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Wolfgang Lubitz - One of the best experts on this subject based on the ideXlab platform.
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suppressing hydrogen peroxide generation to achieve oxygen insensitivity of a NiFe Hydrogenase in redox active films
Nature Communications, 2020Co-Authors: Ute Munchberg, Wolfgang Lubitz, Alaa A Oughli, Darren Buesen, Erik Freier, Nicolas PlumereAbstract:Redox-active films were proposed as protective matrices for preventing oxidative deactivation of oxygen-sensitive catalysts such as Hydrogenases for their use in fuel cells. However, the theoretical models predict quasi-infinite protection from oxygen and the aerobic half-life for Hydrogenase-catalyzed hydrogen oxidation within redox films lasts only about a day. Here, we employ operando confocal microscopy to elucidate the deactivation processes. The hydrogen peroxide generated from incomplete reduction of oxygen induces the decomposition of the redox matrix rather than deactivation of the biocatalyst. We show that efficient dismutation of hydrogen peroxide by iodide extends the aerobic half-life of the catalytic film containing an oxygen-sensitive [NiFe] Hydrogenase to over one week, approaching the experimental anaerobic half-life. Altogether, our data support the theory that redox films make the Hydrogenases immune against the direct deactivation by oxygen and highlight the importance of suppressing hydrogen peroxide production in order to reach complete protection from oxidative stress.
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in search of metal hydrides an x ray absorption and emission study of NiFe Hydrogenase model complexes
Physical Chemistry Chemical Physics, 2016Co-Authors: Stefan Hugenbruch, Frank Neese, Wolfgang Lubitz, Hannah S Shafaat, Katharina Weber, Tobias Kramer, Mario Ulises Delgadojaime, Serena DebeerAbstract:Metal hydrides are invoked as important intermediates in both chemical and biological H2 production. In the [NiFe] Hydrogenase enzymes, pulsed EPR and high-resolution crystallography have argued that the hydride interacts primarily at the Ni site. In contrast, in [NiFe] Hydrogenase model complexes, it is observed that the bridging hydride interacts primarily with the Fe. Herein, we utilize a combination of Ni and Fe X-ray absorption (XAS) and emission (XES) spectroscopies to examine the contribution of the bridging hydride to the observed spectral features in [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3](+). The corresponding data on (dppe)Ni(μ-pdt)Fe(CO)3 are used as a reference for the changes that occur in the absence of a hydride bridge. For further interpretation of the observed spectral features, all experimental spectra were calculated using a density functional theory (DFT) approach, with excellent agreement between theory and experiment. It is found that the iron valence-to-core (VtC) XES spectra reveal clear signatures for the presence of a Fe-H interaction in the hydride bridged model complex. In contrast, the Ni VtC XES spectrum largely reflects changes in the local Ni geometry and shows little contribution from a Ni-H interaction. A stepwise theoretical analysis of the hydride contribution and the Ni site symmetry provides insights into the factors, which govern the different metal-hydride interactions in both the model complexes and the enzyme. Furthermore, these results establish the utility of two-color XES to reveal important insights into the electronic structure of various metal-hydride species.
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models of the ni l and ni sia states of the NiFe Hydrogenase active site
Inorganic Chemistry, 2016Co-Authors: Geoffrey M Chambers, Thomas B Rauchfuss, Mioy T Huynh, Sharon Hammesschiffer, Edward J Reijerse, Wolfgang LubitzAbstract:A new class of synthetic models for the active site of [NiFe]-Hydrogenases are described. The NiI/II(SCys)2 and FeII(CN)2CO sites are represented with (RC5H4)NiI/II and FeII(diphos)(CO) modules, where diphos = 1,2-C2H4(PPh2)2(dppe) or cis-1,2-C2H2(PPh2)2(dppv). The two bridging thiolate ligands are represented by CH2(CH2S)22– (pdt2–), Me2C(CH2S)22– (Me2pdt2–), and (C6H5S)22–. The reaction of Fe(pdt)(CO)2(dppe) and [(C5H5)3Ni2]BF4 affords [(C5H5)Ni(pdt)Fe(dppe)(CO)]BF4 ([1a]BF4). Monocarbonyl [1a]BF4 features an S = 0 NiIIFeII center with five-coordinated iron, as proposed for the Ni-SIa state of the enzyme. One-electron reduction of [1a]+ affords the S = 1/2 derivative [1a]0, which, according to density functional theory (DFT) calculations and electron paramagnetic resonance and Mossbauer spectroscopies, is best described as a NiIFeII compound. The NiIFeII assignment matches that for the Ni-L state in [NiFe]-Hydrogenase, unlike recently reported NiIIFeI-based models. Compound [1a]0 reacts with strong acid...
