Search

Hydrogenases are the most active molecular catalysts for hydrogen production and uptake (1,2), and could therefore facilitate the development of new types of fuel cell (3-5). In [FeFe]-hydrogenases, catalysis takes place at a unique di-iron centre (the [2Fe] subsite), which contains a bridging dithiolate ligand, three CO ligands and two [CN.sup.-] ligands (6,7). Through a complex multienzymatic biosynthetic process, this [2Fe] subsite is first assembled on a maturation enzyme, HydF, and then delivered to the apo-hydrogenase for activation (8). Synthetic chemistry has been used to prepare remarkably similar mimics of that subsite (1), but it has failed to reproduce the natural enzymatic activities thus far. Here we show that three synthetic mimics (containing different bridging dithiolate ligands) can be loaded onto bacterial Thermotoga maritima HydF and then transferred to apo-HydA1, one of the hydrogenases of Chlamydomonas reinhardtii algae. Full activation of HydAl was achieved only when using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of native [FeFe]-hydrogenases (9,10). This is an example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active-site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe]-hydrogenases, with no requirement for the complete maturation machinery., Complexes 1 (11-13), 2 (14) and 3 (15) (Fig. 1a) represent the closest synthetic mimics of the [2Fe] subsite in HydA1. They all share the same primary coordination sphere with [...]

The metalloenzyme [FeFe]-hydrogenase is of interest to future biotechnologies targeting the production of "green" hydrogen (H 2 ). We recently developed a simple two-step functionalized procedure to immobilize the [FeFe]-hydrogenase from Clostridium pasteurianum ("CpI") on mesoporous indium tin oxide (ITO) electrodes to achieve elevated H 2 production with high operational stability and current densities of 8 mA cm -2 . Here, we use a combination of atomic force microscopy (AFM), scanning electron microscopy (SEM) and electrochemical quartz crystal microbalance (EQCM) to understand how mesoporous ITO stabilizes and activates CpI for electroenzymatic H 2 production. Examination of the topography and morphology of the mesoporous ITO surface revealed a hierarchical morphology containing cavities and well-defined nanoparticle agglomerates. Any potential effect of mesoporosity was investigated by comparing the stability and electroenzymatic activity of CpI on mesoporous 'nanoITO' and planar ITO, where we determined that CpI has a higher turnover frequency and adsorbs with greater stability (with respect to electroenzymatic activity over time) to nanoITO surfaces., (© 2024 Wiley-VCH GmbH.)

[FeFe]-hydrogenase is nature's most efficient proton reducing and H 2 -oxidizing enzyme. However, biotechnological applications are hampered by the O 2 sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe 2 (adt)(CO) 6- x (CN) x ] x - and [Fe 2 (pdt)(CO) 6- x (CN) x ] x - ( x = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy. Additionally, we describe a straightforward synthetic recipe for the active site precursor complex Fe 2 (adt)(CO) 6 . Our data indicate that the aminodithiolate (adt) complex, which is the synthetic precursor of the natural active site cofactor, is most oxygen sensitive. This observation highlights the significance of proton transfer in aerobic deactivation, and supported by DFT calculations facilitates an identification of the responsible reactive oxygen species (ROS). Moreover, we show that the ligand environment of the iron ions critically influences the reactivity with O 2 and ROS like superoxide and H 2 O 2 as the oxygen sensitivity increases with the exchange of ligands from CO to CN - . The trends in aerobic deactivation observed for the model complexes are in line with the respective enzyme variants. Based on experimental and computational data, a model for the initial reaction of [FeFe]-hydrogenase with O 2 is developed. Our study underscores the relevance of model systems in understanding biocatalysis and validates their potential as important tools for elucidating the chemistry of oxygen-induced deactivation of [FeFe]-hydrogenase.

