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The synthesis of nanosized covalent organic frameworks (nanoCOFs) with good dispersibility is vital for optical and optoelectronic applications. However, conventional methods rely on surfactants and organic solvents, limiting scalability and water compatibility. Here, we report a facile, scalable, and surfactant-free strategy to synthesize imine-linked nanoCOFs incorporating porphyrin units in aqueous acetic acid. By tuning monomer and catalyst concentrations, we modulated polymerization kinetics to obtain highly crystalline nanoCOFs with controlled sizes (∼50 nm–5 µm), tunable morphologies (nanocubes, nanorods, nanofibers), and good dispersibility. Protonation of porphyrin units during synthesis introduced surface charges, preventing aggregation and enabling excellent water dispersibility. The nanoCOFs exhibited strong and broad light absorption along with high colloidal stability. A proof-of-concept study demonstrated their remarkable photocatalytic activity for oxidative coupling of benzylamines in water, even at an ultra-low loading (0.0074 mol%). This sustainable approach offers a versatile route to high-performance nanoCOFs, advancing COF-based photocatalysis and fundamental studies of their photophysical properties.

Carbon monoxide dehydrogenases (CODHs) catalyse the reversible oxidation of CO to CO₂ and play central roles in microbial carbon metabolism. While well-characterised CODHs from different phylogenetic backgrounds exhibit high bidirectional activity, the enigmatic clade B remains functionally uncharacterised. Here we present the first structural and functional characterisation of a clade B CODH from Ruminococcus flavefaciens (RfCODH). It reveals striking divergence from canonical enzymes. A new anaerobic cryo-EM workflow was developed, carried out entirely under anoxic conditions by manual blotting and plunge freezing. It resulted in a 2.53 Å RfCODH structure. The structure adopts the typical CODH fold, but exhibits blocked gas channels, a compromised proton transfer pathway and disrupted cofactor coordination. This provides a structural rationale for RfCODH’s severely attenuated CO oxidation activity (13 mU/mg vs. 900 U/mg for the well-studied ChCODH-II). EPR spectroscopy reveals unique oxidised C-cluster states not previously characterised in CODHs. Mirror tree analysis hints to co-evolution between clade B CODHs and associated ABC transporter substrate-binding proteins, suggesting these enzymes function in metabolism of substrates imported via the ABC transporter module. All findings indicate evolutionary repurposing of the CODH scaffold for alternative physiological functions.

Hydrogenases are metalloenzymes that play key roles in H₂ metabolism. Beyond their catalytic function, [FeFe] hydrogenases from phylogenetic groups C and D have been proposed to act as H₂ sensors. These putative sensory enzymes contain, in addition to the canonical H₂-activating H-cluster, N-terminal [4Fe-4S] clusters as well as an atypical C-terminal [4Fe-4S] cluster, the latter being further associated with a Per–Arnt–Sim (PAS) domain in group C enzymes. The functional significance of these C-terminal clusters and their influence on enzymatic activity, however, remain poorly understood. Here we studied the accessory [4Fe-4S] clusters in the model group D enzyme from Thermoanaerobacter mathranii (TamHydS), by disrupting cluster formation in the N- and C-terminal domains, respectively. Spectroscopic and biochemical investigations indicated that one of the N-terminal [4Fe-4S] clusters is critical for the structural integrity of TamHydS. In contrast, disrupting the C-terminal [4Fe-4S] cluster through a cysteine to alanine mutation (C379A) resulted in a stable enzyme variant with a modified cluster. The C379A variant retained catalytic activity similar to the WT enzyme, although with a 2-fold enhancement of the H₂ oxidation rate observed under high-driving-force conditions. Based on electron paramagnetic resonance spectroscopy, we found the oxidation state of the C-terminal cluster to be unresponsive to the presence of H₂ gas. We inferred that the H₂-sensing or signaling function is unlikely to involve redox state changes in the C-terminal [4Fe-4S] cluster. Still, highly conserved charged residues around the [4Fe-4S] clusters of group D [FeFe] hydrogenases indicate a functional role of the C-terminal region.