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direkter leistungsvergleich eines bioinspirierten synthetischen ni katalysators und einer NiFe Hydrogenase beide kovalent an eine elektrode gebunden
Angewandte Chemie, 2015Co-Authors: Wolfgang Lubitz, Patricia Rodriguezmacia, Arnab Dutta, Wendy J Shaw, Olaf RudigerAbstract:Das aktive Zentrum von Hydrogenasen inspiriert die Entwicklung molekularer Katalysatoren zur Wasserstoffumsetzung. Ein direkter Vergleich zwischen diesen Katalysatoren und dem Enzym war jedoch bisher nicht moglich, weil verschiedene Techniken zur Bewertung der Katalysatoreigenschaften verwendet wurden. Dies macht es schwierig zu beurteilen, inwieweit die synthetisierten Katalysatoren in ihrer Leistung an das Enzym heranreichen. Hier vergleichen wir die katalytischen Eigenschaften von Ni[(PCy2NGly2)2]+2 mit denen der [NiFe]-Hydrogenase aus Desulfovibrio vulgaris. Beide wurden auf funktionalisierten Elektroden unter identischen Bedingungen immobilisiert. Das Enzym zeigt bei pH 7 eine hohere Aktivitat, geringere Uberspannung und eine bessere Stabilitat, wahrend bei niedrigem pH-Wert der molekulare Katalysator das Enzym in jeder Hinsicht ubertrifft. Dieser erste direkte Vergleich gibt Auskunft uber die Vor- und Nachteile der beiden Systeme und Hinweise auf eine mogliche Verwendung bioinspirierter Komplexe in Brennstoffzellen.
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Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] Hydrogenase
Nature, 2015Co-Authors: Hideaki Ogata, Koji Nishikawa, Wolfgang LubitzAbstract:A sub-angstrom-resolution X-ray crystal structure of [NiFe] Hydrogenase, with direct detection of the products of the heterolytic splitting of dihydrogen into a hydride bridging the Ni and Fe and a proton attached to the sulphur of a cysteine ligand. [NiFe] Hydrogenases use nickel and iron to catalyse the reversible oxidation of molecular hydrogen. They are the focus of much research worldwide because of their potential in biotechnology and in serving as natural models for biomimetic catalysts in the energy sector for hydrogen production and conversion. In protein X-ray crystallography it is notoriously difficult to detect hydrogens, a particularly significant problem in Hydrogenases where hydrogens are involved directly in the reaction. Hideaki Ogata et al. have succeeded in obtaining a sub-angstrom resolution X-ray crystal structure of [NiFe] Hydrogenase leading to detection of most of the hydrogens even close to the metal ions. Using their technique authors were able to detect the products of the heterolytic splitting of dihydrogen: a hydride that bridges the Ni and Fe ions, and a proton that is attached to the sulfur of a cysteine ligand. The enzyme Hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst1. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein2,3,4,5,6. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-angstrom resolution7. Here we report the crystal structure of a standard [NiFe] Hydrogenase (∼91.3 kDa molecular mass) at 0.89 A resolution. The strictly anoxically isolated Hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H−) bridging the Ni and Fe and a proton (H+) attached to the sulphur of a cysteine ligand. The Ni–H− and Fe–H− bond lengths are 1.58 A and 1.78A, respectively. Furthermore, we can assign the Fe–CO and Fe–CN− ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the Hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
Deborah B. Zamble - One of the best experts on this subject based on the ideXlab platform.
