCC 6803, potentials and possible limitations are outlined and realization strategies are presented. In extension, the limits of efficiency of all major biological components can be approached in a semi-artificial system consisting of two electrochemically coupled half-cells without the physiological constraints of a living cell.
Z‐Scheme on wires: The two photosystems of the natural photosynthetic Z‐scheme have been connected by immobilizing them within redox hydrogels on individual electrodes. Upon irradiation, this ...biophotovoltaic device produced photocurrents as a closed and autonomous system. The open‐circuit voltage of the cell corresponds to the potential difference between the two redox hydrogels and indicates the coupling of the two charge separation steps.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy allows a detailed analysis of surface attached molecules, including their secondary structure, orientation, and ...interaction with small molecules in the case of proteins. Here, we present a universal immobilization technique on germanium for all oligo-histidine-tagged proteins. For this purpose, new triethoxysilane derivates were developed: we synthesized a linker-silane with a succinimidyl ester as amine-reactive headgroup and a matrix-silane with an unreactive ethylene glycol group. A new methodology for the attachment of triethoxysilanes on germanium was established, and the surface was characterized by ATR-FTIR and X-ray photoelectron spectroscopy. In the next step, the succinimidyl ester was reacted with aminonitrilotriacetic acid. Subsequently, Ni(2+) was coordinated to form Ni-nitrilotriacetic acid for His-tag binding. The capability of the functionalized surface was demonstrated by experiments using the small GTPase Ras and photosystem I (PS I). The native binding of the proteins was proven by difference spectroscopy, which probes protein function. The function of Ras as molecular switch was demonstrated by a beryllium trifluoride anion titration assay, which allows observation of the "on" and "off" switching of Ras at atomic resolution. Furthermore, the activity of immobilized PS I was proven by light-induced difference spectroscopy. Subsequent treatment with imidazole removes attached proteins, enabling repeated binding. This universal technique allows specific attachment of His-tagged proteins and a detailed study of their function at the atomic level using FTIR difference spectroscopy.
Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of ...their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.
In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the ...dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the 2Fe-2S cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Å resolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.
The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the ...cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.
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