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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]

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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]

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]

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.)

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]

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]