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A whole-cell, high-throughput Hydrogenase assay to identify factors that modulate [NiFe]-Hydrogenase activity
Journal of Biological Chemistry, 2019Co-Authors: Michael J. Lacasse, Stephanie Sebastiampillai, Jean-philippe Côté, Nicholas Hodkinson, Eric D. Brown, Deborah B. ZambleAbstract:[NiFe]-Hydrogenases have attracted attention as potential therapeutic targets or components of a hydrogen-based economy. [NiFe]-Hydrogenase production is a complicated process that requires many associated accessory proteins that supply the requisite cofactors and substrates. Current methods for measuring Hydrogenase activity have low throughput and often require specialized conditions and reagents. In this work, we developed a whole-cell high-throughput Hydrogenase assay based on the colorimetric reduction of benzyl viologen to explore the biological networks of these enzymes in Escherichia coli We utilized this assay to screen the Keio collection, a set of nonlethal single-gene knockouts in E. coli BW25113. The results of this screen highlighted the assay's specificity and revealed known components of the intricate network of systems that underwrite [NiFe]-Hydrogenase activity, including nickel homeostasis and formate deHydrogenase activities as well as molybdopterin and selenocysteine biosynthetic pathways. The screen also helped identify several new genetic components that modulate Hydrogenase activity. We examined one E. coli strain with undetectable Hydrogenase activity in more detail (ΔeutK), finding that nickel delivery to the enzyme active site was completely abrogated, and tracked this effect to an ancillary and unannotated lack of the fumarate and nitrate reduction (FNR) anaerobic regulatory protein. Collectively, these results demonstrate that the whole-cell assay developed here can be used to uncover new information about bacterial [NiFe]-Hydrogenase production and to probe the cellular components of microbial nickel homeostasis.
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Bimodal Nickel-Binding Site on Escherichia coli [NiFe]-Hydrogenase Metallochaperone HypA.
Inorganic Chemistry, 2019Co-Authors: Michael J. Lacasse, Kelly L. Summers, Mozhgan Khorasani-motlagh, Graham N. George, Deborah B. ZambleAbstract:[NiFe]-Hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]...
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bimodal nickel binding site on escherichia coli NiFe Hydrogenase metallochaperone hypa
Inorganic Chemistry, 2019Co-Authors: Michael J. Lacasse, Kelly L. Summers, Graham N. George, Mozhgan Khorasanimotlagh, Deborah B. ZambleAbstract:[NiFe]-Hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]-Hydrogenase requires several accessory proteins to assemble and insert the components of the active site. The penultimate maturation step is the delivery of nickel to a primed Hydrogenase enzyme precursor protein, a process that is accomplished by two nickel metallochaperones, the accessory protein HypA and the GTPase HypB. Recent work demonstrated that nickel is rapidly transferred to HypA from GDP-loaded HypB within the context of a protein complex in a nickel selective and unidirectional process. To investigate the mechanism of metal transfer, we examined the allosteric effects of nucleotide cofactors and partner proteins on the nickel environments of HypA and HypB by using a combination of biochemical, microbiological, computational, and spectroscopic techniques. We observed that loading HypB with either GDP or a nonhydrolyzable GTP analogue resulted in a similar nickel environment. In addition, interaction with a mutant version of HypA with disrupted nickel binding, H2Q-HypA, does not induce substantial changes to the HypB G-domain nickel site. Instead, the results demonstrate that HypB modifies the acceptor site of HypA. Analysis of a peptide maquette derived from the N-terminus of HypA revealed that nickel is predominately coordinated by atoms from the N-terminal Met-His motif. Furthermore, HypA is capable of two nickel-binding modes at the N-terminus, a HypB-induced mode and a binding mode that mirrors the peptide maquette. Collectively, these results reveal that HypB brings about changes in the nickel coordination of HypA, providing a mechanism for the HypB-dependent control of the acquisition and release of nickel by HypA.
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complex formation between the escherichia coli NiFe Hydrogenase nickel maturation factors
Biometals, 2019Co-Authors: Mozhgan Khorasanimotlagh, Meissam Noroozifar, Kagan Kerman, Deborah B. ZambleAbstract:The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-Hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-Hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during Hydrogenase enzyme maturation.