Disruption of hydrogen (H₂) cycling in the gut is linked to gastrointestinal disorders, infections and cancers. However, the mechanisms and microorganisms controlling H2 production in the gut remain unresolved. Here we show that gut H₂ production is primarily driven by the microbial group B [FeFe]-hydrogenase. Metagenomics and metatranscriptomics of stool and tissue biopsy samples show that hydrogenase-encoding genes are widely present and transcribed in gut bacteria. Assessment of 19 taxonomically diverse gut isolates revealed that the group B [FeFe]-hydrogenases produce large amounts of H₂gas and support fermentative growth of Bacteroidetes and Firmicutes. Further biochemical and spectroscopic characterization of purified enzymes show that they are catalytically active, bind a di-iron active site and reoxidize ferredoxin derived from the pyruvate:ferredoxin oxidoreductase reaction. Group B hydrogenase-encoding genes are significantly depleted in favour of other fermentative hydrogenases in patients with Crohn’s disease. Finally, metabolically flexible respiratory bacteria may be the dominant hydrogenotrophs in the gut, rather than acetogens, methanogens and sulfate reducers. These results uncover the enzymes and microorganisms controlling H₂-cycling in the healthy human gut.

[FeFe]-hydrogenases are metalloenzymes catalysing bidirectional hydrogen (H₂) turnover. These enzymes are generally considered to be extremely efficient and fast catalysts. However, [FeFe]-hydrogenases constitute a very diverse enzyme family that can be divided into several distinct phylogenetic groups, denoted as groups A-G. Very little is known about the properties of [FeFe]-hydrogenases outside of the intensively studied group A, but recent studies on putatively sensory group C and D enzymes have revealed distinct differences in reactivity. The variation in structure, reactivity and physiological function observed between phylogenetic groups raises the question if all [FeFe]-hydrogenases follow the same mechanism for H₂ turnover. Here, we provide the first detailed spectroscopic investigation of a slow-acting putatively sensory group D [FeFe]-hydrogenase from Thermoanaerobacter mathranii (TamHydS). Photo-reduction enabled us to characterize redox states in group D [FeFe]-hydrogenase via infrared spectroscopy under catalytic conditions. The sequential population of redox states similar to group A [FeFe]-hydrogenases supports the notion that group A and D [FeFe]-hydrogenases follow a universal catalytic mechanism. However, clear differences between enzymes from different phylogenetic groups become evident when comparing the relative stability and protonation state of suggested key catalytic intermediates. Moreover, the spectroscopic data collected on TamHydS provides new insight into the structure of the reduced active site, lending further support for the notion of a retained bridging CO ligand throughout the entire catalytic cycle.

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

Metal-dependent redox enzymes are central for microbial processing of gases, as exemplified by hydrogenase, nitrogenase, and carbon monoxide dehydrogenase. Due to their remarkable efficiencies and high biotechnological relevance, such gas-processing enzymes are intensively studied. Nevertheless, many of their mechanistic details remain opaque. We herein report a new method for solution assays under reducing conditions based on europium(II) as a terminal reductant and show how it can be employed to gain new insight into hydrogenase kinetics. Compared with the commonly used reductant sodium dithionite, this work shows that Eu(II) can serve as a robust and relatively easy-to-handle alternative electron donor, also providing a larger potential window for catalytic studies. Further, this work clarifies previous discrepancies in the literature regarding the influence of pH on hydrogenase kinetics in these assays. Our study shows that sodium dithionite, most likely due to its decomposition into SO2, alters hydrogenase kinetics in solution assays. Using [FeFe]-hydrogenase I from Clostridium pasteurianum (CpI) as a model system, Eu(II)-based solution assays demonstrated a pH optimum of 5-6 and rates greatly exceeding those observed with sodium dithionite assays. The higher turnover frequencies observed at low pH obtained with Eu(II) align more closely with the electrochemical data. Additionally, a strong driving force dependency was identified. A solution potential change of approximately 180 mV resulted in a 35-fold increase in the catalytic rate, yielding activities far surpassing those of earlier reports on CpI turnover frequencies. These findings provide new insight into the pH dependence and overall kinetic performance of [FeFe]-hydrogenases. More broadly, the report outlines alternative assay methods employing Eu(II) to better understand the enzyme kinetics of hydrogenases and related metalloenzymes.

