Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor.

Identifying the two substrate water sites of nature's water-splitting cofactor (Mn4CaO5 cluster) provides important information toward resolving the mechanism of O-O bond formation in Photosystem II (PSII). To this end, we have performed parallel substrate water exchange experiments in the S1 state of native Ca-PSII and biosynthetically substituted Sr-PSII employing Time-Resolved Membrane Inlet Mass Spectrometry (TR-MIMS) and a Time-Resolved 17O-Electron-electron Double resonance detected NMR (TR-17O-EDNMR) approach. TR-MIMS resolves the kinetics for incorporation of the oxygen-isotope label into the substrate sites after addition of H218O to the medium, while the magnetic resonance technique allows, in principle, the characterization of all exchangeable oxygen ligands of the Mn4CaO5 cofactor after mixing with H217O. This unique combination shows i) that the central oxygen bridge (O5) of Ca-PSII core complexes isolated from Thermosynechococcus vestitus has, within experimental conditions, the same rate of exchange as the slowly exchanging substrate water (WS) in the TR-MIMS experiments and ii) that the exchange rates of O5 and WS are both enhanced by Ca2+→Sr2+ substitution in a similar manner. In the context of previous TR-MIMS results, this shows that only O5 fulfills all criteria for being WS. This strongly restricts options for the mechanism of water oxidation.

A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Photosynthesis re-wired on the pico-second timescale.

Photosystems II and I (PSII, PSI) are the reaction centre-containing complexes driving the light reactions of photosynthesis; PSII performs light-driven water oxidation and PSI further photo-energizes harvested electrons. The impressive efficiencies of the photosystems have motivated extensive biological, artificial and biohybrid approaches to 're-wire' photosynthesis for higher biomass-conversion efficiencies and new reaction pathways, such as H2 evolution or CO2 fixation1,2. Previous approaches focused on charge extraction at terminal electron acceptors of the photosystems3. Electron extraction at earlier steps, perhaps immediately from photoexcited reaction centres, would enable greater thermodynamic gains; however, this was believed impossible with reaction centres buried at least 4 nm within the photosystems4,5. Here, we demonstrate, using in vivo ultrafast transient absorption (TA) spectroscopy, extraction of electrons directly from photoexcited PSI and PSII at early points (several picoseconds post-photo-excitation) with live cyanobacterial cells or isolated photosystems, and exogenous electron mediators such as 2,6-dichloro-1,4-benzoquinone (DCBQ) and methyl viologen. We postulate that these mediators oxidize peripheral chlorophyll pigments participating in highly delocalized charge-transfer states after initial photo-excitation. Our results challenge previous models that the photoexcited reaction centres are insulated within the photosystem protein scaffold, opening new avenues to study and re-wire photosynthesis for biotechnologies and semi-artificial photosynthesis.

A biomimetic assembly of folded photosystem I monolayers for an improved light utilization in biophotovoltaic devices.

In the fabrication of photosystem I (PSI)-based biodevices, the use of multilayered architectures aims to maximize the absorption of incident light that can be converted into high-energy electrons. The challenge in this strategy is to overcome the large driving force imposed by the photoinduced potential difference between the two terminal redox centers that are located at opposite sides of PSI, which translates into charge recombination resulting in sub-optimal performance of commonly implemented systems. The integration of PSI monolayers with electrodes using the Langmuir-Blodgett technique enables a preferential anisotropic orientation of PSI in a tightly packed structure, which minimizes short-circuiting processes and aids to improve the performance of PSI-based biodevices. However, the practical application of PSI monolayer-based biodevices is limited due to the small loading of immobilized PSI molecules, leading to overall low utilization of incident light. Inspired by the stacked arrangements of thylakoids in nature, we demonstrate the fabrication of biomimetic structures using multiple PSI monolayers assembled into a folded architecture to improve light absorption and with that the performance of the overall photoelectrode.

LapB (YciM) orchestrates protein-protein interactions at the interface of lipopolysaccharide and phospholipid biosynthesis.

The outer membrane (OM) of Gram-negative bacteria functions as an essential barrier and is characterized by an asymmetric bilayer with lipopolysaccharide (LPS) in the outer leaflet. The enzyme LpxC catalyzes the first committed step in LPS biosynthesis. It plays a critical role in maintaining the balance between LPS and phospholipids (PL), which are both derived from the same biosynthetic precursor. The essential inner membrane proteins YejM (PbgA, LapC), LapB (YciM), and the protease FtsH are known to account for optimal LpxC levels, but the mechanistic details are poorly understood. LapB is thought to be a bi-functional protein serving as an adaptor for FtsH-mediated turnover of LpxC and acting as a scaffold in the coordination of LPS biosynthesis. Here, we provide experimental evidence for the physical interaction of LapB with proteins at the biosynthetic node from where the LPS and PL biosynthesis pathways diverge. By a total of four in vivo and in vitro assays, we demonstrate protein-protein interactions between LapB and the LPS biosynthesis enzymes LpxA, LpxC, and LpxD, between LapB and YejM, the anti-adaptor protein regulating LapB activity, and between LapB and FabZ, the first PL biosynthesis enzyme. Moreover, we uncovered a new adaptor function of LapB in destabilizing not only LpxC but also LpxD. Overall, our study shows that LapB is a multi-functional protein that serves as a protein-protein interaction hub for key enzymes in LPS and PL biogenesis presumably by virtue of multiple tetratricopeptide repeat (TPR) motifs in its cytoplasmic C-terminal region.

