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Nature chemical biology 19(4), 498 - 506 (2023). doi:10.1038/s41589-022-01226-w, [NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H$_2$ into 2e$^−$ and 2H$^+$ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)$_2$(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN$^−$ molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O$_2$-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)$_2$(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase., Published by Nature Publishing Group, Basingstoke

DFG, 5451160, Untersuchungen der Eignung von Verbindungen mit binaren Untereinheiten aus Elementen der Gruppen 14/16 und 15/16 zum Aufbau ternarer oder quaternarer Anionen durch Reaktionen mit Ubergangsmetallverbindungen; experimentelle und theoretische Studien zu physikalischen Eigenschaften der Produkte

Synthetic biology and especially xenobiology, as emerging new fields of science, have reached an intellectual and experimental maturity that makes them suitable for integration into the university curricula of chemical and biological disciplines. Novel scientific fields that include laboratory work are perfect playgrounds for developing highly motivating research-based teaching modules. We believe that research-based learning enriched by digital tools is the best approach for teaching new emerging essentials of academic education. This is especially true when the scientific field as such is still not canonized with text books and best-practice examples. Our experience shows that iGEM/BIOMOD competitions represent an excellent basis for designing research-based courses in xenobiology. Therefore, we present a report on "iGEM-Synthetic Biology" offered at the Technische Universität Berlin as an example.

Significance The Calvin cycle is the most important carbon fixation pathway in the biosphere. However, its carboxylating enzyme Rubisco also accepts oxygen, thus producing 2-phosphoglycolate. Phosphoglycolate salvage pathways were extensively studied in photoautotrophs but remain uncharacterized in chemolithoautotrophs using the Calvin cycle. Here, we study phosphoglycolate salvage in the chemolithoautotrophic model bacterium Cupriavidus necator H16. We demonstrate that this bacterium mainly reassimilates 2-phosphoglycolate via the glycerate pathway. Upon disruption of this pathway, a secondary route, which we term the malate cycle, supports photorespiration by completely oxidizing 2-phosphoglycolate to CO2. While the malate cycle was not previously known to metabolize 2-phosphoglycolate in nature, a bioinformatic analysis suggests that it may support phosphoglycolate salvage in diverse chemoautotrophic bacteria., 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.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H$_2$ into 2 e$^- $ and 2 H$^+$ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)$_2$(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN$^–$ molecules. Herein, we investigated the sequential assembly of the [NiFe]-cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O$_2$-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)$_2$(CO) fragment, a precursor that contained all cofactor components but remained redox-inactive, and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption, and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

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., ​

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., A multifunctional setup for in situ spectroscopy on gas‐processing metalloenzymes enables the controlled preparation of redox states in various sample forms. This setup allowed the first NRVS characterization of the Nia−C state of [NiFe]‐hydrogenases, provided new insights into the reductive activation of these enzymes, and revealed a so‐far unknown redox state of the oxygen‐tolerant membrane‐bound hydrogenase from R. eutropha.

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of 2-phosphoglycolate, an essential process termed photorespiration, was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, but remains uncharacterized in chemolithoautotrophic bacteria. Here, we study photorespiration in the model chemolithoautotroph Cupriavidus necator (Ralstonia eutropha) 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 photorespiratory pathway. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports photorespiration. In this cycle, glyoxylate is condensed with 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 photorespiration in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so-far unknown photorespiration pathway, highlighting important diversity in microbial carbon fixation metabolism.

The catalytic properties of hydrogenases are nature’s answer to the seemingly simple reaction H2 ⇌ 2H+ + 2e–. Members of the phylogenetically diverse subgroup of [NiFe] hydrogenases generally consist of at least two subunits, where the large subunit harbors the H2-activating [NiFe] site and the small subunit contains iron–sulfur clusters mediating e– transfer. Typically, [NiFe] hydrogenases are susceptible to inhibition by O2. Here, we conducted system minimization by isolating and analyzing the large subunit of one of the rare members of the group of O2-tolerant [NiFe] hydrogenases, namely the preHoxG protein of the membrane-bound hydrogenase from Ralstonia eutropha. Unlike previous assumptions, preHoxG was able to activate H2 as it clearly performed catalytic hydrogen/deuterium exchange. However, it did not execute the entire catalytic cycle described for [NiFe] hydrogenases. Remarkably, H2 activation was performed by preHoxG even in the presence of O2, although the unique [4Fe-3S] cluster located in th...

[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 N10-formyltetrahydrofolate (N10-formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N10-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 N10-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.