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mechanism of selective nickel transfer from hypb to hypa escherichia coli NiFe Hydrogenase accessory proteins
Biochemistry, 2016Co-Authors: Michael J. Lacasse, Colin D Douglas, Deborah B. ZambleAbstract:[NiFe]-Hydrogenase enzymes catalyze the reversible reduction of protons to molecular hydrogen and serve as a vital component of the metabolism of many pathogens. The synthesis of the bimetallic catalytic center requires a suite of accessory proteins, and the penultimate step, nickel insertion, is facilitated by the metallochaperones HypA and HypB. In Escherichia coli, nickel moves from a site in the GTPase domain of HypB to HypA in a process accelerated by GDP. To determine how the transfer of nickel is controlled, the impacts of HypA and nucleotides on the properties of HypB were examined. Integral to this work was His2Gln HypA, a mutant with attenuated nickel affinity that does not support Hydrogenase production in E. coli. This mutation inhibits the translocation of nickel from HypB. H2Q-HypA does not modulate the apparent metal affinity of HypB, but the stoichiometry and stability of the HypB–nickel complex are modulated by the nucleotide. Furthermore, the HypA–HypB interaction was detected by gel fil...
Frank Neese - One of the best experts on this subject based on the ideXlab platform.
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in search of metal hydrides an x ray absorption and emission study of NiFe Hydrogenase model complexes
Physical Chemistry Chemical Physics, 2016Co-Authors: Stefan Hugenbruch, Frank Neese, Wolfgang Lubitz, Hannah S Shafaat, Katharina Weber, Tobias Kramer, Mario Ulises Delgadojaime, Serena DebeerAbstract:Metal hydrides are invoked as important intermediates in both chemical and biological H2 production. In the [NiFe] Hydrogenase enzymes, pulsed EPR and high-resolution crystallography have argued that the hydride interacts primarily at the Ni site. In contrast, in [NiFe] Hydrogenase model complexes, it is observed that the bridging hydride interacts primarily with the Fe. Herein, we utilize a combination of Ni and Fe X-ray absorption (XAS) and emission (XES) spectroscopies to examine the contribution of the bridging hydride to the observed spectral features in [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3](+). The corresponding data on (dppe)Ni(μ-pdt)Fe(CO)3 are used as a reference for the changes that occur in the absence of a hydride bridge. For further interpretation of the observed spectral features, all experimental spectra were calculated using a density functional theory (DFT) approach, with excellent agreement between theory and experiment. It is found that the iron valence-to-core (VtC) XES spectra reveal clear signatures for the presence of a Fe-H interaction in the hydride bridged model complex. In contrast, the Ni VtC XES spectrum largely reflects changes in the local Ni geometry and shows little contribution from a Ni-H interaction. A stepwise theoretical analysis of the hydride contribution and the Ni site symmetry provides insights into the factors, which govern the different metal-hydride interactions in both the model complexes and the enzyme. Furthermore, these results establish the utility of two-color XES to reveal important insights into the electronic structure of various metal-hydride species.
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hydride bridge in NiFe Hydrogenase observed by nuclear resonance vibrational spectroscopy
Nature Communications, 2015Co-Authors: Hideaki Ogata, Frank Neese, Hongxin Wang, Tobias Kramer, David Schilter, Vladimir Pelmenschikov, Maurice Van Gastel, Thomas B RauchfussAbstract:The metabolism of many anaerobes relies on [NiFe]-Hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the (57)Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-Hydrogenase. A unique 'wagging' mode involving H(-) motion perpendicular to the Ni(μ-H)(57)Fe plane was studied using (57)Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)(57)Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe-CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)(57)Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)(57)Fe(CO)3](+) and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H(-) binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe-H moieties in other important natural and synthetic catalysts.
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Hydride bridge in [NiFe]-Hydrogenase observed by nuclear resonance vibrational spectroscopy
Nature Communications, 2015Co-Authors: Hideaki Ogata, Frank Neese, Hongxin Wang, Thomas B Rauchfuss, Tobias Kramer, David Schilter, Vladimir Pelmenschikov, Maurice Van Gastel, Leland B Gee, Aubrey D. ScottAbstract:Understanding the catalytic mechanism of redox-active Hydrogenases is a key to efficient hydrogen production and consumption. Here, the authors use nuclear resonance vibrational spectroscopy to study [NiFe]-Hydrogenase, and observe a bridging hydride structure in an EPR silent intermediate. The metabolism of many anaerobes relies on [NiFe]-Hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the ^57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-Hydrogenase. A unique ‘wagging’ mode involving H^− motion perpendicular to the Ni( μ -H)^57Fe plane was studied using ^57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni( μ -D)^57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni( μ -H/D)^57Fe enzyme mimics [(dppe)Ni( μ -pdt)( μ -H/D)^57Fe(CO)_3]^+ and DFT calculations, which collectively indicate a low-spin Ni( II )( μ -H)Fe( II ) core for Ni-R, with H^− binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.