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO.

The multi-subunit membrane protein complex photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen evolving activity of approx. 6000 µmol O2·(mg Chl·h)-1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen evolving activity of approx. 3000 µmol O2·(mg Chl·h)-1 and a manganese content of 2.7 (±0.2) per reaction center compared to 3.7 (±0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role of this complex in PSII repair.

Structure of cyanobacterial photosystem I complexed with ferredoxin at 1.97 Å resolution.

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning PSI:Fd interface and the possible binding sites of Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and a loosely bound Cyt c6. Side chain interactions at the PSI:Fd interface including bridging water molecules are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC that affect kinetics of Fd binding and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A possible reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

Top-Down Identification and Sequence Analysis of Small Membrane Proteins Using MALDI-MS/MS.

Identification and sequence determination by mass spectrometry have become routine analyses for soluble proteins. Membrane proteins, however, remain challenging targets due to their hydrophobicity and poor annotation. In particular small membrane proteins often remain unnoticed as they are largely inaccessible to Bottom-Up proteomics. Recent advances in structural biology, though, have led to multiple membrane protein complex structures being determined at sufficiently high resolution to detect uncharacterized, small subunits. In this work we offer a guide for the mass spectrometric characterization of solvent extraction-based purifications of small membrane proteins isolated from protein complexes and cellular membranes. We first demonstrate our Top-Down MALDI-MS/MS approach on a Photosystem II preparation, analyzing target protein masses between 2.5 and 9 kDa with high accuracy and sensitivity. Then we apply our technique to purify and sequence the mycobacterial ATP synthase c subunit, the molecular target of the antibiotic drug bedaquiline. We show that our approach can be used to directly track and pinpoint single amino acid mutations that lead to antibiotic resistance in only 4 h. While not applicable as a high-throughput pipeline, our MALDI-MS/MS and ISD-based approach can identify and provide valuable sequence information on small membrane proteins, which are inaccessible to conventional Bottom-Up techniques. We show that our approach can be used to unambiguously identify single-point mutations leading to antibiotic resistance in mycobacteria.

Advances and challenges in photosynthetic hydrogen production.

The vision to replace coal with hydrogen goes back to Jules Verne in 1874. However, sustainable hydrogen production remains challenging. The most elegant approach is to utilize photosynthesis for water splitting and to subsequently save solar energy as hydrogen. Cyanobacteria and green algae are unicellular photosynthetic organisms that contain hydrogenases and thereby possess the enzymatic equipment for photosynthetic hydrogen production. These features of cyanobacteria and algae have inspired artificial and semi-artificial in vitro techniques, that connect photoexcited materials or enzymes with hydrogenases or mimics of these for hydrogen production. These in vitro methods have on their part been models for the fusion of cyanobacterial and algal hydrogenases to photosynthetic photosystem I (PSI) in vivo, which recently succeeded as proofs of principle.

A Straightforward Approach to Synthesize 7-Aminocephalosporanic Acid In Vivo in the Cephalosporin C Producer Acremonium chrysogenum.

The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer Acremonium chrysogenum. Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and ß-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.

Thylakoid attachment to the plasma membrane in Synechocystis sp. PCC 6803 requires the AncM protein.

Thylakoids are the highly specialized internal membrane systems that harbor the photosynthetic electron transport machinery in cyanobacteria and in chloroplasts. In Synechocystis sp. PCC 6803, thylakoid membranes (TMs) are arranged in peripheral sheets that occasionally converge on the plasma membrane (PM) to form thylakoid convergence membranes (TCMs). TCMs connect several thylakoid sheets and form local contact sites called thylapses between the two membrane systems, at which the early steps of photosystem II (PSII) assembly occur. The protein CurT is one of the main drivers of TCM formation known so far. Here, we identify, by whole-genome sequencing of a curT- suppressor strain, the protein anchor of convergence membranes (AncM) as a factor required for the attachment of thylakoids to the PM at thylapses. An ancM- mutant is shown to have a photosynthetic phenotype characterized by reductions in oxygen-evolution rate, PSII accumulation, and PS assembly. Moreover, the ancM- strain exhibits an altered thylakoid ultrastructure with additional sheets and TCMs detached from the PM. By combining biochemical studies with fluorescence and correlative light-electron microscopy-based approaches, we show that AncM is an integral membrane protein located in biogenic TCMs that form thylapses. These data suggest an antagonistic function of AncM and CurT in shaping TM ultrastructure.

Mass spectrometry analysis of the photosystem II assembly factor Psb27 revealed variations in its lipid modification.

The assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

Functional basis of electron transport within photosynthetic complex I.

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme - the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

Cryo-EM photosystem I structure reveals adaptation mechanisms to extreme high light in Chlorella ohadii.

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn's low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 Å, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems.

Structural insights into photosystem II assembly.

Biogenesis of photosystem II (PSII), nature's water-splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28 and Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-haem iron with glutamate, a structural motif found in reaction centres of non-oxygenic photosynthetic bacteria. These results reveal mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water-splitting reaction.

Cryo-EM structure of a functional monomeric Photosystem I from Thermosynechococcus elongatus reveals red chlorophyll cluster.

A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from Thermosynechococcus elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700.

Closing the Gap for Electronic Short-Circuiting: Photosystem†I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices.

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H2 evolution.

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations.

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h-1 L-1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.