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Invasive Gram-negative bacteria often express multiple virulence-associated metal ion chelators to combat host-mediated metal deficiencies. Escherichia coli, Klebsiella, and Yersinia pestis isolates encoding the Yersinia high pathogenicity island (HPI) secrete yersiniabactin (Ybt), a metallophore originally shown to chelate iron ions during infection. However, our recent demonstration that Ybt also scavenges copper ions during infection led us to question whether it might be capable of retrieving other metals as well. Here, we find that uropathogenic E. coli also use Ybt to bind extracellular nickel ions. Using quantitative MS, we show that the canonical metal-Ybt import pathway internalizes the resulting Ni-Ybt complexes, extracts the nickel, and releases metal-free Ybt back to the extracellular space. We find that E. coli and Klebsiella direct the nickel liberated from this pathway to intracellular nickel enzymes. Thus, Ybt may provide access to nickel that is inaccessible to the conserved NikABCDE permease system. Nickel should be considered alongside iron and copper as a plausible substrate for Ybt-mediated metal import by enterobacteria during human infections. [ABSTRACT FROM AUTHOR]

The advancements of quantum chemical methods and computer power allow detailed mechanistic investigations of metalloenzymes. In particular, both quantum chemical cluster and combined QM/MM approaches have been used, which have been proven to successfully complement experimental studies. This review starts with a brief introduction of nickel-dependent enzymes and then summarizes theoretical studies on the reaction mechanisms of these enzymes, including NiFe hydrogenase, methyl-coenzyme M reductase, nickel CO dehydrogenase, acetyl CoA synthase, acireductone dioxygenase, quercetin 2,4-dioxygenase, urease, lactate racemase, and superoxide dismutase.

Lactate racemase (LarA), a new nickel enzyme discovered recently, catalyzes the racemization between d- and l-lactates with a novel nickel pincer cofactor (Ni-PTTMN) derived from nicotinic acid. In this study, by using DFT and a 200-atom active-site model, LarA is revealed to employ a modified proton-coupled hydride-transfer mechanism in which a hydride is transferred to a cofactor pyridine carbon from the substrate α-carbon along with proton transfer from the substrate hydroxy group to a histidine, and then moved back from the opposite side. Tyr294 and Lys298 provide significant acceleration effects by orientating substrates and stabilizing the negative charge developing at the substrate hydroxy oxygen. The barrier was determined to be 12.0 kcal mol−1, which reveals enhanced racemase activity relative to the LarA reaction using NAD+-like cofactors. Compared with NAD+, Ni-PTTMN has a stronger hydride-addition reactivity in moderate and high environmental polarity and may fit perfectly the moderately polar active site of LarA. [ABSTRACT FROM AUTHOR]

Nickel-bound alkaline phosphatase and peroxidase enzymes were used to investigate nickel binding to plasma proteins. Rabbit plasma dilutions to 25,000 were positive by ELISA, while Western blot analysis showed a prominent reaction with histidine-rich glycoprotein (HRG) 1 and lower reaction with fibrinogen (Fgn). To confirm their identities, purified HRG and Fgn were demonstrated to react with the nickel-bound enzymes by Western analysis. With disulfide bonds reduced, HRG and Fgn α-chain reactions were demonstrated. HRG reactions were shown in other species, including human, bovine, chicken and guinea pig, demonstrating general applicability of the detection method. To enhance the purification of rabbit HRG, ammonium sulfate fractionation, immobilized metal ion chromatography and ion-exchange chromatography were optimized. Purified HRG contained trace components larger than HRG that reacted with nickel-enzymes and also with an antibody to HRG by Western analysis, confirming the trace components are related to HRG. These results demonstrate the utility of nickel-enzymes together with antibodies to detect HRG. [ABSTRACT FROM AUTHOR]

