Zu Komplex I verwandte Hydrogenasen in Carboxydothermus hydrogenoformans und Thermoanaerobacter tengcongensis

Die Fähigkeit, Kohlenmonoxid unter anaeroben Bedingungen als Energiesubstrat zu nutzen, ist auf wenige Mikroorganismen beschränkt. Hierzu zählt das Gram-positive thermophile Bakterium Carboxydothermus hydrogenoformans. Die aus der Oxidation von Kohlenmonoxid stammenden Elektronen werden in diesem Organismus auf Protonen unter Bildung von Wasserstoff übertragen. CO + H2O è CO2 + H2 DG°’ = -20 kJ/mol Aus der Membranfraktion dieses Bakteriums wurde nach Solubilisierung mit dem Detergens Dodecyl-b-D-Maltosid ein Enzymkomplex, der als CO-oxidierender/H2-bildender Enzymkomplex bezeichnet wurde, gereinigt und charakterisiert. Mit 5% CO in der Gasphase katalysierte der Enzymkomplex die Umsetzung von CO zu CO2 und H2 mit einer spezifischen Aktivität von etwa 450 U (mg Protein)-1. Höhere CO-Konzentrationen in der Gasphase führten zur Hemmung der Hydrogenase im Enzymkomplex. Der Enzymkomplex konnte sowohl mit CO als auch mit einem starken Reduktionsmittel aktiviert werden. Der gereinigte Enzymkomplex bestand aus acht Untereinheiten, sechs hydrophilen und zwei hydrophoben Polypeptiden. Nach Bestimmung der aminoterminalen Sequenzen konnten die kodierenden Gene, die in zwei direkt aufeinander folgenden Genclustern lokalisiert sind, im vollständig sequenzierten Genom von Ca. hydrogenoformans identifiziert werden. Die biochemische Charakterisierung des Enzymkomplexes und eine Sequenzanalyse der Untereinheiten zeigten, dass der Enzymkomplex aus einer Ni-haltigen Kohlenmonoxid-Dehydrogenase und einer membranständigen [NiFe]-Hydrogenase zusammengesetzt ist. Die Kohlenmonoxid-Dehydrogenase besteht aus der katalytischen Untereinheit CooS und dem Elektronentransferprotein (Polyferredoxin) CooF. Die Hydrogenase setzt sich aus vier hydrophilen Proteinen und zwei integralen Membranproteinen zusammen. Diese besitzen eine hohe Sequenzverwandtschaft zu den korrespondierenden Untereinheiten einer zu Komplex I verwandten Gruppe von [NiFe]-Hydrogenasen. Da Ca. hydrogenoformans mit CO als alleinigem Energiesubstrat wachsen kann, muss die Umsetzung von CO zu CO2 und H2 mit einer Energiekonservierung gekoppelt sein. Es wird angenommen, dass die Hydrogenase in dem Enzymkomplex ähnlich wie Komplex I als Ionenpumpe ( H+ oder Na+) fungiert. Der zweite Teil der vorliegenden Arbeit zielte auf die Charakterisierung der Hydrogenasen in Ta. tengcongensis, einem thermophilen Gram-positives Bakterium, das Stärke oder Glukose zu Acetat, Ethanol, CO2 und H2 fermentiert. In dieser Arbeit wurde gezeigt, dass der Organismus eine einzigartige Kombination von zwei Hydrogenasen besitzt, die die Bildung von H2 während der Fermentation katalysieren. Beide Enzyme, eine Ferredoxin-abhängige membrangebundene-[NiFe]-Hydrogenase und eine heterotetramäre NADH-abhängige [FeFe]-Hydrogenase, wurden gereinigt und charakterisiert. Die membrangebundene Hydrogenase gehört ebenso wie die Hydrogenase aus C. hydrogenogformans zu einer Gruppe von zu Komplex I verwandten [NiFe]-Hydrogenasen. Als physiologisches Substrat dieser Hydrogenase konnte ein Ferredoxin identifiziert werden. Dieses Enzym katalysiert die H2-Bildung mit reduziertem Ferredoxin als Elektronendonor mit einer spezifischen Rate von 38 U (mg Protein)-1. Die aus der löslichen Fraktion von Ta. tengcongensis gereinigte Hydrogenase enthält FMN, mehrere Eisen-Schwefel-Zentren und zeigt im oxidierten Zustand ein EPR-Signal, das charakteristisch für das im aktiven Zentrum von [FeFe]-Hydrogenasen lokalisierte H-Cluster ist. Die biochemische Charakterisierung und Sequenzanalyse der Untereinheiten dieses Enzyms zeigten, dass es sich bei diesem Enzym um eine NADH-abhängige [FeFe]-Hydrogenase handelt. Wurde der H2-Partialdruck (p(H2)) in Ta. Tengcongensis-Kulturen durch Begasung des Fermenters mit N2 niedrig gehalten, wurde 1 mol Glucose zu 2 mol Acetat, 2 mol CO2 und 4 mol H2 umgesetzt. Wenn hingegen H2 in den Kulturen akkumulierte (Zucht in geschlossenen Flaschen), wurde die Fermentation teilweise in Richtung Ethanolbildung verschoben. Während die spezifische Aktivität der Ferredoxin-abhängigen [NiFe]-Hydrogenase nicht durch die Wachstumsbedingungen beeinflusst wurde, war die NAD(H)-abhängige [FeFe]-Hydrogenase-Aktivität etwa 4-fach höher in Zellextrakten aus Fermenter-Kulturen (niedriger p(H2)). Alkohol-Dehydrogenase- und Aldehyd-Dehydrogenase-Aktivitäten waren hingegen etwa 5fach höher in Zellextrakten aus Flaschenkulturen (hoher p(H2)). Dies deutet auf eine Regulation in Abhängigkeit von p(H2) hin

