Biology subjects

Schuller, J.

Publications and source records attributed to Schuller, J..

8 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↗

bioRxiv2026

The anaerobic cryo-EM structure of the methanogenic Mtr complex reveals a nitrogenase-like cluster bound to its active site

Methanogenic archaea conserve energy by coupling methyl-group transfer to the generation of a chemiosmotic sodiumion (Na+) gradient. This central energy-conserving step is catalyzed by the membrane-bound N5-methyl-H4MPT:coenzyme M methyltransferase (Mtr). Here, we present high-resolution cryo-electron microscopy structures of the Mtr complex from Methanosarcina mazei determined under strictly anaerobic conditions. The structures reveal an unexpected, electron-dense metallocluster embedded within the central cavity of the MtrCDE trimer in the membrane plane. Based on the unique topology and density we modeled it as an [Fe8S9C] L-type cluster. It is positioned adjacent to both the coenzyme M substrate and the corrinoid cofactor of MtrA in the MtrA-MtrCDE engaged state, thereby being located right at the catalytic core of the enzyme. We could further show that binding of MtrA to MtrCDE triggers rearrangements within the interface of MtrDE that widen a putative ion-conduction pathway. The proximity of the conserved sodium-binding site to the catalytic center suggests a putative link between methyl-transfer chemistry and Na+ translocation. In a broader context, these findings improve our understanding of how methyl transfer, analogous to redox chemistry, can drive chemiosmotic energy conversion.

biochemistry↗

bioRxiv2026

Electron confurcation drives photosynthetic H2 production in cyanobacteria

Cyanobacteria are major contributors to global photosynthesis and are intensively studied for sustainable green H2 production. Central to this process is the bidirectional [NiFe]-hydrogenase HoxEFUYH, yet its physiological redox partners have remained unresolved. Ferredoxin, NAD(H), and NADP(H) have been proposed as partners, but the lack of active enzyme preparations has prevented a definitive assignment. Here, we purified the intact HoxEFUYH complex from Synechocystis sp. PCC 6803 under strictly anaerobic conditions and reveal its function as both a bifurcating and confurcating hydrogenase. During H2 uptake, HoxEFUYH utilizes NAD+ and oxidized ferredoxin, whereas H2 production strictly requires both NADH and reduced ferredoxin; NADPH does not support either reaction. Combining high-resolution cryo-electron microscopy with biochemical and spectroscopic analyses, our data reveal that an flavin-containing reductase module is electronically connected to the catalytic [NiFe]-hydrogenase core through an extended chain of iron-sulfur clusters, defining the structural basis for bifurcating and confurcating electron flow. These findings fundamentally revise the physiological role of HoxEFUYH by showing that photosynthetic H2 production does not rely solely on photosynthetic electrons but instead couples reduced ferredoxin from the light reaction with NADH derived from "dark" carbohydrate oxidation. This requires reassessment of current strategies for green H2 production in cyanobacteria.

biochemistry↗

bioRxiv2026

Structural Basis of Membrane Potential Coupled Vectorial CO2 Hydration by the DAB2 Complex in Chemolithoautotrophs

The fixation of dissolved inorganic carbon (DIC) such as CO2 and bicarbonate by autotrophic microorganisms is fundamental to the global primary production. Many autotrophs depend on a diversity of CO2-concentrating mechanisms (CCMs) to overcome the inefficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the limited supply of DIC. While cyanobacterial CCMs are well characterized, analogous systems in chemolithoautotrophs, specifically active DIC uptake systems have long been overlooked. Here, we present the first cryo-EM structural analysis of DAB2, an essential membrane-associated protein complex for CO2 uptake in Halothiobacillus neapolitanus. The cytoplasmic subunit DabA2 displays a {beta}-carbonic anhydrase-like fold including a zinc ion, while the transmembrane subunit DabB2 resembles the proton-conducting subunits of respiratory Complex I. Purified DAB2 binds CO2 independent of proton motive force (PMF) however, did not spontaneously hydrate CO2. This suggests that CO2 hydration is PMF-dependent and may involve a gating mechanism. Structural analysis reveals an unconventional deeply buried active site only accessible via gated substrate tunnels, implying that substrate access, product release, and catalytic activation are tightly regulated. A unique transmembrane helix of DabA2 constitutes part of the proton conduction pathway and potentially couples proton translocation to enzymatic turnover. These features define a vectorial CO2 hydration mechanism that prohibits reverse bicarbonate dehydration and requires a proton gradient to initiate catalytic turnover. Our findings establish DAB2 as a prototype of a previously unrecognized family of PMF-driven carbonic anhydrases, elucidating a novel strategy for CO2 capture in non-photosynthetic autotrophs and expanding the mechanistic landscape of bacterial CCMs.

biochemistry↗

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↗

bioRxiv2024

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

evolutionary biology↗

bioRxiv2024

Molecular principles of redox-coupled sodium pumping of the ancient Rnf machinery

The Rnf complex is the primary respiratory enzyme of several anaerobic prokaryotes that transfers electrons from ferredoxin to NAD+ and pumps sodium ions (Na+) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na+-pumping NADH-quinone oxidoreductase (Nqr)1. By running in reverse, Rnf reduces ferredoxin with NADH as reductant at the expense of the transmembrane electrochemical ion gradient and provides low potential electrons for nitrogenases as well as CO2 reductases. Yet, the molecular principles that couple the long-range electron transfer to the Na+ translocation across the membrane remain elusive. Here we resolve key functional states along the electron transfer pathway using redox-controlled cryo-electron microscopy (cryo-EM) that, in combination with biochemical functional assays and atomistic molecular simulations, provide key insight into the redox-driven Na+ pumping mechanism. We show that the reduction of the unique membrane-embedded [2Fe2S] cluster in the vestibule between the RnfA/E subunits electrostatically attracts the sodium ions, and in turn, triggers an inward/outward transition with alternating membrane access driving the Na+ pump and the reduction of NAD+. Our study unveils an ancient mechanism for redox-driven ion pumping, and provides key understanding of the fundamental principles governing energy conversion in biological systems.

biochemistry↗

bioRxiv2024

2-oxoglutarate triggers assembly of active dodecameric Methanosarcina mazei glutamine synthetase

Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 [A] cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on an allosteric binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

microbiology↗