Nickel enzymes allow microorganisms to access chemistry that can be vital for survival and virulence. In this review we highlight recent work on several systems that import nickel ions and deliver them to the active sites of these enzymes. Small molecules, in particular l -His and derivatives, may chelate nickel ions before import at TonB-dependent outer-membrane and ABC-type inner-membrane transporters. Inside the cell, nickel ions are used by maturation factors required to produce nickel enzymes such as [NiFe]-hydrogenase, urease and lactate racemase. These accessory proteins often exhibit metal selectivity and frequently include an NTP-hydrolyzing metallochaperone protein. The research described provides a deeper understanding of the processes that allow microorganisms to access nickel ions from the environment and incorporate them into nickel proteins. [ABSTRACT FROM AUTHOR]

Nickel hydride complexes, defined herein as any molecules bearing a nickel hydrogen bond, are crucial intermediates in numerous nickel-catalyzed reactions. Some of them are also synthetic models of nickel-containing enzymes such as [NiFe]-hydrogenase. The overall objective of this review is to provide a comprehensive overview of this specific type of hydride complexes, which has been studied extensively in recent years. This review begins with the significance and a very brief history of nickel hydride complexes, followed by various methods and spectroscopic or crystallographic tools used to synthesize and characterize these complexes. Also discussed are stoichiometric reactions involving nickel hydride complexes and how some of these reactions are developed into catalytic processes. [ABSTRACT FROM AUTHOR]

Early parental influence led me first to medical school, but after developing a passion for biochemistry and sensing the need for a deeper foundation, I changed to chemistry. During breaks between semesters, I worked in various biochemistry labs to acquire a feeling for the different areas of investigation. The scientific puzzle that fascinated me most was the metabolism of the anaerobic bacterium Clostridium kluyveri, which I took on in 1965 in Karl Decker's lab in Freiburg, Germany. I quickly realized that little was known about the biochemistry of strict anaerobes such as clostridia, methanogens, acetogens, and sulfate-reducing bacteria and that these were ideal model organisms to study fundamental questions of energy conservation, CO2 fixation, and the evolution of metabolic pathways. My passion for anaerobes was born then and is unabated even after 50 years of study. [ABSTRACT FROM AUTHOR]

Urease is a Ni(II) enzyme present in every domain of life, in charge for nitrogen recycling through urea hydrolysis. Its activity requires the presence of two Ni(II) ions in the active site. These are delivered by the concerted action of four accessory proteins, named UreD, UreF, UreG and UreE. This process requires protein flexibility at different levels and some disorder-to-order transition events that coordinate the mechanism of protein-protein interaction. In particular, UreG, the GTPase in charge of nucleotide hydrolysis required for urease activation, presents a significant degree of intrinsic disorder, existing as a conformational ensemble featuring characteristics that recall a molten globule. Here, the folding properties of UreG were explored in Archaea hyperthermophiles, known to generally feature significantly low level of structural disorder in their proteome. UreG proteins from Methanocaldococcus jannaschii ( Mj) and Metallosphaera sedula ( Ms) were structurally and functionally analyzed by integrating circular dichroism, NMR, light scattering and enzymatic assays. Metal-binding properties were studied using isothermal titration calorimetry. The results indicate that, as the mesophilic counterparts, both proteins contain a significant amount of secondary structure but maintain a flexible fold and a low GTPase activity. As opposed to other UreGs, secondary structure is lost at high temperatures (68 and 75 °C, respectively) with an apparent two-state mechanism. Both proteins bind Zn(II) and Ni(II), with affinities two orders of magnitude higher for Zn(II) than for Ni(II). No major modifications of the average conformational ensemble are observed, but binding of Zn(II) yields a more compact dimeric form in MsUreG. [ABSTRACT FROM AUTHOR]

The article presents a study on nonredox nickel enzymes. It highlights their reaction mechanisms, biochemistries and structures along with the exclusion of isolated proteins in proteomics facilities. It also notes the reason on the selection of nickel as a catalysis for biological reactions due to its adaptable coordination geometry.

