Biology subjects

Nowaczyk, M. M.

Publications and source records attributed to Nowaczyk, M. M..

11 recordsLinked to original sources

bioRxiv2026

Structural Basis of Redox-Coupled CO2 hydration by the Cyanobacterial NDH-1MS' complex

The NAD(P)H dehydrogenase-like complex, NDH-1MS, is a constitutively expressed CO2-concentrating mechanism critical for sustaining CO2 fixation in cyanobacteria. This complex has been proposed to function as a vectorial carbonic anhydrase and accumulates intracellular bicarbonate against chemical equilibrium by coupling CO2 hydration to the photosynthetic cyclic electron flow. Using cryo-electron microscopy, we determined the structure of NDH-1MS from Thermosynechococcus vestitus. Contrary to its inducible counterpart NDH-1MS, our data suggest that a candidate CO2 hydration site may reside in the carbon-concentrating subunit CupB instead of the CupB-NdhF4 interface previously proposed. The active-site region partially resembles metal ion-independent iota-carbonic anhydrases and may bind CO2 or bicarbonate. We further observe features consistent with a Grotthuss-type proton transfer network connecting the active-site region to the antiporter-like subunits NdhF4 and NdhD4; however, unlike complex I, the proton export channel in NdhF4 appears to be blocked by bulky hydrophobic amino acids. Structural comparison between the oxygenic photosynthesis-specific subunit NdhV-bound state and dissociated state further reveals a correlation between plastoquinone stability and NdhV association. Taken together, we propose a revised working model in which NDH-1MS may function as a metal ion-independent vectorial carbonic anhydrase, with CO2 hydration coupled to proton transfer events in the antiporter-like subunits across the thylakoid membrane. SignificancePhotosynthetic complex I-like CO2-uptake systems are central to cyanobacterial carbon fixation, yet the molecular basis for coupling CO2 hydration to electron transfer has remained unresolved. Here, structures of NDH-1MS in NdhV-bound and NdhV-free states revise the architectural framework for catalysis and coupling in the constitutive CO2-uptake module. The data identify a CupB-centered candidate active-site region, reveal a non-canonical terminal antiporter-like subunit with an apparently blocked P-side exit, and link NdhV association to altered plastoquinone stability. Together, these findings refine current models for vectorial carbonic anhydrase activity in cyanobacterial NDH-1 and provide a structural basis for understanding how photosynthetic redox chemistry may be coupled to inorganic carbon concentration.

plant biology↗

bioRxiv2025

Structural insights into late-stage photosystem II assembly by Psb32

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 Mn4O5Ca cluster, which catalyzes the unique water splitting reaction in mature PSII, remains unclear. Using cryo-electron microscopy, we have determined the structure of two novel late-stage PSII assembly intermediates from Thermosynechococcus vestitus BP-1. In contrast to previous studies, the resulting monomeric PSII complexes contain both PsbJ and Psb27 and exhibit a fully mature acceptor side, while the oxygen evolving complex (OEC) is still in an immature state. The second complex additionally associates with the late-acting assembly factor Psb32 and the extrinsic subunit PsbV. While Psb32 has received little attention, its proposed role in the complex challenges the previous assumption that all extrinsic subunits associate spontaneously, as well as the notion that PsbO initiates binding and solely drives OEC formation. Our structures of the Psb27-PSII and Psb32-PSII intermediates provide novel insights, how structural changes of C-termini of the D1 and D2 core proteins regulate maturation of the OEC and how the catalytic side is prepared for binding of the Mn4O5Ca cluster. The Psb32-PSII complex potentially represents the final PSII assembly intermediate that precedes the incorporation and photoactivation of the Mn4O5Ca cluster, allowing us to explain the final steps in the PSII biogenesis and assembly pipeline in great detail, as only the two extrinsic subunits PsbO and PsbU are missing.

plant biology↗

bioRxiv2025

Dissecting a biological electron transport network with electrochemistry

Photosynthetic membranes contain complex networks of redox proteins and molecules which direct electrons along various energy-to-chemical interconversion reactions important for sustaining life on Earth. Analysing and disentangling the mechanisms, regulation and interdependencies of these electron transfer pathways is extremely difficult, owing to the large number of interacting components in the native membrane environment. Whilst electrochemistry is well established for studying electron transfer in purified proteins, it has proven difficult to directly wire into proteins within their native membrane environments, and even harder to probe on a systems-level the electron transfer networks they are entangled within. Here, we show how photosynthetic membranes from cyanobacteria can be directly wired to electrodes to access their complex electron transfer networks. Measurements of native membranes with structured electrodes revealed distinctive electrochemical signatures, enabling analysis from the scale of individual proteins to entire biochemical pathways, as well as their interplay. This includes measurements of overlapping photosynthetic and respiratory pathways, the redox activities of membrane-bound quinones, along with validation using in operando spectroscopic measurements. Importantly, we further demonstrated direct extraction of electrons from native membrane-bound Photosystem I at -600 mV, which is [~]1 V more negative than from purified photosystems. This finding opens up opportunities for biotechnologies for solar electricity, fuel and chemical generation. We foresee this electrochemical method being adapted to analyze other photosynthetic and non-photosynthetic membranes, as well as aiding the development of new biocatalytic, biohybrid and biomimetic systems.

