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Iron–sulfur (Fe–S) centers are versatile building blocks in biological electron transfer chains because their redox potentials may cover a wide potential range depending on the type of the cluster and the specific protein environment. Resonance Raman (RR) spectroscopy is widely used to analyze structural properties of such cofactors, but it remains still a challenge to disentangle the overlapping signals of metalloproteins carrying several Fe–S centers. In this work, we combined RR spectroscopy with protein engineering and X‐ray crystallography to address this issue on the basis of the oxygen‐tolerant membrane‐bound hydrogenase from Ralstonia eutropha that catalyzes the reversible conversion of hydrogen into protons and electrons. Besides the NiFe‐active site, this enzyme harbors three different Fe–S clusters constituting an electron relay with a distal [4Fe–4S], a medial [3Fe–4S], and an unusual proximal [4Fe–3S] cluster that may carry a hydroxyl ligand in the superoxidized state. RR spectra were measured from protein crystals by varying the crystal orientation with respect to the electric field vector of the incident laser to achieve a preferential RR enhancement for individual Fe–S clusters. In addition to spectral discrimination by selective reduction of the proximal cluster, protein engineering allowed for transforming the proximal and medial cluster into standard cubane‐type [4Fe–4S] centers in the C19G/C120G and P242C variants, respectively. The latter variant was structurally characterized for the first time in this work. Altogether, the entirety of the RR data provided the basis for identifying the vibrational modes characteristic of the various cluster states in this "model" enzyme as a prerequisite for future studies of complex (FeS)‐based electron transfer chains. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2‐tolerant [NiFe]‐hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X‐ray crystallographic data with spectroscopic analyses. [ABSTRACT FROM AUTHOR]

In this work, we demonstrate that diazonium electrografting of biocompatible interfaces on transparent conducting oxide indium tin oxide (ITO) can be controlled and optimized to achieve low charge transfer resistance, allowing highly efficient electron transfer to an immobilized model enzyme, the oxygentolerant [NiFe]-hydrogenase from Ralstonia eutropha. The use of a radical scavenger enables control of the interface thickness, and thus facilitates maximization of direct electron transfer processes between the enzyme's active center and the electrode. Using this approach, amine and carboxylic acid functionalities were grafted on ITO, allowing enzyme immobilization both under moderate electrostatic control and covalently via amide bond formation. Despite an initial decrease in catalytic activity, covalent immobilization led to an improvement in current stability compared to just electrostatically immobilized enzyme. Given the superior stability of electrografted interfaces in comparison to adsorbed or self-assembled interfaces, we propose electrografting as an alternative approach for the functional immobilization of redox-active enzymes on transparent conducting oxide (TCO) electrodes in bioelectronic devices. [ABSTRACT FROM AUTHOR]

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term "phosphoglycolate salvage." Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2. We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism. [ABSTRACT FROM AUTHOR]

[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2tolerance of the enzyme. [ABSTRACT FROM AUTHOR]

Multilayered or stacked lipid membranes are a common principle in biology and have various functional advantages compared to single-lipid membranes, such as their ability to spatially organize processes, compartmentalize molecules, and greatly increase surface area and hence membrane protein concentration. Here, a supramolecular assembly of a multilayered lipid membrane system is reported in which poly- l-lysine electrostatically links negatively charged lipid membranes. When suitable membrane enzymes are incorporated, either an ubiquinol oxidase (cytochrome bo3 from Escherichia coli) or an oxygen tolerant hydrogenase (the membrane-bound hydrogenase from Ralstonia eutropha), cyclic voltammetry (CV) reveals a linear increase in biocatalytic activity with each additional membrane layer. Electron transfer between the enzymes and the electrode is mediated by the quinone pool that is present in the lipid phase. Using atomic force microscopy, CV, and fluorescence microscopy it is deduced that quinones are able to diffuse between the stacked lipid membrane layers via defect sites where the lipid membranes are interconnected. This assembly is akin to that of interconnected thylakoid membranes or the folded lamella of mitochondria and has significant potential for mimicry in biotechnology applications such as energy production or biosensing. [ABSTRACT FROM AUTHOR]

Hydrogenases are nature's key catalysts involved in both microbial consumption and production of molecular hydrogen. H2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H2, the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry--that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H2 oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O2, employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N10-formyl-tetrahydrofolate (N10-CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N10-CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates. [ABSTRACT FROM AUTHOR]