Highlights: [•] The function, structure, and mechanisms are described for nickel-dependent enzymes. [•] Nickel active sites range from simple mononuclear centers to complex multi-metal complexes. [•] Methods to establish nickel specificity are discussed. [ABSTRACT FROM AUTHOR]

Nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori. Indeed, H. pylori possesses two nickel-enzymes that are essential for in vivo colonization, [NiFe] hydrogenase and urease, an abundant virulence factor that contains 24 nickel ions per active complex. Because of these two enzymes, survival of H. pylori relies on an important supply of nickel, implying a tight control of its distribution and storage. In this review, we will present the pathways of activation of the nickel enzymes as well as original mechanisms found in H. pylori for the uptake, trafficking and distribution of nickel between the two enzymes. These include (i) an outer-membrane nickel uptake system, the FrpB4 TonB-dependent transporter, (ii) overlapping protein complexes and interaction networks involved in nickel trafficking and distribution between urease and hydrogenase and, (iii) Helicobacter specific nickel-binding proteins that are involved in nickel storage and can play the role of metallo-chaperones. Finally, we will discuss the implication of the nickel trafficking partners in virulence and propose them as novel therapeutic targets for treatments against H. pylori infection. [ABSTRACT FROM AUTHOR]

Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview. [ABSTRACT FROM AUTHOR]

Abstract: Background: The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni2+ into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH. Methods: Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG. Results: Using both methods, affinity constants were found to be 5nM and 13nM for HypA–UreE and 8μM and 14μM for UreG-UreE. Neither Zn2+ nor Ni2+ had an effect on the kinetics or stability of the HypA–UreE complex. By contrast, addition of Zn2+, but not Ni2+, altered the kinetics and greatly increased the stability of the UreE–UreG complex, likely due in part to Zn2+-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition. General significance: Factors influencing the pathogen''s nickel budget are important to understand pathogenesis and for future drug design. [Copyright &y& Elsevier]

Urease, a Ni-containing metalloenzyme, features an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme (jack bean), K and k, measured at different temperatures and pressures. Such an analysis is useful as it provides information on the molecular nature of the intermediate and transition states of the catalytic reaction. We measured the parameters in a noninteracting buffer using a stopped-flow technique in the temperature range 15-35 °C and in the pressure range 5-132 MPa, the pressure-dependent measurements being the first of their kind performed for urease. While temperature enhanced the activity of urease, pressure inhibited the enzyme; the inhibition was biphasic. Analyzing K provided the characteristics of the formation of the ES complex, and analyzing k, the characteristics of the activation of ES. From the temperature-dependent measurements, the energetic parameters were derived, i.e. thermodynamic Δ H and Δ S for ES formation, and kinetic Δ H and Δ S for ES activation, while from the pressure-dependent measurements, the binding Δ V and activation $$ \Updelta V_{\rm cat}^{ \ne } $$ volumes were determined. The thermodynamic and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al. ( Structure 7:205-216; 1999), in which the Ni-Ni bridging hydroxide-not the terminal hydroxide-is the nucleophile in the catalytic reaction. Graphical abstract: [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This article reports on the activation of dioxygen on nickel(I) dispersed inside the nanopores of the ZSM-5 zeolite, which can be regarded as a heterogeneous mimetic system (zeozyme) for Ni-bearing enzymes. The side-on η²-coordination of the resulting nickel-bound superoxo adduct was ascertained by detailed analysis of the EPR spectra of both 16O2 and 17O2 species supported by computer simulations of the spectra and relativistic DFT calculations of the EPR signatures. Molecular analysis of the g and A(17O) tensors (gxx = 2.0635, gyy = 2.0884, gzz = 2.1675; ∣Axx∣ ≈ 1.0 mT, ∣Ayy∣ = 5.67 mT, ∣Azz∣ ≈ 1.3 mT) and quantum chemical modeling revealed an unusual electronic and magnetic structure of the observed adduct (with gzz(gmax) > gyy(gmid) > gxx(gmin) and the largest O-17 hyperfine splitting along the gmid direction) in comparison to the known homogeneous and enzymatic nickel-superoxo systems. It is best described as a mixed metalloradical with two supporting oxygen donor ligands and even triangular spin-density redistribution within the η2-{NiO2}11 magnetophore. The semioccupied molecular orbital (SOMO) is constituted by highly covalent δ overlap between the out-of-plane 2p(πg*) MO of dioxygen and the 3dx²-y² MO of nickel. By means of the extended transition state-natural orbitals for the chemical valence approach (ETS-NOCV), three distinct orbital channels (associated with σ,π, and δ overlap) of congruent and incongruent charge and spin density flows within the η²-{NiO2}11 unit, contributing jointly to activation of the attached dioxygen, were identified. Their individual energetic relevance was quantified, which allowed for explaining the oxygen binding mechanism with unprecedented accuracy. The nature and structure sensitivity of the g tensor was rationalized in terms of the contributions due to the magnetic field-induced couplings of the relevant molecular orbitals that control the g-tensor anisotropy. The calculated O-17 hyperfine coupling constants correspond well with the experimental parameters, supporting assignment of the adduct. To the best of our knowledge, the η²-{NiO2}11 superoxo adducts have not been observed yet for digonal mononuclear nickel(I) centers supported by oxygen donor ligands. [ABSTRACT FROM AUTHOR]