The active site cofactor of [FeFe]-hydrogenases consists of a cubane [4Fe-4S]-cluster and a unique [2Fe-2S]-cluster, harboring unusual CO- and CN - -ligands. The biosynthesis of the [2Fe-2S]-cluster requires three dedicated maturation enzymes called HydG, HydE and HydF. HydG and HydE are both involved in synthesizing a [2Fe-2S]-precursor, still lacking parts of the azadithiolate (adt) moiety that bridge the two iron atoms. This [2Fe-2S]-precursor is then finalized within the scaffold protein HydF, which binds and transfers the [2Fe-2S]-precursor to the hydrogenase. However, its exact binding mode within HydF is still elusive. Herein, we identified the binding location of the [2Fe-2S]-precursor by altering size and charge of a highly conserved protein pocket via site directed mutagenesis (SDM). Moreover, we identified two serine residues that are essential for binding and assembling the [2Fe-2S]-precursor. By combining SDM and molecular docking simulations, we provide a new model on how the [2Fe-2S]-cluster is bound to HydF and demonstrate the important role of one highly conserved aspartate residue, presumably during the bioassembly of the adt moiety.

The exact mechanisms and the neural circuits involved in anesthesia induced unconsciousness are still not fully understood. To elucidate them valid animal models are necessary. Since the most commonly used species in neuroscience are mice, we established a murine model for commonly used anesthetics/sedatives and evaluated the epidural electroencephalographic (EEG) patterns during slow anesthesia induction and emergence. Forty-four mice underwent surgery in which we inserted a central venous catheter and implanted nine intracranial electrodes above the prefrontal, motor, sensory, and visual cortex. After at least one week of recovery, mice were anesthetized either by inhalational sevoflurane or intravenous propofol, ketamine, or dexmedetomidine. We evaluated the loss and return of righting reflex (LORR/RORR) and recorded the electrocorticogram. For spectral analysis we focused on the prefrontal and visual cortex. In addition to analyzing the power spectral density at specific time points we evaluated the changes in the spectral power distribution longitudinally. The median time to LORR after start anesthesia ranged from 1080 [1st quartile: 960; 3rd quartile: 1080]s under sevoflurane anesthesia to 1541 [1455; 1890]s with ketamine. Around LORR sevoflurane as well as propofol induced a decrease in the theta/alpha band and an increase in the beta/gamma band. Dexmedetomidine infusion resulted in a shift towards lower frequencies with an increase in the delta range. Ketamine induced stronger activity in the higher frequencies. Our results showed substance-specific changes in EEG patterns during slow anesthesia induction. These patterns were partially identical to previous observations in humans, but also included significant differences, especially in the low frequencies. Our study emphasizes strengths and limitations of murine models in neuroscience and provides an important basis for future studies investigating complex neurophysiological mechanisms., (© 2024. The Author(s).)

Hydrogenases are a diverse group of metalloenzymes that catalyze the conversion of H 2 into protons and electrons and the reverse reaction. A subgroup is formed by the [FeFe]‑hydrogenases, which are the most efficient enzymes of microbes for catalytic H 2 conversion. We have determined the stability and activity of two [FeFe]‑hydrogenases under high temperature and pressure conditions employing FTIR spectroscopy and the high-pressure stopped-flow methodology in combination with fast UV/Vis detection. Our data show high temperature stability and an increase in activity up to the unfolding temperatures of the enzymes. Remarkably, both enzymes reveal a very high pressure stability of their structure, even up to pressures of several kbars. Their high pressure-stability enables high enzymatic activity up to 2 kbar, which largely exceeds the pressure limit encountered by organisms in the deep sea and sub-seafloor on Earth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O 2 ) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4Fe H ) and a unique diiron moiety (2Fe H ). Previous attempts to prevent O 2 -induced damage have focused on enhancing the protein's sieving effect for O 2 by blocking the hydrophobic gas channels that connect the protein surface and the 2Fe H . In this study, we aimed to block an O 2 diffusion pathway and shield 4Fe H instead. Molecular dynamics (MD) simulations identified a novel water channel (W H ) surrounding the H-cluster. As this hydrophilic path may be accessible for O 2 molecules we applied site-directed mutagenesis targeting amino acids along W H in proximity to 4Fe H to block O 2 diffusion. Protein film electrochemistry experiments demonstrate increased O 2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O 2 in the environment of the 4Fe H in both cases. The results strongly suggest that, in wild type proteins, O 2 diffuses from the 4Fe H to the 2Fe H . These results reveal new strategies for improving the O 2 stability of [FeFe]-hydrogenases by focusing on the O 2 diffusion network near the active site., (© 2023 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