biochemistry↗

bioRxiv2024

Complexome profiling of the Chlamydomonas psb28 mutant reveals THYLAKOID ENRICHED FRACTION 5 as an early photosystem II assembly factor

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, while overaccumulating RCII, CP43mod and D1mod. The mutant had less PSI and more Cytb6f and showed fewer thylakoid stacks and distorted chloroplast morphology. Complexome profiling of the psb28 mutant revealed that TEF5, the homolog of Arabidopsis PSB33/LIL8, co-migrated particularly with RCII. TEF5 also interacted with PSI. A Chlamydomonas tef5 null mutant is also severely impaired in PSII assembly and overaccumulates RCII and CP43mod. RC47 was not detectable in the light-grown tef5 mutant. Our data suggest a possible role for TEF5 in facilitating the assembly of CP47mod into RCII. Both the psb28 and tef5 mutants exhibited decreased synthesis of CP47 and PsbH, suggesting negative feedback regulation possibly exerted by the accumulation of RCII and/or CP43mod in both mutants. The strong effects of missing auxiliary factors on PSII assembly in Chlamydomonas suggest a more effective protein quality control system in this alga than in land plants and cyanobacteria. One-sentence summaryThe Chlamydomonas psb28 mutant is severely impaired in PSII assembly which via complexome profiling allowed identifying TEF5 as a novel PSII assembly factor that likely facilitates CP47 assembly. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Michael Schroda (m.schroda@rptu.de).

plant biology↗

bioRxiv2022

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 b6f 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 E. 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.

biochemistry↗

bioRxiv2022

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. 6,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen-evolving activity of approx. 3,000 {micro}mol O2{middle dot} (mg Chl{middle dot}h)-1 and 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 in the repair of the complex.

plant biology↗

bioRxiv2022

Proteomic identification of the interactome of stalled ribosome nascent chain complexes translating the thylakoid membrane protein D1

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, and targeting to prevent misfolding or aggregation and to ensure efficient transfer of the nascent chain to the insertion machinery. However, little is known about the regulatory network underlying these processes. To identify factors specifically involved in the cotranslational biogenesis of the reaction center protein D1 of photosystem II we established a chloroplast-derived in vitro translation method that allows the production and affinity purification of stalled RNCs bearing nascent chains of D1 of different defined lengths. Stalled RNCs translating the soluble ribosomal subunit uS2c were affinity-purified for comparison. Quantitative tandem-mass spectrometry revealed a set of about 120 proteins specifically associated with D1 RNCs. The interactome includes proteins with broad functions in protein processing, biogenesis and metabolic pathways, such as chlorophyll biosynthesis. We identified STIC2 as a new factor specifically associated with D1 RNCs. Furthermore, our results demonstrated that the interaction of STIC2 with the thylakoid insertase Alb3 and its homologue Alb4 is mediated by the conserved motif III within the C-terminal regions of Alb3 and Alb4. Our data suggest that STIC2 is involved in cotranslational substrate delivery at the thylakoid membrane by coordinating the binding of the D1 RNCs to the insertase machinery.

plant biology↗

bioRxiv2022

Structure of cyanobacterial photosystem I complexed with Cytochrome c6 and Ferredoxin at 1.97 A 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 the binding of Fd and the bound Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and Cyt c6. In our structure Cyt c6 is loosely bound in a non-productive site on the periphery of the complex. Side chain interactions at the PSI:Fd interface are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC 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 reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

plant biology↗

bioRxiv2021

OJIP chlorophyll fluorescence induction profiles and plastoquinone binding affinity of the Photosystem II assembly intermediate PSII-I from Thermosynechococcus elongatus

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-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.

plant biology↗

bioRxiv2021

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, and is fundamental to bioenergetics in photosynthetic bacteria and some higher plant cell types. 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, and unambiguously assign them to the structure using double electron-electron resonance (DEER). 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.

biophysics↗

bioRxiv2020

How to build a water-splitting machine: structural insights into photosystem II assembly

Biogenesis of photosystem II (PSII), natures 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 [A] resolution. It contains three assembly factors (Psb27, Psb28, 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 bicarbonate with glutamate as a ligand of the non-heme iron, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel 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. One Sentence HighlightThe high-resolution Cryo-EM structure of the photosystem II assembly intermediate PSII-I reveals how natures water splitting catalyst is assembled, protected and prepared for photoactivation by help of the three assembly factors Psb27, Psb28 and Psb34.

plant biology↗