Abstract: Urea is a plant metabolite derived either from root uptake or from catabolism of arginine by arginase. In agriculture, urea is intensively used as a nitrogen fertilizer. Urea nitrogen enters the plant either directly, or in the form of ammonium or nitrate after urea degradation by soil microbes. In recent years various molecular players of plant urea metabolism have been investigated: active and passive urea transporters, the nickel metalloenzyme urease catalyzing the hydrolysis of urea, and three urease accessory proteins involved in the complex activation of urease. The degradation of ureides derived from purine breakdown has long been discussed as a possible additional metabolic source for urea, but an enzymatic route for the complete hydrolysis of ureides without a urea intermediate has recently been described for Arabidopsis thaliana. This review focuses on the proteins involved in plant urea metabolism and the metabolic sources of urea but also addresses open questions regarding plant urea metabolism in a physiological and agricultural context. The contribution of plant urea uptake and metabolism to fertilizer urea usage in crop production is still not investigated although globally more than half of all nitrogen fertilizer is applied to crops in the form of urea. Nitrogen use efficiency in crop production is generally well below 50% resulting in economical losses and creating ecological problems like groundwater pollution and emission of nitric oxides that can damage the ozone layer and function as greenhouse gasses. Biotechnological approaches to improve fertilizer urea usage bear the potential to increase crop nitrogen use efficiency. [ABSTRACT FROM AUTHOR]

Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O2 ·−). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel–thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O2 ·−, demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, ). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys → Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni2+ ion in each mutant adopts a high-spin ( S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin ( S = 0) configuration observed for the four-coordinate Ni2+ center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys → Ser mutant possesses an essentially six-coordinate Ni2+ center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys → Ser NiSOD mutants are explored. [ABSTRACT FROM AUTHOR]

Iron- and 2-oxoglutarate-dependent dioxygenases are a diverse family of non-heme iron enzymes that catalyze various important oxidations in cells. A key structural motif of these dioxygenases is a facial triad of 2-histidines-1-carboxylate that coordinates the Fe(II) at the catalytic site. Using histone demethylase JMJD1A and DNA repair enzyme ABH2 as examples, we show that this family of dioxygenases is highly sensitive to inhibition by carcinogenic nickel ions. We find that, with iron, the 50% inhibitory concentrations of nickel (IC50 [Ni(II)]) are 25 μM for JMJD1A and 7.5 μM for ABH2. Without iron, JMJD1A is 10 times more sensitive to nickel inhibition with an IC50 [Ni(II)] of 2.5 μM, and approximately one molecule of Ni(II) inhibits one molecule of JMJD1A, suggesting that nickel causes inhibition by replacing the iron. Furthermore, nickel-bound JMJD1A is not reactivated by excessive iron even up to a 2 mM concentration. Using x-ray absorption spectroscopy, we demonstrate that nickel binds to the same site in ABH2 as iron, and replacement of the iron by nickel does not prevent the binding of the cofactor 2-oxoglutarate. Finally, we show that nickel ions target and inhibit JMJD1A in intact cells, and disruption of the iron-binding site decreases binding of nickel ions to ABH2 in intact cells. Together, our results reveal that the members of this dioxygenase family are specific targets for nickel ions in cells. Inhibition of these dioxygenases by nickel is likely to have widespread impacts on cells (e.g. impaired epigenetic programs and DNA repair) and may eventually lead to cancer development. [ABSTRACT FROM AUTHOR]

