Marc M Nowaczyk

• Ruhr University Bochum

Photosystem II (PSII) assembly is a stepwise process in which intermediate complexes with auxiliary proteins are transiently formed to allow efficient de novo biogenesis or repair of damaged PSII. In particular, the role of extrinsic PSII subunits (PsbO, PsbU, PsbV) and auxiliary proteins such as Psb27 for the formation and photoactivation of the M...

Electron transport chains (ETCs) drive fundamental biological processes, including respiration and photosynthesis. Methodology for studying biological electron transport on a systems-level is lacking, owing to ETCs forming complex overlapping networks of electron transfer, akin to cellular metabolism. Here, we demonstrate the use of electrochemistr...

Fixation of CO2 into the organic compound formate by formate dehydrogenases (FDHs) is regarded as the oldest autotrophic process on Earth. It has been proposed that an FDH-dependent CO2 fixation module could support CO2 assimilation even in photoautotrophic organisms. In the present study, we characterized FDH from Clostridium carboxidivorans (ccFD...

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled rib...

Several auxiliary factors are required for the assembly of photosystem (PS) II, one of which is Psb28. While the absence of Psb28 in cyanobacteria has little effect on PSII assembly, we show here that the Chlamydomonas psb28 -null mutant is severely impaired in PSII assembly, showing drastically reduced PSII supercomplexes, dimers and monomers, whi...

Identifying the two substrate water sites of nature’s water-splitting cofactor (Mn 4 CaO 5 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 S 1 state of native Ca-PSII and biosynthetically substit...

Biocatalytic asymmetric reduction of C=C and C=O bonds is highly attractive to produce valuable (chiral) chemicals for the fine and pharmaceutical industry, yet occurs at the expense of reduced nicotinamide adenine dinucleotide coenzyme NADPH that requires recycling. Established methods each have their challenges. Here we developed a light‐driven a...

Transient absorption (TA) spectroscopy has proved fundamental to our understanding of energy and charge transfer in biological systems, allowing measurements of photoactive proteins on sub-picosecond timescales. Recently, ultrafast TA spectroscopy has been applied in vivo, providing sub-picosecond measurements of photosynthetic light harvesting and...

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 b6f complex towards photosystem I (PSI). This membrane...

The recent increase of interest in photocatalysis spread to biocatalysis and triggered a rush for the development of light‐dependent enzyme‐mediated or enzyme‐coupled processes. After several years of intense research on photobiocatalysis, it is time to evaluate the state of the field in a structured manner. In this Perspective, we suggest to group...

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-...

Photosynthetic hydrogen (photoH2) production is an elegant approach to storing solar energy. The most efficient strategy is to couple the hydrogen‐producing enzyme, the hydrogenase (H2ase), directly to photosystem I (PSI), which is a light‐driven nanomachine found in photosynthetic organisms. PSI–H2ase fusions have been tested in vivo and in vitro....

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 b 6 f complex towards photosystem I (PSI). This membra...

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 ar...

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 cofa...

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 ce...

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c 6 (Cyt c 6 ) 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 c 6 . Here we describe the high resolution cryo-EM structure...

The use of photosynthetic biohybrids to drive redox reactions in vitro is a promising strategy due to the natural abundance and high quantum efficiency of the bio-components. Here, we present different tools for the fabrication of photosystem I-based biohybrid devices by using redox-active polymers, 3D-structured electrodes as well as additional li...

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 advanc...

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 cofa...

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 hyd...

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...

The synthesis of multi-span thylakoid membrane proteins initiates at ribosomes off the membrane. Subsequently, the ribosome nascent chain complexes (RNCs) are transferred to the translocase machinery in the thylakoid membrane for cotranslational protein insertion. These steps require finely tuned mechanisms for protein processing, quality control,...

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c 6 (Cyt c 6 ) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning the binding of Fd and the bound Cyt c 6 . Here we describe the high resolution cryo-EM structure of Thermosynechococ...

Photosystems II and I (PSII and PSI) are the reaction centre complexes that drive the light reactions of photosynthesis. PSII performs light-driven water oxidation (quantum efficiencies and catalysis rates of up to 80% and 1000 $e^{-}\text{s}^{-1}$, respectively) and PSI further photo-energises the harvested electrons (quantum efficiencies of ~100%...

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. Howeve...

Photosystem I (PSI), a robust and abundant biomolecule capable of delivering high‐energy photoelectrons, has a great potential for the fabrication of light‐driven semi‐artificial bioelectrodes. Although possibilities have been explored in this regard, the true capabilities of this technology have not been achieved yet, particularly for their use as...

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 (T...

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 atte...

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 understo...

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 c...

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 re...

Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupt...

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...

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, and is fundamental to bioenergetics in photosynthetic bacteria and so...

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 re...

The highly optimized natural process of oxygenic photosynthesis leads to the formation of redox equivalents, such as NADPH, that can be used to fuel heterologous biotransformations in phototrophic microorganisms. We investigated the reduction of 2-methylmaleimide by the ene-reductase YqjM in the cyanobacterium Synechocystis sp. PCC 6803 and doubled...

