Biology subjects

Murphy, B. J.

Publications and source records attributed to Murphy, B. J..

9 recordsLinked to original sources

bioRxiv2026

Structural basis of sulfide production in dissimilatory sulfur metabolism

The membrane-bound DsrMK(JOP) complex is central to dissimilatory sulfur metabolism, including in sulfate-reducing microbes (SRM), a group that plays important roles in shaping planetary and human health. Despite this global importance, the mechanism of sulfide production and its links to energy conservation remain unclear. Here, we present high-resolution cryo-EM structures of DsrMKJOP from Archaeoglobus fulgidus, alone, with menadiol and with the sulfur-carrying substrate DsrC-trisulfide, complemented by physiological and biochemical studies. The results clarify how SRM control the reactivity of sulfur to selectively achieve sulfide production. While DsrC-trisulfide is highly stable in isolation, interaction with the DsrK subunit facilitates its hydrolytic activation, triggering a conformational change. This brings a perthiosulfenate sulfur intermediate into the catalytic pocket of DsrK for reduction at a single non-cubane [4Fe-4S] cluster, likely supported by a conserved non-ligating cysteine. DsrM harbors a structural quinone-binding site, but seems not to catalyze menaquinol oxidation, although this likely occurs in DsrMK complexes from different sulfur-metabolizing organisms. In DsrMKJOP, trisulfide reduction by DsrK is linked to quinol oxidation at DsrP, releasing protons to the periplasm to generate a proton-motive force.

microbiology↗

bioRxiv2026

Challenges in reconstituting the peroxiredoxin 2:STAT3 transient redox-relay complex in vitro

Peroxiredoxin 2 (Prdx2) mediates redox signaling by transferring oxidative equivalents to target proteins such as STAT3, a redox-sensitive transcription factor implicated in inflammation and cancer. Although this interaction has been demonstrated in cells, reconstituting the Prdx2:STAT3 complex in vitro remains challenging due to its transient and redox-dependent nature. Here we test various conditions to stabilize the complex between taggless Prdx2 and the core fragment of STAT3 (CF-STAT3), including oxidants, detergents, the facilitator Annexin A2, anaerobic environments, and CovalX crosslinking. Complex formation was assessed via mass photometry, analytical size-exclusion chromatography (SEC), SEC-MALS, and electron microscopy (EM). No stable complex was observed under standard conditions. Anaerobic environments briefly stabilized the interaction, but cryo-EM could not resolve the structure. CovalX crosslinking yielded short-lived but homogeneous complexes. We found that Prdx2 is highly susceptible to hyperoxidation at its peroxidatic cysteine, particularly in the presence of DTT or excess H2O2, resulting in loss of function. Maintaining non-reducing conditions during purification preserved Prdx2 in an oxidation-competent state, promoting formation of the disulfide bond between the peroxidatic and resolving cysteines and thereby enabling reproducible detection of a weak complex with CF-STAT3. Our findings establish a framework for studying redox-relay protein complexes in vitro and highlight the importance of oxidation state management during protein handling.

molecular biology↗

bioRxiv2024

Snapshots of acetyl-CoA synthesis, the final step of CO2 fixation in the Wood-Ljungdahl pathway

In the ancient microbial Wood-Ljungdahl pathway, CO2 is fixed in a multi-step process ending with acetyl-CoA synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). Here, we present catalytic snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction including electron transfer to the CODH for CO2 reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site and acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme crucial for CO2 fixation in anaerobic organisms. One-Sentence Summary: Structural description of key states of CO2 fixation by the carbon monoxide dehydrogenase/acetyl-CoA synthase complex.

biophysics↗

bioRxiv2024

Structural evidence for two-stage binding of mitochondrial ferredoxin 2 to the core iron-sulfur cluster assembly complex

Iron-sulfur (FeS) clusters are ubiquitous metallocofactors that are essential for life. In eukaryotes, FeS cluster biosynthesis begins with the de novo assembly of a [2Fe-2S] cluster by the core iron-sulfur cluster assembly (ISC) complex in the mitochondrial matrix. This complex comprises the scaffold protein ISCU2, the cysteine desulfurase subcomplex NFS1-ISD11-ACP1, the allosteric activator frataxin (FXN) and the electron donor ferredoxin 2 (FDX2). The interaction of FDX2 with the complex remains unclear. Here, we present cryo-EM structures of the FDX2-bound core ISC complex and show that FDX2 and FXN compete for overlapping binding sites during [2Fe-2S] cluster biosynthesis. FDX2 binds in two conformations; in the distal conformation, helix F of FDX2 shows loose electrostatic interaction with an arginine patch of NFS1, while in the proximal conformation this interaction tightens and the FDX2-specific C terminus forms contacts with NFS1; in this conformation, the [2Fe-2S] cluster of FDX2 is close enough to the ISCU2 FeS cluster assembly site for rapid electron transfer.