Abstract: X-ray crystallographic studies [Ogata et al., J. Am. Chem. Soc. 124 (2002) 11628–11635] have shown that carbon monoxide binds to the nickel ion at the active site of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F and inhibits its catalytic function. In the present work spectroscopic aspects of the CO inhibition for this bacterial organism are reported for the first time and enable a direct comparison with the existing crystallographic data. The binding affinity of each specific redox state for CO is probed by FTIR spectro-electrochemistry. It is shown that only the physiological state Ni–SIa reacts with CO. The CO-inhibited product state is EPR-silent (Ni2+) and exists in two forms, Ni–SCO and Ni–SCOred. At very negative potentials, the exogenous CO is electrochemically detached from the active site and the active Ni–R states are obtained. At temperatures below 100 K, photodissociation of the extrinsic CO from the Ni–SCO state results in Ni–SIa that is identified to be the only light-induced state. In the dark, rebinding of CO takes place; the recombination rate constants are of biexponential character and the activation barrier is determined to be approximately 9 kJ mol−1. In addition, formation of a paramagnetic CO-inhibited state (Ni–CO) was observed that results from the interaction of carbon monoxide with the Ni–L state. It is proposed that the nickel in Ni–CO is in a formal monovalent state (Ni1+). [Copyright &y& Elsevier]

The article presents an overview of the pathways that contribute to nickel homeostasis and nickel regulation. It discusses the structures of nickel enzymes to address the chemistry of intracellular nickel. It offers information on the membrane transporters that are used in cellular pathways, which include the NikABCDE, NiCoT, and the CbiMNQO. It notes that nickel is assembled on protein scaffolds by accessory proteins, including HypD and HypC.

Of the eight known nickel enzymes, all but glyoxylase I catalyze the use and/or production of gases central to the global carbon, nitrogen, and oxygen cycles. Nickel appears to have been selected for its plasticity in coordination and redox chemistry and is able to cycle through three redox states (1+, 2+, 3+) and to catalyze reactions spanning ∼1.5 V. This minireview focuses on the catalytic mechanisms of nickel enzymes, with an emphasis on the role(s) of the metal center. The metal centers vary from mononuclear to complex metal clusters and catalyze simple hydrolytic to multistep redox reactions. [ABSTRACT FROM AUTHOR]

Abstract: Ureases (urea amidohydrolases, EC 3.5.1.5) are a group of highly proficient enzymes, widely distributed in nature, whose catalytic function is to catalyze the hydrolysis of urea, its final products being carbonic acid and ammonia. The products and the resulting increase in pH of the reaction environment are consequential characteristics of the action of ureases. Apart from its natural significance, ureases-catalyzed hydrolysis of urea is important in that it has great potential for practical applications. In view of this importance, this article offers a review of the properties of the enzymes, where in addition to the established knowledge, the recent findings are presented. Special emphasis is put on the functional and practical properties of ureases that can be customized and exploited in a diversity of important applications, notably medical, analytical, environmental and engineering. [Copyright &y& Elsevier]

In the presence of excess nickel, Escherichia coli NikR regulates cellular nickel uptake by suppressing the transcription of the nik operon, which encodes the nickel uptake transporter, NikABCDE. Previously published in vitro studies have shown that NikR is capable of binding a range of divalent transition metal ions in addition to Ni2+, including Co2+, Cu2+, Zn2+, and Cd2+. To understand how the high-affinity nickel binding site of NikR is able to accommodate these other metal ions, and to improve our understanding of NikR's mechanism of binding to DNA, we have determined structures of the metal-binding domain (MBD) of NikR in the apo form and in complex with Cu2+ and Zn2+ ions and compared them with the previously published structures with Ni2+. We observe that Cu2+ ions bind in a manner very similar to that of Ni2+, with a square planar geometry but with longer bond lengths. Crystals grown in the presence of Zn2+ reveal a protein structure similar to that of apo MBD with a disordered α3 helix, but with two electron density peaks near the Ni2+ binding site corresponding to two Zn2+ ions. These structural findings along with biochemical data on NikR support a hypothesis that ordering of the α3 helix is important for repressor activation. [ABSTRACT FROM AUTHOR]