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 cyanobacteria...

In any given organism, approximately one-third of all proteins have a yetunknown function. A widely distributed domain of unknown function is DUF1127. Approximately 17,000 proteins with such an arginine-rich domain are found in 4,000 bacteria. Most of them are single-domain proteins, and a large fraction qualifies as small proteins with fewer than...

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 with 2.94 Å resolution. It contains three assembly factors (...

Im Hinblick auf die Entwicklung verbesserter biophotovoltaischer Anordnungen für die Umwandlung von Sonnenenergie ermöglicht eine gemischte Monoschicht aus Trimeren und Monomeren des Photosystems I die Herstellung hocheffizienter Biophotoelektroden, indem elektronische Kurzschlussprozesse minimiert werden und gleichzeitig eine hohe Dichte photoakti...

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 with 2.94 Å resolution. It contains three assembly factors (...

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-y...

Old Yellow Enzymes (OYEs) selectively reduce carbon‑carbon double bonds of a broad range of substrates with excellent stereoselectivity. Current challenges of their application in bio-based industrial processes are the cost-efficient regeneration of the nicotinamide co-substrate and expanding the range of industrially relevant substrates. Microalga...

The fabrication and electrochemical evaluation of transparent photoelectrodes consisting of Photosystem I (PSI) or Photosystem II (PSII) is described, which are embedded and electrically wired by a redox polymer. The fabrication process is performed by an automated airbrush‐type spray coating system, which ensures controlled and scalable electrode...

Interfacing photosynthetic protein complexes with electrodes is frequently used for the identification of electron transfer mechanisms and the fabrication of biosensors. Binding of herbicide compounds to the terminal plastoquinone QB at photosystem II (PSII) causes disruption of electron flow that is associated with a diminished performance of the...

Photosystem II (PSII) is the only enzyme that catalyzes light-induced water oxidation being the basis for its application as a biophotoanode in various bio-photovoltaics and photo-bioelectrochemical cells. However, the absorption spectrum of PSII limits the quantum efficiency in the range of visible light, due to a gap in the green absorption regio...

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the...

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 funct...

Photosynthetic organisms capture light energy to drive their energy metabolism, and employ the chemical reducing power to convert carbon dioxide (CO2) into organic molecules. Photorespiration, however, significantly reduces the photosynthetic yields. To survive under low CO2 concentrations, cyanobacteria evolved unique carbon-concentration mechanis...

Photosynthetic protein complexes are attractive building blocks for the fabrication of semi-artificial energy conversion devices. However, limitations in the efficiency of the implemented biophotovoltaic systems prevent their use in practical applications. In particular, the effective minimization of recombination processes in photosystem I (PSI) m...

Key proteins of the photosynthetic complexes are encoded on the chloroplast genome and co-translationally inserted into the thylakoid membrane. However, the molecular details of this process are largely unknown. Here, we demonstrate by ribosome profiling that the conserved chloroplast signal recognition particle subunit, cpSRP54, is required for ef...

Significance Recent results have shown that nature’s water splitting catalyst inserts an additional water molecule into what appears to be a solvent inaccessible site late in its reaction cycle. The emerging consensus of the field is that this water molecule is one of the substrates of the reaction. Here, we show that this water molecule does not c...

Substrate channeling is a widespread mechanism in metabolic pathways to avoid decomposition of unstable intermediates, competing reactions, and to accelerate catalytic turnover. During the biosynthesis of light‐harvesting phycobilins in cyanobacteria, two members of the ferredoxin‐dependent bilin reductases are involved in the reduction of the open...

Following inspiration by natural photosynthesis, the design and fabrication of semi-artificial biophotoelectrochemical devices able to harvest solar energy and aiming on the implementation of green and sustainable energy conversion systems is presently an important field of research. Here we present the development of a fully light-driven biosuperc...

Plugging into the pump Photosynthetic organisms use light to fix carbon dioxide in a process that requires both chemical reducing equivalents and adenosine triphosphate (ATP). Balancing the ratio of these inputs is accomplished by a short circuit in electron flow through photosynthetic complex I, a proton pump that contributes to ATP production but...

Solar-driven coupling of water oxidation with CO2 reduction sustains life on our planet and is of high priority to contempo-rary energy research. Here, we report a photoelectrochemical tandem device, which performs photocatalytic CO2 reduction to formate. We employ a semi-artificial design, which wires a W-dependent formate dehydrogenase (FDH) cath...

Natural photosynthesis stores sunlight in chemical energy carriers, but it has not evolved for the efficient synthesis of fuels, such as H2. Semi-artificial photosynthesis combines the strengths of natural photosynthesis with synthetic chemistry and materials science to develop model systems that overcome nature’s limitations, such as low-yielding...