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Mössbauer and computational investigation of a functional [NiFe] Hydrogenase model complex.
Chemical Communications, 2015Co-Authors: Amélie Kochem, Eckhard Bill, Frank NeeseAbstract:Developing biomimetic complexes that model the active site of [NiFe] Hydrogenase enzymes in order to catalyze the activation of H2 is a topic of major interest. A functional [NiFe] Hydrogenase model complex has recently been described by Ogo et al. (Science, 2013, 339, 682–683). Here, we report a Mossbauer and computational investigation of this model complex. This study affords deeper understanding of the electronic structure, the reactivity and the mechanism of H2 activation by this complex.
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key hydride vibrational modes in NiFe Hydrogenase model compounds studied by resonance raman spectroscopy and density functional calculations
Inorganic Chemistry, 2012Co-Authors: Hannah S Shafaat, Frank Neese, Katharina Weber, Taras Petrenko, Wolfgang LubitzAbstract:Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. While many enzymatic states of the [NiFe] Hydrogenase have been studied extensively, there are multiple catalytically relevant EPR-silent states that remain poorly characterized. Analysis of model compounds using new spectroscopic techniques can provide a framework for the study of these elusive states within the protein. We obtained optical absorption and resonance Raman (RR) spectra of (dppe)Ni(μ-pdt)Fe(CO)3 and [(dppe)Ni(μ-pdt)(μ-H)Fe(CO)3][BF4], which are structural and functional model compounds for the EPR-silent Ni–SI and Ni–R states of the [NiFe] Hydrogenase active site. The studies presented here use RR spectroscopy to probe vibrational modes of the active site, including metal–hydride stretching vibrations along with bridging ligand–metal and Fe–CO bending vibrations, with isotopic substitution used to identify key metal–hydride modes. The metal–hydride vibrations are essentially uncoupled and...
Hideaki Ogata - One of the best experts on this subject based on the ideXlab platform.
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Hydride bridge in [NiFe]-Hydrogenase observed by nuclear resonance vibrational spectroscopy
Nature Communications, 2015Co-Authors: Hideaki Ogata, Frank Neese, Hongxin Wang, Thomas B Rauchfuss, Tobias Kramer, David Schilter, Vladimir Pelmenschikov, Maurice Van Gastel, Leland B Gee, Aubrey D. ScottAbstract:Understanding the catalytic mechanism of redox-active Hydrogenases is a key to efficient hydrogen production and consumption. Here, the authors use nuclear resonance vibrational spectroscopy to study [NiFe]-Hydrogenase, and observe a bridging hydride structure in an EPR silent intermediate. The metabolism of many anaerobes relies on [NiFe]-Hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the ^57Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-Hydrogenase. A unique ‘wagging’ mode involving H^− motion perpendicular to the Ni( μ -H)^57Fe plane was studied using ^57Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni( μ -D)^57Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe–CO/CN bands. Spectra have been interpreted by comparison with Ni( μ -H/D)^57Fe enzyme mimics [(dppe)Ni( μ -pdt)( μ -H/D)^57Fe(CO)_3]^+ and DFT calculations, which collectively indicate a low-spin Ni( II )( μ -H)Fe( II ) core for Ni-R, with H^− binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe–H moieties in other important natural and synthetic catalysts.