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H2). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN)2CO metallo-center and a small subunit with three iron–sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN)2CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor- containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional “O2-sensitive” [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., “O2-tolerant” [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the “unready” Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis

Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)2CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN− ligands as well as CO2. Metal and sulphide analyses revealed that along with the [4Fe–4S]2+ cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)2CO cofactor

The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: benzyl viologen oxidoreductase activity

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>synthesizes three membrane-bound molybdenum- and selenocysteine-containing formate dehydrogenases, as well as up to four membrane-bound [NiFe]-hydrogenases. Two of the formate dehydrogenases (Fdh-N and Fdh-O) and two of the hydrogenases (Hyd-1 and Hyd-2) have their respective catalytic subunits located in the periplasm and these enzymes have been shown previously to oxidize formate and hydrogen, respectively, and thus function in energy metabolism. Mutants unable to synthesize the [NiFe]-hydrogenases retain a H₂: benzyl viologen oxidoreductase activity. The aim of this study was to identify the enzyme or enzymes responsible for this activity.</p> <p>Results</p> <p>Here we report the identification of a new H₂: benzyl viologen oxidoreductase enzyme activity in <it>E. coli </it>that is independent of the [NiFe]-hydrogenases. This enzyme activity was originally identified after non-denaturing polyacrylamide gel electrophoresis and visualization of hydrogen-oxidizing activity by specific staining. Analysis of a crude extract derived from a variety of <it>E. coli </it>mutants unable to synthesize any [NiFe]-hydrogenase-associated enzyme activity revealed that the mutants retained this specific hydrogen-oxidizing activity. Enrichment of this enzyme activity from solubilised membrane fractions of the hydrogenase-negative mutant FTD147 by ion-exchange, hydrophobic interaction and size-exclusion chromatographies followed by mass spectrometric analysis identified the enzymes Fdh-N and Fdh-O. Analysis of defined mutants devoid of selenocysteine biosynthetic capacity or carrying deletions in the genes encoding the catalytic subunits of Fdh-N and Fdh-O demonstrated that both enzymes catalyze hydrogen activation. Fdh-N and Fdh-O can also transfer the electrons derived from oxidation of hydrogen to other redox dyes.</p> <p>Conclusions</p> <p>The related respiratory molybdo-selenoproteins Fdh-N and Fdh-O of <it>Escherichia coli </it>have hydrogen-oxidizing activity. These findings demonstrate that the energy-conserving selenium- and molybdenum-dependent formate dehydrogenases Fdh-N and Fdh-O exhibit a degree of promiscuity with respect to the electron donor they use and identify a new class of dihydrogen-oxidizing enzyme.</p

The [NiFe]-hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron- and carbon dioxide-binding proteins.