In the development of semi-artificial biophotovoltaic assemblies, deeper understanding of electrochemical processes is required to achieve functional and efficient devices. Evaluation of photosystem 2 embedded in an Os-complex modified redox polymer using scanning photoelectrochemical microscopy (SPECM) provides insight into the intricate electroch...

Interfacing photosynthetic proteins specifically photosystem 1 (PS1) with electrodes enables light-induced charge separation processes for powering semiartificial photobiodevices with, however, limited long-term stability. Here, we present the in-depth evaluation of a PS1/Os-complex-modified redox polymer-based biocathode by means of scanning photo...

Photosystem I (PSI), a large protein complex located in the thylakoid membrane, mediates the final step in light-driven electron transfer to the stromal electron carrier protein ferredoxin (Fd). Here, we report the first structural description of the PSI-Fd complex from Thermosynechococcus elongatus. The trimeric PSI complex binds three Fds in a no...

Redox polymer/protein biophotoelectrochemistry was used to analyse forward electron transfer of isolated PSII complexes with natural PsbA-variants. PsbA1- or PsbA3-PSII was embedded in a redox hydrogel that allows diffusion-free electron transfer to the electrode surface and thus measurement of an immediate photocurrent response. The initial photoc...

Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts...

Electrochemical communication between two photobioelectrochemical half-cells based on photosystem 1 and photo-system 2 is investigated in operando. The driving force for the electron transfer reactions is applied in a wireless mode using bipolar electrochemistry with the actual electrode potentials being self-regulated by the redox processes. Four...

In the development of photosystem-based energy conversion devices, the in-depth understanding of electron transfer processes involved in photocurrent generation and possible charge recombination is essential as a basis for the development of photo-bioelectrochemical architectures with increased efficiency. The evaluation of a bio-photocathode based...

The development of a versatile microbiosensor for hydrogen detection is reported. Carbon-based microelectrodes were modified with a [NiFe]-hydrogenase embedded in a viologen-modified redox hydrogel for the fabrication of a sensitive hydrogen biosensor By integrating the microbiosensor in a scanning photoelectrochemical microscope, it was capable of...

Cyanobacterial photosynthesis can be regarded as the blueprint for the photosynthesis of green algae and higher plants: While most of the principal reactions on the molecular level are preserved, their structural arrangement is compact and intimately connected with the respiratory chain. This allows conclusions on the evolution of special functiona...

Interfacing photosynthetic proteins and electrodes for investigating light-induced charge separation remains challenging. The discovery of a competing charge transfer pathway through the light-harvesting antenna defines new design requirements for electrode modification.

The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b...

Supplemental MST-measurements to Fig 4 (left panel, evaluated affinity; right panel, raw MST traces). (TIF)

Determination of the binding affinity of Chaetosphaeridium globosum cpSRP54M/cpSRP43D189Y complex formation using microscale thermophoresis (left panel, evaluated affinity; right panel, raw MST traces). (TIF)

Amino acid sequence of Chaetosphaeridium globosum cpSRP54M and cpSRP43 corresponding to materials and methods ´Plasmids and plasmid construction´. (PDF)

Supplemental MST-measurements to Fig 6 (left panel, evaluated affinity; right panel, raw MST traces). (TIF)

Photosynthetic microorganisms have received considerable attention as production organisms for chemicals. An investigation of the photosynthetic NADPH supply for enantioselective biotransformations with recombinant cyanobacteria showed that the specific activity of the cells is comparable to heterotrophic organisms. Light-dispersion of the cells, h...

Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-lin...

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 towards it to form biogenesis centers. The initial steps in photosystem II (PSII) assembly are thought to ta...

This chapter presents biophotoelectrochemical systems where one of nature's photosynthetic proteins, such as photosystem 1 (PS1), photosystem 2 (PS2), or bacterial reaction centers, are employed to create devices for technological applications. We use recent advances in biophotoelectrodes for energy conversion and sensing to illustrate the fundamen...

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...

A recombinant enoate reductase was expressed in cyanobacteria and used for the light-catalyzed, enantioselective reduction of C=C bonds. The coupling of oxidoreductases to natural photosynthesis allows asymmetric syntheses fueled by the oxidation of water. Bypassing the addition of sacrificial cosubstrates as electron donors significantly improves...

In plants, algae, and cyanobacteria, photosystem 2 (PS2) catalyzes the light driven oxidation of water. The main products of this reaction are protons and molecular oxygen. In vitro, however, it was demonstrated that reactive oxygen species like hydrogen peroxide are obtained as partially reduced side products. The transition from oxygen to hydroge...

Eine in Cyanobakterien exprimierte, rekombinante Enoat-Reduktase wurde für die lichtkatalysierte, enantioselektive Reduktion von C=C-Bindungen genutzt. Dabei ermöglicht die Kopplung von Oxidoreduktasen an die natürliche Photosynthese eine durch Wasseroxidation angetriebene asymmetrische Synthese. Durch den Verzicht auf zusätzliche Cosubstrate als E...