Probing the Influence of the Protein Scaffold on H-Cluster Reactivity via Gain-of-Function Studies─Improved H₂ Evolution and O₂ Tolerance through Rational Design of [FeFe] Hydrogenase

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 27, 2025

[FeFe] hydrogenases make up a structurally diverse family of metalloenzymes that catalyze proton/dihydrogen interconversion. They can be classified into phylogenetically distinct groups denoted A-G, which differ in structure and reactivity. Prototypical Group A have high turnover rates remarkable energy efficiency. As compared to enzymes, the putatively sensory D hydrogenase from Thermoanaerobacter mathranii (TamHydS) has thousand-fold lower H2 evolution rate overpotential requirement drive catalysis (irreversible) but shows increased inhibitor tolerance. This divergence activity between makes them ideal models for studying second (active-site environment) outer (e.g., substrate transport) coordination sphere effects on metal cofactors. Herein, we generated three TamHydS-based variants, each mimicking proposed key structural features hydrogenase: "active site" (AS), "proton-transfer pathway" (PTP), "combined" (CM = AS + PTP) variant. fourth single-point variant, A137C, introduces critical cysteine active site, was characterized as reference. No change isolation resulted A-like behavior; i.e., no positive impact catalytic performance observed. The CM however, showed retained requirement. Additionally, variant improved already relatively stability TamHydS against O2 CO inhibition. These findings show rates, (ir)reversibility, susceptibility gaseous inhibitors are decoupled. Moreover, results highlight importance exploring diversity path toward understanding factors enable outstanding properties hydrogenases.

Language: Английский

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A [FeFe] Hydrogenase–Rubrerythrin Chimeric Enzyme Functions to Couple H₂ Oxidation to Reduction of H₂O₂ in the Foodborne Pathogen Clostridium perfringens

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: March 6, 2025

[FeFe] hydrogenases are a diverse class of H2-activating enzymes with wide range utilities in nature. As H2 is promising renewable energy carrier, exploration the increasingly realized functional diversity instrumental for understanding how these remarkable can benefit society and inspire new technologies. In this work, we uncover properties highly unusual natural chimera composed hydrogenase rubrerythrin as single polypeptide. The unique combination rubrerythrin, an enzyme that functions H2O2 detoxification, raises question whether catalytic reactions, such oxidation reduction, functionally linked. Herein, express purify representative from Clostridium perfringens (termed CperHydR) apply various electrochemical spectroscopic approaches to determine its activity confirm presence each proposed metallocofactors. cumulative data demonstrate contains surprising array metallocofactors: site termed H-cluster, two [4Fe-4S] clusters, rubredoxin Fe(Cys)4 centers, hemerythrin-like diiron site. absence H2-evolution current protein film voltammetry highlights exceptional bias toward greatest extent has been observed hydrogenase. Here, CperHydR uses H2, catalytically split by domain, reduce Structural modeling suggests homodimeric nature protein. Overall, study demonstrates H2-dependent reductase. Equipped information, discuss possible role part oxygen-stress response system, proposing constitutes pathway mitigation.

Language: Английский

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Protein Dynamics Affect O₂-Stability of Group B [FeFe]-Hydrogenase from Thermosediminibacter oceani

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: April 23, 2025

In the pursuit of sustainable "green" energy generation, [FeFe]-hydrogenases have attracted significant attention due to their ability catalyze hydrogen production. However, sensitivity these enzymes O2 is a major obstacle for application as biocatalysts in conversion technologies. search an O2-stable [FeFe]-hydrogenase, we identified hydrogenase ToHydA from Thermosediminibacter oceani that belongs rarely characterized Group B (M2a) [FeFe]-hydrogenases. Our findings demonstrate exhibits remarkable O2-stability, even under prolonged exposure. By characterizing site-directed mutagenesis variants, found highly conserved proton-transporting active site cysteine residue protects H-cluster O2-induced degradation by forming Hinact state. The additional TSCCCP motif ToHydA, feature unique [FeFe]-hydrogenases, enhances flexibility and facilitates formation Moreover, possesses features, including unusual resting state distinguishes enzyme other atomistic molecular dynamics simulations reveal previously unrecognized cluster hydrophobic residues centered around cysteine-bearing loop. This structural appears be common characteristic hydrogenases form O2-protected exploiting features future research can aim rationally design combine high catalytic activity with enhanced O2-stability develop more efficient durable catalysts.

Language: Английский

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Engineered Living Energy Materials

Interdisciplinary materials, Journal Year: 2025, Volume and Issue: unknown

Published: April 29, 2025

ABSTRACT To foster sustainable development, a pivotal trend lies in harnessing energy supplies that propel modern economic and societal progress. Recent advancements living materials for applications have sparked groundbreaking research area: engineered (ELEMs), which seamlessly integrate biological artificial systems efficient conversion storage. consolidate this area, herein, we summarize delve into the evolution of ELEMs. Firstly, provide an overview structural features mechanisms employed by bio‐modules spanning proteins, organelles, entire organisms. They can be directly used as components constructing ELEMs or inspirations design such entities. Then, comprehensively review latest strides based on their distinct modes. Finally, discuss challenges confronting envision future trajectories. The progress holds immense potential to catalyze interdisciplinary endeavors encompassing medicine, environmental science, technologies.