Several species of microalgae can convert light energy into molecular hydrogen (H 2 ) by employing enzymes of early phylogenetic origin, [FeFe]-hydrogenases, coupled to the photosynthetic electron transport chain. Bacterial [FeFe]-hydrogenases consist of a conserved domain that harbors the active site cofactor, the H-domain, and an additional domain that binds electron-conducting FeS clusters, the F-domain. In contrast, most algal hydrogenases characterized so far have a structurally reduced, so-termed M1-type architecture, which consists only of the H-domain that interacts directly with photosynthetic ferredoxin PetF as an electron donor. To date, only a few algal species are known to contain bacterial-type [FeFe]-hydrogenases, and no M1-type enzymes have been identified in these species. Here, we show that the chlorophycean alga Uronema belkae possesses both bacterial-type and algal-type [FeFe]-hydrogenases. Both hydrogenase genes are transcribed, and the cells produce H 2 under hypoxic conditions. The biochemical analyses show that the two enzymes show features typical for each of the two [FeFe]-hydrogenase types. Most notable in the physiological context is that the bacterial-type hydrogenase does not interact with PetF proteins, suggesting that the two enzymes are integrated differently into the alga's metabolism.

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN - ligands. The biogenesis of this diiron site, termed [2Fe H ], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2Fe H ] precursor, termed [2Fe P ], to the [FeFe]-hydrogenase. The binding site of [2Fe P ] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2Fe P ]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2Fe P ]. Surprisingly, our results suggest that binding or transfer of [2Fe P ] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Hydrogenases are H 2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN - ) ligands. Extrinsic CO and CN - , however, inhibit hydrogenases. The mechanism by which CN - binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN - -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN - and CO ligands and to show that extrinsic CN - binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN - treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Old Yellow Enzymes (OYEs) are flavin-containing ene-reductases that have been intensely studied with regard to their biotechnological potential for sustainable chemical syntheses. OYE-encoding genes are found throughout the domains of life, but their physiological role is mostly unknown, one reason for this being the promiscuity of most ene-reductases studied to date. The unicellular green alga Chlamydomonas reinhardtii possesses four genes coding for OYEs, three of which we have analyzed biochemically before. Ene-reductase CrOYE3 stood out in that it showed an unusually narrow substrate scope and converted N -methylmaleimide (NMI) with high rates. This was recapitulated in a C. reinhardtii croye3 mutant that, in contrast to the wild type, hardly degraded externally added NMI. Here we show that CrOYE3-mediated NMI conversion depends on electrons generated photosynthetically by photosystem II (PSII) and that the croye3 mutant exhibits slightly decreased photochemical quenching in high light. Non-photochemical quenching is strongly impaired in this mutant, and it shows enhanced oxidative stress. The phenotypes of the mutant suggest that C. reinhardtii CrOYE3 is involved in the protection against photooxidative stress, possibly by converting reactive carbonyl species derived from lipid peroxides or maleimides from tetrapyrrole degradation., Competing Interests: The Authors did not report any conflict of interest., (© 2023 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

The high turnover rates of [FeFe]-hydrogenases under mild conditions and at low overpotentials provide a natural blueprint for the design of hydrogen catalysts. However, the unique active site (H-cluster) degrades upon contact with oxygen. The [FeFe]-hydrogenase from Clostridium beijerinckii (CbA5H) is characterized by the flexibility of its protein structure, which allows a conserved cysteine to coordinate to the active site under oxidative conditions. Thereby, intrinsic cofactor degradation induced by dioxygen is minimized. However, the protection from O 2 is only partial, and the activity of the enzyme decreases upon each exposure to O 2 . By using site-directed mutagenesis in combination with electrochemistry, ATR-FTIR spectroscopy, and molecular dynamics simulations, we show that the kinetics of the conversion between the oxygen-protected inactive state (cysteine-bound) and the oxygen-sensitive active state can be accelerated by replacing a surface residue that is very distant from the active site. This sole exchange of methionine for a glutamate residue leads to an increased resistance of the hydrogenase to dioxygen. With our study, we aim to understand how local modifications of the protein structure can have a crucial impact on protein dynamics and how they can control the reactivity of inorganic active sites through outer sphere effects., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)