[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

The catalytic properties of hydrogenases are nature's answer to the seemingly simple reaction H

Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation. Consequently, the heterogeneous charge-transfer resistance can be reduced sufficiently to enhance the direct electron transfer between an immobilized redox-active enzyme and the electrode. This was demonstrated for the oxygen-tolerant [NiFe] hydrogenase from the “Knallgas” bacterium Ralstonia eutropha by relating its electrochemical activity for hydrogen oxidation to the interface properties.

[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.

[NiFe]-Hydrogenasen katalysieren die reversible heterolytische Spaltung von Wasserstoff in Protonen und Elektronen. Das tief im Protein liegende aktive Zentrum wird dabei von gasformigen Substraten und Inhibitoren uber Tunnel erreicht. Hier wird die Proteinstruktur der O2-toleranten, membrangebundenen [NiFe]-Hydrogenase von Ralstonia eutropha (ReMBH) anhand von Proteinkristallen beschrieben, die mit Krypton derivatisiert wurden. Die Positionen der Kryptonatome ermoglichen eine umfassende Beschreibung des Gastunnelnetzwerks. Eine detaillierte Ubersicht von Grose, Lange und Route der Tunnel wurde mithilfe von Rechnungen erstellt. Vergleicht man die Tunneleigenschaften der ReMBH mit Kristallstrukturen anderer O2-toleranter und O2-sensitiver [NiFe]-Hydrogenasen hinsichtlich der Grose und Anzahl der hydrophoben Gastunnel, ergeben sich wesentliche Unterschiede zwischen beiden Gruppen. Einige sind womoglich auf die bemerkenswerte Eigenschaft der Sauerstofftoleranz zuruckzufuhren.

Dioxygen-tolerant [NiFe]-hydrogenases are defined by their ability to catalyze the reaction, H2⇌2H++2e- even in the presence of O2. 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 O2-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 H2, O2, and CO2. 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 O2-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.

Significance Tracking the route of substrates, intermediates, and inhibitors in proteins is fundamental in understanding their specific function. However, following the route of gases like molecular oxygen within enzymes has always been challenging. In protein X-ray crystallography, gases can be mimicked using krypton or xenon (with a higher electron count); however, these have a different physical behavior compared to true substrates/inhibitors. In our crystal structure of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha, we were able to show the direct path of molecular oxygen between the enzyme exterior and the active site with the “soak-and-freeze” derivatization method. This technique might be useful to detect O2 traveling routes in many other enzymes., [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 O2 tolerance 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 O2 molecules 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 O2 tolerance of the enzyme.

Scientific Reports 7: Article number: 44628; published online: 16 March 2017; updated: 21 June 2017. In this Article, Henry N. Chapman is incorrectly listed as being affiliated with ‘Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA’ and an additional affiliation was omitted.

Hydrogenases catalyze the reversible oxidation of H(2) into protons and electrons and are usually readily inactivated by O(2). However, a subgroup of the [NiFe] hydrogenases, including the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha, has evolved remarkable tolerance toward O(2) that enables their host organisms to utilize H(2) as an energy source at high O(2). 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 H(2) oxidation at high O(2). 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.

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.

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

The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.

In chloroplasts and bacteria, the Tat (twin-arginine translocation) system is engaged in transporting folded passenger proteins across the thylakoid and cytoplasmic membranes, respectively. To date, three membrane proteins (TatA, TatB, and TatC) have been identified to be essential for Tat-dependent protein translocation in the plant system, whereas soluble factors seem not to be required. In contrast, in the bacterial system, several cytosolic chaperones were described to be involved in Tat transport processes. Therefore, we have examined whether stromal or peripherally associated membrane proteins also play a role in Tat transport across the thylakoid membrane. Analyzing both authentic precursors as well as the chimeric 16/23 protein, which allows us to study each step of the translocation process individually, we demonstrate that a soluble form of TatA is present in the chloroplast stroma, which significantly improves the efficiency of Tat-dependent protein transport. Furthermore, this soluble TatA is able to reconstitute the Tat transport properties of thylakoid membranes that are transport-incompetent due to extraction with solutions of chaotropic salts.

The tolerance towards oxic conditions of O-2-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.

We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Nir-B [Ni(III)] and Nir-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Nia-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Nia-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H2-reduced active site states.

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.

The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the 'export' of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways.

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 coli. 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 Asp164 (by Asn) and Lys167 (by Arg or Gln) 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.

The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode.