[NiFe]-hydrogenase accessory proteins HypC and HypD form a complex that binds a Fe–(CN)2CO moiety and CO2. In this study two HypC homologues from Escherichia coli were purified under strictly anaerobic conditions and both contained sub-stoichiometric amounts of iron (approx. 0.3 mol Fe/mol HypC). Infrared spectroscopic analysis identified a signature at 2337 cm−1 indicating bound CO2. Aerobically isolated HypC lacked both Fe and CO2. Exchange of either of the highly conserved amino acid residues Cys2 or His51 abolished both Fe- and CO2-binding. Our results suggest that HypC delivers CO2 bound directly to Fe for reduction to CO by HypD

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from <i>E. coli</i>

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional &#8220;O2-sensitive&#8222; [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., &#8220;O2-tolerant&#8222; [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the &#8220;unready&#8222; Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis

Identification of an Isothiocyanate on the HypEF Complex Suggests a Route for Efficient Cyanyl-Group Channeling during [NiFe]-Hydrogenase Cofactor Generation.

[NiFe]-hydrogenases catalyze uptake and evolution of H2 in a wide range of microorganisms. The enzyme is characterized by an inorganic nickel/ iron cofactor, the latter of which carries carbon monoxide and cyanide ligands. In vivo generation of these ligands requires a number of auxiliary proteins, the so-called Hyp family. Initially, HypF binds and activates the precursor metabolite carbamoyl phosphate. HypF catalyzes removal of phosphate and transfers the carbamate group to HypE. In an ATP-dependent condensation reaction, the C-terminal cysteinyl residue of HypE is modified to what has been interpreted as thiocyanate. This group is the direct precursor of the cyanide ligands of the [NiFe]-hydrogenase active site cofactor. We present a FT-IR analysis of HypE and HypF as isolated from E. coli. We follow the HypF-catalyzed cyanation of HypE in vitro and screen for the influence of carbamoyl phosphate and ATP. To elucidate on the differences between HypE and the HypEF complex, spectro-electrochemistry was used to map the vibrational Stark effect of naturally cyanated HypE. The IR signature of HypE could ultimately be assigned to isothiocyanate (-N=C=S) rather than thiocyanate (-S-C≡N). This has important implications for cyanyl-group channeling during [NiFe]-hydrogenase cofactor generation

Crystal structure of HypE–SCN from <i>T</i>. <i>kodakarensis</i> in the ‘inward’ conformation.

<p>The αβ domains A and B are colored orange and green, respectively. The <i>C</i>–terminal loop region (330–338) is shown in blue. The loop ends with the conserved PR(V/I)C motif and the cysteine (C338) is modified to a thiocyanate in the published model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133118#pone.0133118.ref027" target="_blank">27</a>]. Inset: domains A and B form a surface cleft that localizes the C–terminus close to a magnesium–sequestered ATP molecule. Drawn after pdb coordinates 3WQJ.</p

SEIRAS probes the vibrational Stark effect of HypE.

<p><b>(A)</b> Amide I (▲, 1658 cm^–1) and amide II (●, 1549 cm^–1) band formation over time after injection of HypE onto the bare gold surface. Additionally, the increase of the peak at 2118 cm^–1 (<b>□</b>) is followed. Kinetics are consistent with the Boltzmann model for a sigmoidal fit (R² as given in cursive brackets does not include the 18 h signal for 2118 cm^–1). <b>(B)</b> SEIRAS spectrum of HypE from 2250 to 2000 cm^–1 without external potential including the thiocyanate vibration at 2118 cm^–1 (a). The peak fits best with contributions at 2119 and 2106 cm^–1. Sequentially setting the cell potential to (b) +300 mV and (c) –300 mV vs. SHE gives rise to difference bands illustrating the vibrational Stark effect on HypE. Positive contributions are marked in bold. See text for details. Spectra (b) and (c) fit with three Gaussians to R = 8 x 10^-6 and 9 x 10^-6, respectively.</p