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[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]

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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]

Energy-coupling factor transporters are a large group of importers for trace nutrients in prokaryotes. The in vivo oligomeric state of their substrate-specific transmembrane proteins (S units) is a matter of debate. Here we focus on the S unit BioY of Rhodobacter capsulatus, which functions as a low-affinity biotin transporter in its solitary state. To analyze whether oligomerization is a requirement for function, a tail-to-head-linked BioY dimer was constructed. Monomeric and dimeric BioY conferred comparable biotin uptake activities on recombinant Escherichia coil Fluorophore-tagged variants of the dimer were shown by fluorescence anisotropy analysis to oligomerize in vivo. Quantitative mass spectrometry identified biotin in the purified proteins at a stoichiometry of 1:2 for the BioY monomer and 1:4 (referring to single BioY domains) for the dimer. Replacement of the conserved Asp 164 (by Asn) and Lys167 (by Arg or Gin) in the monomer and in both halves of the dimer inactivated the proteins. The presence of those mutations in one half of the dimers only slightly affected biotin binding but reduced transport activity to 25% (Asp164Asn and Lys167Arg) or 75% (Lys167Gln). Our data (i) suggest that intermolecular interactions of domains from different dimers provide functionality, (ii) confirm an oligomeric architecture of BioY in living cells, and (iii) demonstrate an essential role of the last transmembrane helix in biotin recognition. [ABSTRACT FROM AUTHOR]

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising-strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via the widely used Strep-tag II affinity peptide. Electrochemistry, surface-sensitive spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tag labeled enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology., (© 2024 Wiley‐VCH GmbH.)

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O 2 -tolerant hydrogenase. It catalyzes the oxidation of H 2 into 2e - and 2H + in the presence of high O 2 concentrations. This characteristic trait is intimately linked to the unique Cys 6 [4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys 5 [4Fe-4S] or Cys 4 [4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O 2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O 2 protection mechanism that detoxifies O 2 to H 2 O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O 2 binding under electron-deficient conditions., Competing Interests: The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to P. S. (patrick.scheerer@charite.de), O. L. (oliver.lenz@tu-berlin.de), and I. Z. (ingo.zebger@tu-berlin.de)., (This journal is © The Royal Society of Chemistry.)

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 O 2 -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 57 Fe 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., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN - ) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe(CN) 2 (CO) cofactor requires the activity of at least six maturation proteins, designated HypA-F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivo data indicated that HypX releases CO using N 10 -formyltetrahydrofolate ( N 10 -formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N 10 -formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitro evidence that purified HypX first transfers the formyl group of N 10 -formyl-THF to produce formyl-coenzyme A (formyl-CoA) as a central reaction intermediate. In a second step, formyl-CoA is decarbonylated, resulting in free CoA and carbon monoxide. Purified HypX proved to be metal-free, which makes it a unique catalyst among the group of CO-releasing enzymes.

The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H 2 ⇌ 2H + + 2e - . Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H 2 -activating [NiFe] site and the small subunit contains iron-sulfur clusters mediating e - transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O 2 . Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O 2 -tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H 2 as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H 2 activation was performed by preHoxG even in the presence of O 2 , although the unique [4Fe-3S] cluster located in the small subunit and described to be crucial for tolerance toward O 2 was absent. These findings challenge the current understanding of O 2 tolerance of [NiFe] hydrogenases. The applicability of this minimal hydrogenase in basic and applied research is discussed.

Dioxygen-tolerant [NiFe]-hydrogenases are defined by their ability to catalyze the reaction, H 2 ⇌2H + +2e - even in the presence of O 2 . Catalytic and probably also noncatalytic mechanisms protect their active sites from being inactivated by reactive oxygen species, which makes them attractive subjects of investigation from both fundamental and applied perspectives. Prominent representatives of the O 2 -tolerant [NiFe]-hydrogenases have been isolated from the chemolithoautotrophic model organism Ralstonia eutropha H16, which can thrive in a simple mineral medium supplemented with the gases H 2 , O 2 , and CO 2 . In this chapter, we describe methods for cultivation and genetic manipulation of R. eutropha, both of which are prerequisites for the reproducible manufacturing of high-quality hydrogenase preparations. The purification procedures for two different O 2 -tolerant [NiFe]-hydrogenases from R. eutropha are described in detail, as well as the corresponding biochemical procedures used for the determination of the catalytic properties of these sophisticated enzymes., (© 2018 Elsevier Inc. All rights reserved.)

This corrects the article DOI: 10.1038/srep44628.

[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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the thylakoid membrane of chloroplasts as well as in the cytoplasmic membrane of bacteria, the DeltapH/Tat-dependent protein transport pathway is responsible for the translocation of folded proteins. Using the chimeric 16/23 protein as model substrate in thylakoid transport experiments, we dissected the transport process into several distinct steps that are characterized by specific integral translocation intermediates. Formation of the early translocation intermediate Ti-1, which still exposes the N and the C terminus to the stroma, is observed with thylakoids pretreated with (i) solutions of chaotropic salts or alkaline pH, (ii) protease, or (iii) antibodies raised against TatA, TatB, or TatC. Membrane insertion takes place even into liposomes, demonstrating that proteinaceous components are not required. This suggests that Tat-dependent transport may be initiated by the unassisted insertion of the substrate into the lipid bilayer, and that interaction with the Tat translocase takes place only in later stages of the process.