Language: Английский

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Secondary structure changes as the potential H2 sensing mechanism of Group D [FeFe]-hydrogenases

Chemical Communications, Journal Year: 2024, Volume and Issue: 60(78), P. 10914 - 10917

Published: Jan. 1, 2024

[FeFe]-hydrogenases function as both H

Language: Английский

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Biogeochemical Fe-Redox Cycling in Oligotrophic Deep-Sea Sediment

Water, Journal Year: 2024, Volume and Issue: 16(19), P. 2740 - 2740

Published: Sept. 26, 2024

Biogeochemical redox cycling of iron (Fe) essentially governs various geochemical processes in nature. However, the mechanistic underpinnings Fe-redox deep-sea sediments remain poorly understood, due to limited access environment. Here, abyssal sediment collected from a depth 5800 m Pacific Ocean was characterized for its elemental, mineralogical, and biological properties. The sedimentary environment determined be oligotrophic with nutrition, yet contained considerable amount trace elements. reactions progressed through an initial lag phase, followed by fast Fe(II) reduction extended period Fe(III) oxidation before achieving equilibrium after 58 days. presence external H2 electron donor significantly increased extent bio-reduction 7.73% relative amendment-free control under high pressure MPa. A similar enhancement 11.20% observed following lactate amendment atmospheric pressure. bio-oxidation occurred 16 days’ anaerobic culturing, coupled nitrate reduction. During Fe bio-redox reactions, microbial community composition shaped presence/absence donor, while hydrostatic levels were controlling factor. Shewanella spp. emerged as primary Fe(III)-reducing microorganisms, stimulated supplemented lactate. Marinobacter hydrocarbonoclasticus predominant Fe(II)-oxidizing microorganism across all conditions. Our findings illustrate continuous occurring environment, coexisting microorganisms determining oscillation valence states within sediment.

Language: Английский

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Cold-subduction biogeodynamics boost deep energy delivery to the forearc

Geochimica et Cosmochimica Acta, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 1, 2024

Language: Английский

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Deciphering the chemical landscape and potential ecological function of RiPPs from the untapped Archaea domain

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 8, 2024

Abstract Chemical communication is crucial in ecosystems with complex microbial communities. However, the difficulties inherent to cultivation of archaea have led a limited understanding their chemical language, especially regarding structure diversity and function secondary or specialized metabolites (SMs). Our comprehensive investigation into biosynthetic potential archaea, combined metabolic analyses first report heterologous expression has unveiled previously unexplored capabilities archaeal ribosomally synthesized post-translationally modified peptides (RiPPs). We identified twenty-four new lanthipeptides RiPPs exhibiting unique characteristics, including novel subfamily featuring an type diamino-dicarboxylic (DADC) termini, largely expanding landscape SMs. This sheds light on novelty emphasizes as untapped resource for natural product discovery. Additionally, demonstrate specific antagonistic activity against haloarchaea, mediating biotic interaction halophilic niche. Furthermore, they showcase ecological role enhancing host’s motility by inducing rod-shaped cell morphology upregulating archaellin gene expression, facilitating abiotic environments. These discoveries broaden our language provide promising prospects future exploration SM-mediated interaction. Figure

Language: Английский

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Gassy archaea

Nature Reviews Microbiology, Journal Year: 2024, Volume and Issue: 22(8), P. 456 - 456

Published: June 18, 2024

Language: Английский

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A widespread hydrogenase drives fermentative growth of gut bacteria in healthy people

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Aug. 15, 2024

Abstract Molecular hydrogen (H 2 ) is among the most central, but least understood, metabolites in human gastrointestinal tract (gut). H gas produced large quantities during bacterial fermentation and consumed as an energy source by bacteria archaea. Disruption of cycling linked to disorders, infections, cancers, with used indicator gut dysfunction through breath tests. Despite this, microorganisms, pathways, enzymes mediating production remain unresolved. Here we show that a previously uncharacterised enzyme, group B [FeFe]-hydrogenase, drives fermentative gut. Analysis stool, biopsy, isolate (meta)genomes (meta)transcriptomes this hydrogenase encoded highly expressed. Through analysis 19 taxonomically diverse isolates, [FeFe]-hydrogenase produces amounts supports growth both Bacteroidetes Firmicutes. Bacteroides particularly dominate production. Biochemical spectroscopic characterisation shows purified [FeFe]-hydrogenases are catalytically active bind di-iron site. These hydrogenases enriched guts healthy individuals, significantly depleted favour other Crohn’s disease. Furthermore, metabolically flexible respiratory abundant oxidizers gut, not sulfate reducers, methanogens, acetogens thought. This combination enzymatic, cellular, ecosystem-level provides first detailed understanding reveals new links between microbiota function health.

Language: Английский

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