Fe^III complexes based on the [Fe^III(ImP)₂]⁺ motif (ImP = bis(2,6-bis(3-methylimidazol-2-ylidene-1-yl)phenylene)), where the ligand contains both carbene and cyclometalated moieties, are a promising class of photoactive materials made from this abundant metal. In this work, it is shown that bromo or furanyl substituents attached to the cyclometalating moiety of the ImP ligands stabilize the ²LMCT excited state to very different extent resulting in opposing effects on the ²LMCT lifetime. For [Fe^III(ImPBr)₂]⁺, the lifetime (255 ps) of its moderately stabilized ²LMCT state (1.85 eV) is slightly increased compared to the parent complex (1.90 eV, 240 ps) pointing to an increased barrier for deactivation via the ⁴MC state and enabling applications as photoredox catalyst. In contrast, the ²LMCT energy of [Fe^III(ImPFur)₂]⁺ is lowered substantially to a value of 1.63 eV due to the extended π-system of the ligands and the reduced energy gap favors internal conversion directly to the ground state resulting in a considerably reduced ²LMCT lifetime of 59 ps. These findings have general implications for design of ligand modifications aiming at extended LMCT lifetimes and/or modified ground and excited state potentials.

As a new class of organic photocatalysts, polymer dots show a potential application in photocatalytic hydrogen peroxide production coupled with chemical oxidation such as methanol oxidation. However, the poor methanol oxidation ability by polymer dots still inhibits the overall photocatalytic reaction occurring in the neutral condition. In this work, an organic molecular catalyst 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl radical is covalently linked to a fluorene unit in a polymer skeleton, eventually enabling photocatalytic hydrogen peroxide production coupled with methanol oxidation in the neutral condition. By conducting various spectroscopic measurements, charge transfer between components in this molecular catalyst-immobilized polymer dots system is studied and found to be very efficient for hydrogen peroxide production coupled with alcohol oxidation. This work proves a strategy for designing polymer dots photocatalysts with molecular catalysts, facilitating their future development and potential applications in other fields such as water splitting, CO₂ reduction, photoredox catalysis and photodynamic therapy.

Two iron complexes featuring the bidentate, nonconjugated N-heterocyclic carbene (NHC) 1,1′-methylenebis(3-methylimidazol-2-ylidene) (mbmi) ligand, where the two NHC moieties are separated by a methylene bridge, have been synthesized to exploit the combined influence of geometric and electronic effects on the ground- and excited-state properties of homoleptic Fe^III-hexa-NHC [Fe(mbmi)₃](PF₆)₃ and heteroleptic Fe^II-tetra-NHC [Fe(mbmi)₂(bpy)](PF₆)₂ (bpy = 2,2′-bipyridine) complexes. They are compared to the reported Fe^III-hexa-NHC [Fe(btz)₃](PF₆)₃ and Fe^II-tetra-NHC [Fe(btz)₂(bpy)](PF₆)₂ complexes containing the conjugated, bidentate mesoionic NHC ligand 3,3′-dimethyl-1,1′-bis(p-tolyl)-4,4′-bis(1,2,3-triazol-5-ylidene) (btz). The observed geometries of [Fe(mbmi)₃](PF₆)₃ and [Fe(mbmi)₂(bpy)](PF₆)₂ are evaluated through L–Fe–L bond angles and ligand planarity and compared to those of [Fe(btz)₃](PF₆)₃ and [Fe(btz)₂(bpy)](PF₆)₂. The Fe^II/Fe^III redox couples of [Fe(mbmi)₃](PF₆)₃ (−0.38 V) and [Fe(mbmi)₂(bpy)](PF₆)₂ (−0.057 V, both vs Fc^+/0) are less reducing than [Fe(btz)₃](PF₆)₃ and [Fe(btz)₂(bpy)](PF₆)₂. The two complexes show intense absorption bands in the visible region: [Fe(mbmi)₃](PF₆)₃ at 502 nm (ligand-to-metal charge transfer, ²LMCT) and [Fe(mbmi)₂(bpy)](PF₆)₂ at 410 and 616 nm (metal-to-ligand charge transfer, ³MLCT). Lifetimes of 57.3 ps (²LMCT) for [Fe(mbmi)₃](PF₆)₃ and 7.6 ps (³MLCT) for [Fe(mbmi)₂(bpy)](PF₆)₂ were probed and are somewhat shorter than those for [Fe(btz)₃](PF₆)₃ and [Fe(btz)₂(bpy)](PF₆)₂. [Fe(mbmi)₃](PF₆)₃ exhibits photoluminescence at 686 nm (²LMCT) in acetonitrile at room temperature with a quantum yield of (1.2 ± 0.1) × 10^–4, compared to (3 ± 0.5) × 10^–4 for [Fe(btz)₃](PF₆)₃.