Abstract: This article, dedicated to Edward Stiefel, reviews three nickel enzymes that play important roles in the carbon cycle: CO dehydrogenase, acetyl-CoA synthase, and methyl-coenzyme M reductase. After a short discussion of the carbon cycle, the structures of the active centers of the proteins and their proposed mechanisms are discussed. A brief description of future research areas is presented for each enzyme system. A short perspective on future research on nickel enzymes ends this contribution. [Copyright &y& Elsevier]

The article provides a research on the maturation of Nickel enzyme in Helicobacter hepaticus. Helicobacter hepaticus is a causative agent of hepatocellular carcinoma in mice and chronic hepatitis. It possesses a hydrogenase and a urease. These two enzymes contain nickel enzymes. Helicobacter hepaticus is analyzed to have full set of accessory genes. These genes are required for the nickel maturation of each enzyme in other micro- organisms. Further, the study discusses the role of accessory proteins in hydrogenase and urease activities.

[NiFe] hydrogenases are metalloenzymes involved in many biological processes concerning the metabolism of hydrogen. The maturation of the large subunit of these hydrogenases requires the cleavage of a peptide at the C terminus by an endopeptidase before the final formation of the [NiFe] metallocenter. HycI is an endopeptidase of the M52 family and responsible for the C-terminal cleavage of the large subunit of hydrogenase 3 in Escherichia coli. Although extensive studies were performed, the molecular mechanism of recognition and cleavage of hydrogenase 3 remains elusive. Herein, we report the solution structure of E. coli HycI determined by high resolution nuclear magnetic resonance spectroscopy. This is the first solution structure of the apo form of endopeptidase of the M52 family reported thus far. The overall structure is similar to the crystal structure of holo-HybD in the same family. However, significant diversity was observed between the two structures. Especially, HycI shows an open conformation at the putative nickel-binding site, whereas HybD adopts a closed conformation. In addition, we performed backbone dynamic studies to probe the motional properties of the apo form of HycI. Furthermore, the metal ion titration experiments provide insightful information on the substrate recognition and cleavage processes. Taken together, our current structural, biochemical, and dynamic studies extend the knowledge of the M52 family proteins and provide novel insights into the biological function of HycI. [ABSTRACT FROM AUTHOR]

Abstract: NikR proteins are bacterial metallo-regulatory transcription factors that control the expression of the nickel uptake system and/or nickel containing enzymes such as urease, and are involved in the acid stress response. Here, a comparative study is reported on NikR from Helicobacter pylori (HpNikR) and Escherichia coli (EcNikR), as well as the Q2E mutant of EcNikR. Most attention was focused on the Ni(II) binding properties of these proteins, as a function of pH. The influence of the pH on the Ni(II) binding and aggregation properties was studied using gel filtration analysis and UV–visible absorption spectroscopy in the presence of an increasing concentration of nickel. Q2E and wt EcNikR are identical in Ni(II) binding but the Q2E mutant is impaired to some extent in DNA-binding. For EcNikR it is shown that between pH 6 and 8, addition of Ni(II) above 1 equiv. induces mass aggregation and precipitation, concomitant with binding of Ni(II) up to a maximum of 5–8 Ni(II) ions per monomer. The Ni(II) site with highest affinity is the well-described square planar site with three histidines and one cysteine ligands. Aggregation is complete in the presence of less than 1 extra equiv. of Ni(II) and aggregation is fully reversible and precipitates are rapidly solubilized by addition of EDTA. The sensitivity of EcNikR to aggregation decreases with decreasing pH, concurrent with histidines being the main ligands of the site responsible for aggregation. HpNikR does not display aggregation except at alkaline pH, where 3 Ni(II) equiv. are needed. The participation of a cluster consisting of surface-exposed histidines present in EcNikR but not in HpNikR, is proposed to be involved in aggregation. Our results on HpNikR are compatible with the crystallographic data and with the ability of this protein to bind more than one nickel. [Copyright &y& Elsevier]