Hydrogenases are metalloprotein enzymes that catalyse the reversible oxidation of dihydrogen to protons and electrons, a critical pathway in anaerobic metabolism. This reaction is of particular interest for hydrogen-based applications, in fuel cells for instance, but many applications are hindered by the high oxygen sensitivity that is an intrinsic feature of most hydrogenases. Two groups report the structures of oxygen-tolerant hydrogenases, one from the soil bacterium Ralstonia eutropha and the other from the marine bacterium Hydrogenovibrio marinus. The structures shed light on how redox-sensitive active-site intermediates are protected from destruction. Both enzymes feature a novel iron-sulphur centre at the active site, coordinated by a group of cysteine residues. Hydrogenases are abundant enzymes that catalyse the reversible interconversion of H2 into protons and electrons at high rates1. 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 production2. Despite comprehensive genetic, biochemical, electrochemical and spectroscopic investigations3,4,5,6,7,8, 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 A 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 potentials7,8,9. 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.

Bislang konnten vier Wege identifiziert werden, auf denen ein Protein in die chloroplastidäre Thylakoidmembran integriert bzw. über diese Membran transportiert werden kann. Einer dieser Wege ist der sogenannte Tat-Weg (twin-arginine translocation). Dieser Transportweg, der auch in der Cytoplasmamembran von Bakterien und Archaeen gefunden wird, zeichnet sich insbesondere durch die Fähigkeit aus, Proteine in vollständig gefalteter Konformation über eine Membran, welche einen Protonengradienten aufrechterhält, transportieren zu können. Gleichzeitig ist der Transport energetisch unabhängig von Nukleosidtriphosphaten wie ATP und benötigt lediglich einen Protonengradienten (DpH) bzw. einen elektrochemischen Gradienten (DY), der an der Membran anliegt. Vermittelt wird der Transport durch Signalpeptide, die sich am N-Terminus des zu transportierenden Proteins befinden und ein sogenanntes Zwillingsarginin-Motiv (engl. twin-arginine motif) enthalten. Da dieses Motiv in den entsprechenden Signalpeptiden sehr stark konserviert ist, trug es auch zur Benennung des Transportwegs bei. Nach Abschluss des Transports werden die Signalpeptide im Allgemeinen durch eine Prozessierungspeptidase abgespalten. Bis heute wurden drei Proteine gefunden, die für den Tat-abhängigen Transport an der Thylakoidmembran essentiell sind und als TatA, TatB und TatC bezeichnet werden. Da der Tat-Weg in der Lage ist gefaltete Proteine zu transportieren, ist davon auszugehen, dass die Translokation mechanistisch gesehen grundlegend anders ablaufen muss als z.B. der Transport über den Sec-Weg, welcher Proteine ausschließlich in ungefalteter Form transportiert. Daher lag der Fokus der vorliegenden Arbeit vorwiegend auf der genaueren Charakterisierung von Teilschritten des Tat-abhängigen Proteintransports, mit dem Ziel die Aufklärung des zu Grunde liegenden Mechanismus voranzutreiben. Die Analyse von Einzelschritten des Tat-Wegs wurde durch den Einsatz eines bestimmten chimären Transportsubstrats, dem sogenannten 16/23-Protein, ermöglicht. Der Transport dieses Proteins über den Tat-Weg ist derart verlangsamt, dass zwei Translokationsintermediate detektiert werden können. Durch die Verwendung des 16/23-Proteins ist es also jederzeit möglich zu verfolgen, inwiefern sich Veränderungen am Transportsystem auf den Fortgang des Transportprozesses auswirken. So wurde im ersten Teil der Arbeit festgestellt, dass die Proteine der Translokase für die Initiation des Transportvorgangs nicht essentiell sind. Stattdessen muss lediglich eine Membran vorhanden sein, damit das erste Translokationsintermediat ausgebildet werden kann. Im zweiten Teil der Arbeit stellte sich heraus, dass das Protein TatA in hochmolekularen Komplexen in löslicher Form im Stroma von Chloroplasten vorkommt. Bislang wurde es aber hauptsächlich als membranständiger Teil der Tat-Translokase beschrieben. Die lösliche Form ist, wie es zuvor für die membranständige Form beschrieben wurde, in der Lage den eigentlichen Translokationsschritt, in dem der reife Teil des Transportsubstrats über die Membran transportiert wird, zu vermitteln. Im letzten Teil der Arbeit wurde nach der besonderen Eigenschaft gesucht, die zur Verlangsamung des Transportprozesses des 16/23-Proteins führt. Dabei zeigte sich, dass eine Erhöhung der Hydrophobizität im Bereich der Prozessierungsstelle zu einer verlängerten Bindung an einen Komplex aus den Proteinen TatB und TatC führt. Dies hat wiederum insgesamt eine Verlangsamung des Transportprozesses zur Folge. Des Weiteren stellte sich heraus, dass die Dissoziation des Transportsubstrats von dem genannten Komplex aus TatB und TatC auch eine Voraussetzung für die finale Prozessierung zum reifen Protein und somit für den Abschluss des Transportprozesses darstellt., To date four protein transport pathways have been identified at the thylakoid membrane of chloroplasts. One of these pathways, which also operates at the cytoplasmic membranes of bacteria and archaea, is referred to as Tat pathway (twin-arginine translocation) and mediates the transport of folded proteins across energised membranes. The Tat pathway acts independent from nucleoside triphosphates like ATP but depends on a proton gradient (DpH) or electric potential (DY) across the membrane, respectively. The transport of passenger proteins is mediated by N-terminal extensions, the so-called signal peptides, which carry a highly conserved twin-arginine motif. This motif in turn led to the term twin-arginine translocation (Tat). Upon completion of the transport process signal peptides are generally removed by a corresponding signal peptidase. To date three proteins, namely TatA, TatB, and TatC, have been identified to be essential for Tat-dependent protein translocation in the plant system. Since the Tat pathway is capable of transporting folded proteins a mechanism can be assumed differing from other pathways like the Sec pathway, which exclusively transports proteins in an unfolded conformation. Therefore, the present study focussed primarily on the characterisation of distinct steps of the translocation process aiming for the elucidation of the underlying mechanism. Analysis of single steps of the transport process was facilitated by using a chimeric protein referred to as 16/23 protein. Tat-dependent translocation of this protein is drastically slowed down so that two translocation intermediates can be detected. Thus, any manipulation of the transport system can be traced back to a certain step of the transport process. In the first part of the present study this system revealed that proteins of the Tat translocase are dispensable for the initiation of the transport process. Instead, the presence of a membrane is sufficient for the formation of the first translocation intermediate. In the second part the protein TatA was found to be present in high molecular weight complexes within the chloroplast stroma. So far TatA was assumed to be an integral membrane component of the Tat translocase exclusively located within the thylakoid membrane. Soluble TatA is able to mediate the translocation step in particular, like it was previously described for membrane-integral TatA. The last part of the present study concentrated on the special feature of the 16/23 protein which leads to its slowed transport. It turned out that an increase in hydrophobicity within the processing site of the precursor protein leads to prolonged association with a complex composed of the proteins TatB and TatC leading to a retardation of the transport process. In addition, dissociation of the precursor protein from the above mentioned complex is a prerequisite for terminal processing of the precursor protein to its mature form.