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hydride bridge in NiFe Hydrogenase observed by nuclear resonance vibrational spectroscopy
Nature Communications, 2015Co-Authors: Hideaki Ogata, Frank Neese, Hongxin Wang, Tobias Kramer, David Schilter, Vladimir Pelmenschikov, Maurice Van Gastel, Thomas B RauchfussAbstract:The metabolism of many anaerobes relies on [NiFe]-Hydrogenases, whose characterization when bound to substrates has proven non-trivial. Presented here is direct evidence for a hydride bridge in the active site of the (57)Fe-labelled fully reduced Ni-R form of Desulfovibrio vulgaris Miyazaki F [NiFe]-Hydrogenase. A unique 'wagging' mode involving H(-) motion perpendicular to the Ni(μ-H)(57)Fe plane was studied using (57)Fe-specific nuclear resonance vibrational spectroscopy and density functional theory (DFT) calculations. On Ni(μ-D)(57)Fe deuteride substitution, this wagging causes a characteristic perturbation of Fe-CO/CN bands. Spectra have been interpreted by comparison with Ni(μ-H/D)(57)Fe enzyme mimics [(dppe)Ni(μ-pdt)(μ-H/D)(57)Fe(CO)3](+) and DFT calculations, which collectively indicate a low-spin Ni(II)(μ-H)Fe(II) core for Ni-R, with H(-) binding Ni more tightly than Fe. The present methodology is also relevant to characterizing Fe-H moieties in other important natural and synthetic catalysts.
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Hydrogens detected by subatomic resolution protein crystallography in a [NiFe] Hydrogenase
Nature, 2015Co-Authors: Hideaki Ogata, Koji Nishikawa, Wolfgang LubitzAbstract:A sub-angstrom-resolution X-ray crystal structure of [NiFe] Hydrogenase, with direct detection of the products of the heterolytic splitting of dihydrogen into a hydride bridging the Ni and Fe and a proton attached to the sulphur of a cysteine ligand. [NiFe] Hydrogenases use nickel and iron to catalyse the reversible oxidation of molecular hydrogen. They are the focus of much research worldwide because of their potential in biotechnology and in serving as natural models for biomimetic catalysts in the energy sector for hydrogen production and conversion. In protein X-ray crystallography it is notoriously difficult to detect hydrogens, a particularly significant problem in Hydrogenases where hydrogens are involved directly in the reaction. Hideaki Ogata et al. have succeeded in obtaining a sub-angstrom resolution X-ray crystal structure of [NiFe] Hydrogenase leading to detection of most of the hydrogens even close to the metal ions. Using their technique authors were able to detect the products of the heterolytic splitting of dihydrogen: a hydride that bridges the Ni and Fe ions, and a proton that is attached to the sulfur of a cysteine ligand. The enzyme Hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst1. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein2,3,4,5,6. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-angstrom resolution7. Here we report the crystal structure of a standard [NiFe] Hydrogenase (∼91.3 kDa molecular mass) at 0.89 A resolution. The strictly anoxically isolated Hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H−) bridging the Ni and Fe and a proton (H+) attached to the sulphur of a cysteine ligand. The Ni–H− and Fe–H− bond lengths are 1.58 A and 1.78A, respectively. Furthermore, we can assign the Fe–CO and Fe–CN− ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the Hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis.
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observation of the fe cn and fe co vibrations in the active site of NiFe Hydrogenase by nuclear resonance vibrational spectroscopy
Angewandte Chemie, 2013Co-Authors: Saeed Kamali, Hideaki Ogata, Wolfgang Lubitz, Hongxin Wang, Devrani Mitra, Brian C Manor, Thomas B Rauchfuss, Deborah Byrne, Violaine BonnefoyAbstract:Nuclear inelastic scattering of (57)Fe labeled [NiFe] Hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes.
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intermediates in the catalytic cycle of NiFe Hydrogenase functional spectroscopy of the active site
ChemPhysChem, 2010Co-Authors: Mariaeirini Pandelia, Hideaki Ogata, Wolfgang LubitzAbstract:The [NiFe] Hydrogenase from the anaerobic sulphate reducing bacterium Desulfovibrio vulgaris Miyazaki F is an excellent model for constructing a mechanism for the function of the so-called 'oxygen-sensitive' Hydrogenases. The present review focuses on spectroscopic investigations of the active site intermediates playing a role in the activation/deactivation and catalytic cycle of this enzyme as well as in the inhibition by carbon monoxide or molecular oxygen and the light-sensitivity of the Hydrogenase. The methods employed include magnetic resonance and vibrational (FTIR) techniques combined with electrochemistry that deliver information about details of the geometrical and electronic structure of the intermediates and their redox behaviour. Based on these data a mechanistic scheme is developed.
