TNNI3 encoding cTnI, the inhibitory subunit of the troponin complex, is the main target for mutations leading to restrictive cardiomyopathy (RCM). Here we investigate two cTnI-R170G/W amino acid ...replacements, identified in infantile RCM patients, which are located in the regulatory C-terminus of cTnI. The C-terminus is thought to modulate the function of the inhibitory region of cTnI. Both cTnI-R170G/W strongly enhanced the Ca2+-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn, whereas binding to actin was either strengthened (R170G) or weakened (R170W). Furthermore, the stability of reconstituted thin filaments was reduced as revealed by electron microscopy. Filaments containing R170G/W appeared wavy and showed breaks. Decoration of filaments with myosin subfragment S1 was normal in the presence of R170W, but was irregular with R170G. Surprisingly, both mutants did not affect the Ca2+-dependent activation of reconstituted cardiac thin filaments. In the presence of the N-terminal fragment of cardiac myosin binding protein C (cMyBPC-C0C2) cooperativity of thin filament activation was increased only when the filaments contained wildtype cTn. No effect was observed in the presence of cTn containing R170G/W. cMyBPC-C0C2 significantly reduced binding of wildtype troponin to actin/tropomyosin, but not of both mutant cTn. Moreover, we found a direct troponin/cMyBPC-C0C2 interaction using microscale thermophoresis and identified cTnI and cTnT, but not cTnC as binding partners for cMyBPC-C0C2. Only cTn containing cTnI-R170G showed a reduced affinity towards cMyBPC-C0C2. Our results suggest that the RCM cTnI variants R170G/W impair the communication between thin and thick filament proteins and destabilize thin filaments.
Photosynthetic complex I enables cyclic electron flow around photosystem I, a regulatory mechanism for photosynthetic energy conversion. We report a 3.3-angstrom-resolution cryo-electron microscopy ...structure of photosynthetic complex I from the cyanobacterium
The model reveals structural adaptations that facilitate binding and electron transfer from the photosynthetic electron carrier ferredoxin. By mimicking cyclic electron flow with isolated components in vitro, we demonstrate that ferredoxin directly mediates electron transfer between photosystem I and complex I, instead of using intermediates such as NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). A large rate constant for association of ferredoxin to complex I indicates efficient recognition, with the protein subunit NdhS being the key component in this process.
Photosystem II (PS II) is a multi-subunit complex localized in the thylakoid membrane that performs the light-dependent photosynthetic charge separation. The PS II reaction centre comprises, among ...others, the D1 protein. De novo synthesis and repair of PS II require efficient mechanisms for transport and insertion of plastid encoded D1 into the thylakoid membrane. To elucidate the process of D1 insertion, we used an in vitro translation system derived from pea chloroplasts to reconstitute the D1 insertion. Thereby, truncated D1 encoding psbA mRNAs lacking a stop codon were translated in the presence of thylakoid membranes and the translation was stalled by addition of chloramphenicol. The generated ribosome nascent chain complexes (RNCs) were tightly associated with the thylakoids. Subsequently, these D1 insertion intermediates were enriched from solubilized thylakoids by sucrose cushion centrifugation. Immunological analyses demonstrated the presence of the cpSec translocase, Alb3, cpFtsY, cpSRP54 and Vipp1 (vesicle-inducing protein in plastids 1) in the enriched D1 insertion intermediates. A complex formation between cpSecY, Alb3, cpFtsY and Vipp1 in thylakoid membranes was shown by gel filtration chromatography, BN (Blue Native)/SDS-PAGE and co-immunoprecipitation experiments. Furthermore, a stimulating effect of recombinant Vipp1 on the formation of a D1 insertion intermediate was observed in vitro. These results suggest a co-operative function of these proteins in D1 insertion.
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.
Photosynthetic organisms capture light energy to drive their energy metabolism, and employ the chemical reducing power to convert carbon dioxide (CO
) into organic molecules. Photorespiration, ...however, significantly reduces the photosynthetic yields. To survive under low CO
concentrations, cyanobacteria evolved unique carbon-concentration mechanisms that enhance the efficiency of photosynthetic CO
fixation, for which the molecular principles have remained unknown. We show here how modular adaptations enabled the cyanobacterial photosynthetic complex I to concentrate CO
using a redox-driven proton-pumping machinery. Our cryo-electron microscopy structure at 3.2 Å resolution shows a catalytic carbonic anhydrase module that harbours a Zn
active site, with connectivity to proton-pumping subunits that are activated by electron transfer from photosystem I. Our findings illustrate molecular principles in the photosynthetic complex I machinery that enabled cyanobacteria to survive in drastically changing CO
conditions.
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.
Biotechnological solar hydrogen production has the aim to utilize electrons from the light reactions of photosynthesis. Recently, cyanobacterial and algal H2ases, the enzymes that catalyze production of H2, were successfully fused to PSI. They intercept electrons from the photosynthetic electron transport chain.Both engineered strains produce photosynthetic hydrogen for prolonged periods. They maintained their ability to grow photoautotrophically.The processes do not exclusively rely upon water oxidation as desired, which is mostly due to the fact that the H2ases are sensitive to oxygen.
Photosynthesis occurs in thylakoids, a highly specialized membrane system. In the cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis 6803), the thylakoids are arranged parallel to the ...plasma membrane and occasionally converge toward it to form biogenesis centers. The initial steps in PSII assembly are thought to take place in these regions, which contain a membrane subcompartment harboring the early assembly factor PratA and are referred to as PratA-defined membranes (PDMs). Loss of CurT, the Synechocystis 6803 homolog of Arabidopsis thaliana grana-shaping proteins of the CURVATURE THYLAKOID1 family, results in disrupted thylakoid organization and the absence of biogenesis centers. As a consequence, PSII is less efficiently assembled and accumulates to only 50% of wild-type levels. CurT induces membrane curvature in vitro and is distributed all over the thylakoids, with local concentrations at biogenesis centers. There it forms a sophisticated tubular network at the cell periphery, as revealed by live-cell imaging. CurT is part of several high molecular mass complexes, and Blue Native/SDS-PAGE and isoelectric focusing demonstrated that different isoforms associate with PDMs and thylakoids. Moreover, CurT deficiency enhances sensitivity to osmotic stress, adding a level of complexity to CurT function. We propose that CurT is crucial for the differentiation of membrane architecture, including the formation of PSII-related biogenesis centers, in Synechocystis 6803.
The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn ₄O ₅Ca) 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 Mn ₄O ₅Ca 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 (Mn A₄-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the Mn A₄-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.
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