biophysics↗

bioRxiv2024

Localising elements in single-particle reconstructions by REEL-EM: Reconstructed Electron Energy-Loss - Elemental Mapping

For structures determined by single particle cryo-EM, no technique currently exists for mapping elements to defined locations, leading to errors in the assignment of metals and other ions, cofactors, substrates, inhibitors, and lipids that play essential roles in activity and regulation. Elemental mapping in the electron microscope is well established for dose-tolerant samples but is challenging for biological samples, especially in a cryo-preserved state. Here, we combine electron energy-loss spectroscopy (EELS) with single-particle image processing to allow elemental mapping in cryo-preserved macromolecular complexes. Proof-of-principle data show that our method, REEL-EM, allows 3D reconstruction of EELS data, such that a high total electron dose is accumulated across many copies of a complex. Working with two test samples, we demonstrate that we can reliably localise abundant elements. We discuss the current limitations of the method and potential future developments.

biophysics↗

bioRxiv2023

Structural and mechanistic insights into Streptococcus pneumoniae NADPH oxidase

NADPH oxidases (NOXs) play a major role in the physiology of eukaryotic cells by mediating the production of reactive oxygen species (ROS). Evolutionarily distant proteins sharing the NOX catalytic core have been recently described in Bacteria. Among them, the Streptococcus pneumoniae NOX (SpNOX) has been proposed as a model for the study of NOXs due to its high activity and stability in detergent micelles. Here, we report high-resolution cryo-EM structures of substrate-free and stably reduced NADH-bound SpNOX, and of the NADPH-bound SpNOX and a Phe397Ala mutant under turnover conditions. In combination with structure-guided mutagenesis and biochemical analyses, we provide the structural basis for constitutive activity, the lack of substrate specificity towards NADPH and the electron transfer pathway. Additionally, we shed light on the catalytic regulation by the C-terminal tail residue Phe397 and the potential in vivo function of this protein.

biophysics↗

bioRxiv2022

Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli

The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogen lyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H2 during fermentation, its reversibility, allowing H2-dependent CO2 reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically- and anaerobically-isolated forms at resolutions reaching 2.6 [A]. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in preventing reverse activity in vivo, and evaluate possible structural determinants of the bias toward hydrogen production over its oxidation.

biophysics↗

bioRxiv2021

Structure of the hexameric fungal plasma membrane proton pump in its auto-inhibited state

The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 [A] resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

biochemistry↗

bioRxiv2019

Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling

F1Fo-ATP synthases play a central role in cellular metabolism, making the energy of the proton-motive force across a membrane available for a large number of energy-consuming processes. We determined the single-particle cryo-EM structure of active dimeric ATP synthase from mitochondria of Polytomella sp. at 2.7- 2.8 [A] resolution. Separation of 13 well-defined rotary substates by 3D classification provides a detailed picture of the molecular motions that accompany c-ring rotation and result in ATP synthesis. Crucially, the F1 head rotates along with the central stalk and c-ring rotor for the first ~30{degrees} of each 120{degrees} primary rotary step. The joint movement facilitates flexible coupling of the stoichiometrically mismatched F1 and Fo subcomplexes. Flexibility is mediated primarily by the interdomain hinge of the conserved OSCP subunit, a well-established target of physiologically important inhibitors. Our maps provide atomic detail of the c-ring/a-subunit interface in the membrane, where protonation and deprotonation of c-ring cGlu111 drives rotary catalysis. An essential histidine residue in the lumenal proton access channel binds a strong non-peptide density assigned to a metal ion that may facilitate c-ring protonation, as its coordination geometry changes with c-ring rotation. We resolve ordered water molecules in the proton access and release channels and at the gating aArg239 that is critical in all rotary ATPases. We identify the previously unknown ASA10 subunit and present complete de novo atomic models of subunits ASA1-10, which make up the two interlinked peripheral stalks that stabilize the Polytomella ATP synthase dimer.

biochemistry↗