Yoshiki Higuchi - One of the best experts on this subject based on the ideXlab platform.
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kinetic analysis of inactivation and enzyme reaction of oxygen tolerant NiFe Hydrogenase at direct electron transfer bioanode
Bulletin of the Chemical Society of Japan, 2014Co-Authors: Yuki Kitazumi, Yoshiki Higuchi, Hirofumi Nishihara, Osamu Shirai, Kouhei Kurita, Kenji KanoAbstract:Membrane-bound [NiFe]-Hydrogenase (MBH) from Hydrogenovibrio marinus is an O2-tolerant enzyme and allows direct-electron-transfer (DET) bioelectrocatalysis for H2-oxidation. MBH is a promising bioe...
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structural basis for a 4fe 3s cluster in the oxygen tolerant membrane bound NiFe Hydrogenase
Nature, 2011Co-Authors: Yasuhito Shomura, Hirofumi Nishihara, Ki Seok Yoon, Yoshiki HiguchiAbstract:Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-Hydrogenase
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single crystal epr studies of the reduced active site of NiFe Hydrogenase from desulfovibrio vulgaris miyazaki f
Journal of the American Chemical Society, 2003Co-Authors: Hideaki Ogata, Yoshiki Higuchi, Matthias Stein, Stefanie Foerster, Marc Brecht, Wolfgang LubitzAbstract:In the catalytic cycle of [NiFe] Hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the g tensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J. Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H(-)) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(I) oxidation state is assumed for this species with a proton (H(+)) removed from the bridge between the nickel and the iron atom.
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structural studies of the carbon monoxide complex of NiFe Hydrogenase from desulfovibrio vulgaris miyazaki f suggestion for the initial activation site for dihydrogen
Journal of the American Chemical Society, 2002Co-Authors: Hideaki Ogata, Kunio Miki, Shun Hirota, Yasutaka Mizoguchi, Nobuhiro Mizuno, Shinichi Adachi, Noritake Yasuoka, Tatsuhiko Yagi, Osamu Yamauchi, Yoshiki HiguchiAbstract:The carbon monoxide complex of [NiFe]Hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]Hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2−1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni−Fe active site. The CO was not replaced with H2 in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni−C distances and Ni−C−O angles were about 1.77 A and 160°, respectively, except for one case (1.72 A and 135°), in which an additional electron density peak between the CO and Sγ(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sγ(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sγ(Cys546) atoms suggest that these two atoms at the Ni−Fe act...
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removal of the bridging ligand atom at the ni fe active site of NiFe Hydrogenase upon reduction with h2 as revealed by x ray structure analysis at 1 4 a resolution
Structure, 1999Co-Authors: Yoshiki Higuchi, Kunio Miki, Hideaki Ogata, Noritake Yasuoka, Tatsuhiko YagiAbstract:Abstract Background: The active site of [NiFe] Hydrogenase, a heterodimeric protein, is suggested to be a binuclear Ni–Fe complex having three diatomic ligands to the Fe atom and three bridging ligands between the Fe and Ni atoms in the oxidized form of the enzyme. Two of the bridging ligands are thiolate sidechains of cysteinyl residues of the large subunit, but the third bridging ligand was assigned as a non-protein monatomic sulfur species in Desulfovibrio vulgaris Miyazaki F Hydrogenase. Results: The X-ray crystal structure of the reduced form of D. vulgaris Miyazaki F [NiFe] Hydrogenase has been solved at 1.4 A resolution and refined to a crystallographic R factor of 21.8%. The overall structure is very similar to that of the oxidized form, with the exception that the third monatomic bridge observed at the Ni–Fe site in the oxidized enzyme is absent, leaving this site unoccupied in the reduced form. Conclusions: The unusual ligand structure found in the oxidized form of D. vulgaris Miyazaki F [NiFe] Hydrogenase was confirmed in the reduced form of the enzyme, with the exception that the electron density assigned to the monatomic sulfur bridge had almost disappeared. On the basis of this finding, as well as the observation that H 2 S is liberated from the oxidized enzyme under an atmosphere of H 2 in the presence of its electron carrier, it was postulated that the monatomic sulfur bridge must be removed for the enzyme to be activated. A possible mechanism for the catalytic action of the Hydrogenase is proposed.