Abstract: The nickel enzymes CODH and ACS (carbon monoxide dehydrogenase and acetyl-CoA synthase), as found in for example Moorella thermoacetica (formerly Clostridium thermoaceticum), play an important role in carbon cycling: the enzymes convert carbon dioxide through to cell carbon. CODH and ACS are neither structurally nor functionally related to NiFe-hydrogenase, other than that all three enzymes contain assemblies of iron, nickel and sulfur at their active sites. However, the coordination chemistry of synthetic compounds that show structural or functional analogy to the enzyme active sites has been developed in parallel. Herein will be described mono-, di- and multi-nuclear metallic complexes of relevance to the metallocentres of CODH and ACS. [Copyright &y& Elsevier]

Methyl-coenzyme M reductase (MCR) catalyses the formation of methane from methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) in methanogenic archaea. The enzyme has an α2 β2 γ2 subunit structure forming two structurally interlinked active sites each with a molecule F430 as a prosthetic group. The nickel porphinoid must be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-based electron paramagnetic resonance (EPR) signal and a UV–vis spectrum with an absorption maximum at 385 nm. This state is called the MCR-red1 state. In the presence of coenzyme M (HS-CoM) and coenzyme B the MCR-red1 state is in part converted reversibly into the MCR-red2 state, which shows a rhombic Ni(I)-based EPR signal and a UV–vis spectrum with an absorption maximum at 420 nm. We report here for MCR from Methanothermobacter marburgensis that the MCR-red2 state is also induced by several coenzyme B analogues and that the degree of induction by coenzyme B is temperature-dependent. When the temperature was lowered below 20°C the percentage of MCR in the red2 state decreased and that in the red1 state increased. These changes with temperature were fully reversible. It was found that at most 50% of the enzyme was converted to the MCR-red2 state under all experimental conditions. These findings indicate that in the presence of both coenzyme M and coenzyme B only one of the two active sites of MCR can be in the red2 state (half-of-the-sites reactivity). On the basis of this interpretation a two-stroke engine mechanism for MCR is proposed. [ABSTRACT FROM AUTHOR]

Electron nuclear double resonance (ENDOR) and hyperfine sublevel correlation spectroscopy (HYSCORE) are applied to study the active site of catalytic [NiFe]-hydrogenase from Desulfovibrio vulgaris Miyazaki F in the reduced Ni-C state. These techniques offer a powerful tool for detecting nearby magnetic nuclei, including a metal-bound substrate hydrogen, and for mapping the spin density distribution of the unpaired electron at the active site. The observed hyperfine couplings are assigned via comparison with structural data from X-ray crystallography and knowledge of the complete g-tensor in the Ni-C state (Foerster et al. (2003) J Am Chem Soc 125:83–93). This is found to be in good agreement with density functional theory calculations. The two most strongly coupled protons ( aiso=13.7, 11.8 MHz) are assigned to the β-CH2 protons of the nickel-coordinating cysteine 549, and a third proton ( aiso=8.9 MHz) is assigned to a β-CH2 proton of cysteine 546. Using D2O exchange experiments, the presence of a hydride in the bridging position between the nickel and iron—recently been detected for a regulatory hydrogenase (Brecht et al. (2003) J Am Chem Soc 125:13075–13083)—is experimentally confirmed for the first time for catalytic hydrogenases. The hydride exhibits a small isotropic hyperfine coupling constant ( aiso=−3.5 MHz) since it is bound to Ni in a direction perpendicular to the z-axis of the Ni % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX! % MathType!MTEF!2!1!+- % feaafeart1ev1aaatCvAUfeBSn0BKvguHDwzZbqefeKCPfgBGuLBPn % 2BKvginnfarmWu51MyVXgatuuDJXwAK1uy0HwmaeHbfv3ySLgzG0uy % 0Hgip5wzaebbnrfifHhDYfgasaacH8srps0lbbf9q8WrFfeuY-Hhbb % f9v8qqaqFr0xc9pk0xbba9q8WqFfea0-yr0RYxir-Jbba9q8aq0-yq % -He9q8qqQ8frFve9Fve9Ff0dmeaabaqaciGacaGaaeqabaWaaeWaea % aakeaadaqadaqaaiaaiodacaWGKbWaaSbaaSqaaiaadQhadaahaaad % beqaaiaaikdaaaaaleqaaaGccaGLOaGaayzkaaaaaa!3ED1! $$ {\left( {3d_{{z^{2} }} } \right)} $$ orbital. Nitrogen signals that belong to the nitrogen Nε of His-88 have been identified. This residue forms a hydrogen bond with the spin-carrying Ni-coordinated sulfur of Cys-549. Comparison with other hydrogenases reveals that the active site is essentially the same in all proteins, including a regulatory hydrogenase. [ABSTRACT FROM AUTHOR]