In bacteria and chloroplasts, the Tat (twin arginine translocation) system is capable of translocating folded passenger proteins across the cytoplasmic and thylakoidal membranes, respectively. Transport depends on signal peptides that are characterized by a twin pair of arginine residues. The signal peptides are generally removed after transport by specific processing peptidases, namely the leader peptidase and the thylakoidal processing peptidase. To gain insight into the prerequisites for such signal peptide removal, we mutagenized the vicinity of thylakoidal processing peptidase cleavage sites in several thylakoidal Tat substrates. Analysis of these mutants in thylakoid transport experiments showed that the amino acid composition of both the C-terminal segment of the signal peptide and the N-terminal part of the mature protein plays an important role in the maturation process. Efficient removal of the signal peptide requires the presence of charged or polar residues within at least one of those regions, whereas increased hydrophobicity impairs the process. The relative extent of this effect varies to some degree depending on the nature of the precursor protein. Unprocessed transport intermediates with fully translocated passenger proteins are found in membrane complexes of high molecular mass, which presumably represent Tat complexes, as well as free in the lipid bilayer. This seems to indicate that the Tat substrates can be laterally released from the complexes prior to processing and that membrane transport and terminal processing of Tat substrates are independent processes.

Zur Untersuchung von Metalloenzymen haben wir einen Aufbau für die Präparation katalytischer Intermediate und deren anschließende Charakterisierung mit spektroskopischen Techniken entwickelt. Mit diesem können Redoxreaktionen in Enzymen in Form von Lyophilisat, gelöst oder als Kristall in einem großen Temperaturbereich IR-spektroskopisch in situ verfolgt werden. Zwei sauerstofftolerante [NiFe]-Hydrogenasen wurden als Modellenzyme untersucht. Zunächst wurde der Aufbau zur Herstellung von komprimiertem Lyophilisat einer Hydrogenase in einem paramagnetischen Zustand mit verbrückendem Hydrid genutzt. Dies erleichterte die Charakterisierung durch 57Fe-kernresonante inelastische Streuung und erlaubte es, in Kombination mit DFT, die schwingungsspektroskopischen Merkmale dieses katalytischen Intermediats zu detektieren. Der In-situ-IR-Aufbau lieferte zusammen mit Resonanz-Raman-Untersuchungen auch Einblicke in die Redoxchemie von Proteinkristallen. Eine Ergänzung röntgenkristallographischer Daten durch komplementäre spektroskopische Analysen ist daher essentiell.