Oxygen-tolerant [NiFe] hydrogenases are attractive biocatalysts for utilization in H2/O2 fuel cells, which thereby reduces the amount of platinum-based catalysts. The O2-tolerant membrane-bound hydrogenases isolated from Ralstonia eutropha and Aquifex aeolicus were previously studied at planar electrodes. The design of a powerful enzymatic fuel cell, however, requires a considerable increase in enzyme loading. Herein, we immobilized the two hydrogenases on carbon nanotubes, and we demonstrated that the enzyme binding and electron-transfer properties on the 3D networks relied on the same surface chemistry as that of the planar electrodes. We evaluated how the intrinsic properties of each hydrogenase, that is, temperature and O2 tolerance, were affected by immobilization on different electrode surfaces. The role of the detergent used for protein purification was especially emphasized. We also demonstrated that O2 reduction products affected more seriously the enzyme activity than molecular O2. If immobilized on pyrene-modified carbon nanotubes, both enzymes were used for the first time in a mild-temperature, membraneless H2/O2 enzymatic fuel cell, fed with O2-rich gas mixture, opening new avenues toward the development of alternative energy supplies. [ABSTRACT FROM AUTHOR]

[NiFe] hydrogenases are metalloenzymes catalyzing the reversible heterolytic cleavage of hydrogen into protons and electrons. Gas tunnels make the deeply buried active site accessible to substrates and inhibitors. Understanding the architecture and function of the tunnels is pivotal to modulating the feature of O2 tolerance in a subgroup of these [NiFe] hydrogenases, as they are interesting for developments in renewable energy technologies. Here we describe the crystal structure of the O2-tolerant membrane-bound [NiFe] hydrogenase of Ralstonia eutropha (ReMBH), using krypton-pressurized crystals. The positions of the krypton atoms allow a comprehensive description of the tunnel network within the enzyme. A detailed overview of tunnel sizes, lengths, and routes is presented from tunnel calculations. A comparison of the ReMBH tunnel characteristics with crystal structures of other O2-tolerant and O2-sensitive [NiFe] hydrogenases revealed considerable differences in tunnel size and quantity between the two groups, which might be related to the striking feature of O2 tolerance. [ABSTRACT FROM AUTHOR]

Copyright of Angewandte Chemie is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The tolerance towards oxic conditions of O2-tolerant [NiFe] hydrogenases has been attributed to an unusual [4Fe–3S] cluster that lies proximal to the [NiFe] active site. Upon exposure to oxygen, this cluster converts to a superoxidised (5+) state, which is believed to secure the formation of the so-called Ni–B state that is rapidly reactivated under reducing conditions. Here, the reductive reactivation of the membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha in a native-like lipid membrane was characterised and compared to a variant that instead carries a typical [4Fe–4S] proximal cluster. Reactivation from the Ni–B state was faster in the [4Fe–4S] variant, suggesting that the reactivation rate in MBH is limited by the reduction of the superoxidised [4Fe–3S] cluster. We propose that the [4Fe–3S] cluster plays a major role in protecting MBH by blocking the reversal of electron transfer to the [NiFe] active site, which would produce damaging radical oxygen species. [ABSTRACT FROM AUTHOR]

Hydrogenases catalyze the reversible oxidation of H2 into protons and electrons and are usually readily inactivated by O2. However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O2 that enables their host organisms to utilize H2 as an energy source at high O2. This feature is crucially based on a unique six cysteine-coordinated [4Fe-3S] cluster located close to the catalytic center, whose properties were investigated in this study using a multidisciplinary approach. The [4Fe-3S] cluster undergoes redox-dependent reversible transformations, namely iron swapping between a sulfide and a peptide amide N. Moreover, our investigations unraveled the redox-dependent and reversible occurence of an oxygen ligand located at a different iron. This ligand is hydrogen bonded to a conserved histidine that is essential for H2 oxidation at high O2. We propose that these transformations, reminiscent of those of the P-cluster of nitrogenase, enable the consecutive transfer of two electrons within a physiological potential range. [ABSTRACT FROM AUTHOR]

Insights in active sites: Hydrogen‐conversion by hydrogenase is mediated by a sophisticated, metal‐containing catalytic center. Resonance Raman spectroscopy is used for the first time in the characterization of the active site of these biocatalysts. An integrated spectroscopic and computational approach gives insights into structural and photochemical properties of the active site of an oxygen‐tolerant [NiFe] hydrogenase. [ABSTRACT FROM AUTHOR]

Mithilfe eines optimierten Metallzentrums ermöglichen Hydrogenasen die Umsetzung von Wasserstoff. Resonanz ‐ Raman ‐ Spektroskopie wird als neue Methode zur Charakterisierung des aktiven Zentrums dieser Biokatalysatoren vorgestellt. Ein kombinierter spektroskopischer und theoretischer Ansatz gibt Einblicke in die Struktur und die photochemischen Eigenschaften des [NiFe] ‐ Zentrums einer sauerstofftoleranten Hydrogenase. [ABSTRACT FROM AUTHOR]

Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H2 into protons and electrons at high rates. Those hydrogenases maintaining their activity in the presence of O2 are considered to be central to H2-based technologies, such as enzymatic fuel cells and for light-driven H2 production. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations, the molecular background allowing a structural interpretation of how the catalytic centre is protected from irreversible inactivation by O2 has remained unclear. Here we present the crystal structure of an O2-tolerant [NiFe]-hydrogenase from the aerobic H2 oxidizer Ralstonia eutropha H16 at 1.5?Å resolution. The heterodimeric enzyme consists of a large subunit harbouring the catalytic centre in the H2-reduced state and a small subunit containing an electron relay consisting of three different iron-sulphur clusters. The cluster proximal to the active site displays an unprecedented [4Fe-3S] structure and is coordinated by six cysteines. According to the current model, this cofactor operates as an electronic switch depending on the nature of the gas molecule approaching the active site. It serves as an electron acceptor in the course of H2 oxidation and as an electron-delivering device upon O2 attack at the active site. This dual function is supported by the capability of the novel iron-sulphur cluster to adopt three redox states at physiological redox potentials. The second structural feature is a network of extended water cavities that may act as a channel facilitating the removal of water produced at the [NiFe] active site. These discoveries will have an impact on the design of biological and chemical H2-converting catalysts that are capable of cycling H2 in air. [ABSTRACT FROM AUTHOR]

Redox-Intermediate von [NiFe]-Hydrogenasen durch einen experimentellen Ansatz für solvatisierte, lyophilisierte und kristallisierte Metalloenzyme. Frontispiz: Ein neuer Aufbau zur Untersuchung der Struktur und Funktion von solvatisierten, lyophilisierten und kristallinen Metalloenzymen - veranschaulicht anhand von [NiFe]-Hydrogenasen Keywords: [NiFe]-Hydrogenasen; Biokatalyse; In-situ-Spektroskopie; Metalloenzyme; Schwingungsspektroskopie DE [NiFe]-Hydrogenasen Biokatalyse In-situ-Spektroskopie Metalloenzyme Schwingungsspektroskopie 1 1 1 07/10/21 20210712 NES 210712 B Metalloenzyme b Im Forschungsartikel auf S. 15988 erforschen Christian Lorent, Marius Horch, Lars Lauterbach, Ingo Zebger et al. [Extracted from the article]

Keywords: [NiFe]-hydrogenase; biocatalysis; in situ spectroscopy; metalloenzymes; vibrational spectroscopy EN [NiFe]-hydrogenase biocatalysis in situ spectroscopy metalloenzymes vibrational spectroscopy 1 1 1 07/10/21 20210712 NES 210712 B Metalloenzymes b In their Research Article on page 15854, Christian Lorent, Marius Horch, Lars Lauterbach, Ingo Zebger et al. explore redox intermediates of [NiFe]-hydrogenases by an advanced experimental approach for solvated, lyophilized, and crystallized metalloenzymes. Frontispiece: Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes [NiFe]-hydrogenase, biocatalysis, in situ spectroscopy, metalloenzymes, vibrational spectroscopy. [Extracted from the article]

In article number 1606265 Lars J. C. Jeuken and co‐workers use a layer‐by‐layer assembly of lipid bilayers to multiply the surface concentration of electroactive membrane enzymes at electrodes. The interconnected membrane multilayers, akin to those of thylakoid membranes, create a material that exhibits a linear increase in bioelectrocatalytic activity with each additional enzyme‐containing membrane layer (containing either ubiquinol oxidase or an oxygen‐tolerant hydrogenase). [ABSTRACT FROM AUTHOR]

The biological conversion of hydrogen is catalyzed by [NiFe] hydrogenases utilizing a tailored bimetallic center. In their Communication on page 5162 ff. P. Hildebrandt, I. Zebger, M. Horch, and co‐workers use resonance Raman spectroscopy for the first time to characterize this active site by directly probing Fe–CO/CN vibrational modes. Applying an integrated spectroscopic and computational approach, this method provides new insights into structural and photochemical aspects of the [NiFe] site. [ABSTRACT FROM AUTHOR]

[NiFe] ‐ Hydrogenasen katalysieren die biologische Umwandlung von Wasserstoff mithilfe eines maßgeschneiderten bimetallischen Zentrums. In ihrer Zuschrift auf S. 5267 ff. nutzen P. Hildebrandt, I. Zebger, M. Horch et al. die Resonanz ‐ Raman ‐ Spektroskopie als neue Charakterisierungstechnik für dieses aktive Zentrum, die eine Möglichkeit zur direkten Beobachtung der Fe ‐ CO/CN ‐ Schwingungsmoden bietet. Ihr kombinierter spektroskopischer und theoretischer Ansatz gibt Einblicke in die Struktur und die photochemischen Eigenschaften des [NiFe] ‐ Zentrums. [ABSTRACT FROM AUTHOR]