This report describes a new ELISA procedure based on end-point titrations. This end-point ELISA takes advantage of the change of color intensity that occurs when peroxidase-containing wells of an ELISA plate are revealed with diaminobenzidine–nickel and further intensification with silver: as antibody concentration and, therefore, peroxidase concentration, decreased, the color became stronger in some wells and, afterwards(i.e., at lower antibody and peroxidase concentrations), the color faded toward clear background. It is proposed that the reciprocal of the sample dilution at which the color intensifies can be used as a measure of the sample antibody content. This report verifies the validity and precision of that procedure. [ABSTRACT FROM AUTHOR]

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F430 as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR fromMethanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparentKM=20 mM; apparentVmax=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparentKM=5 mM; apparentVmax=30 U/mg). Propyl-coenzyme M (apparentKi=2 mM) and allyl-coenzyme M (apparentKi=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I]0.5 V=2 µM), cyano-coenzyme M ([I]0.5 V=0.2 mM), 3-bromopropionate ([I]0.5 V=3 mM), seleno-coenzyme M ([I]0.5 V=6 mM) and trifluoromethyl-coenzyme M ([I]0.5 V=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D2O. The signal was abolished upon exposure of the enzyme to O2. 3-Bromopropanesulfonate ([I]0.5 V=0.1 µM), 3-iodopropanesulfonate ([I]0.5 V=1 µM), and 4-bromobutyrate also inactivated... [ABSTRACT FROM AUTHOR]

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. It contains the nickel porphinoid F430, a prosthetic group that has been proposed to be directly involved in the catalytic cycle by the direct binding and subsequent reduction of the substrate methyl-coenzyme M. The active enzyme (MCRred1) can be generated in vivo and in vitro by reduction from MCRox1, which is an inactive form of the enzyme. Both the MCRred1 and MCRox1 forms have been proposed to contain F430 in the Ni(I) oxidation state on the basis of EPR and ENDOR data. In order to further address the oxidation state of the Ni center in F430, variable-temperature, variable-field magnetic circular dichroism (VTVH MCD), coupled with parallel absorption and EPR studies, have been used to compare the electronic and magnetic properties of MCRred1, MCRox1, and various EPR silent forms of MCR, with those of the isolated penta-methylated cofactor (F430M) in the +1, +2 and +3 oxidation states. The results confirm Ni(I) assignments for MCRred1 and MCRred2 forms of MCR and reveal charge transfer transitions involving the Ni d orbitals and the macrocycle π orbitals that are unique to Ni(I) forms of F430. Ligand field transitions associated with S=1 Ni(II) centers are assigned in the near-IR MCD spectra of MCRox1-silent and MCR-silent, and the splitting in the lowest energy d–d transition is shown to correlate qualitatively with assessments of the zero-field splitting parameters determined by analysis of VTVH MCD saturation magnetization data. The MCD studies also support rationalization of MCRox1 as a tetragonally compressed Ni(III) center with an axial thiolate ligand or a coupled Ni(II)-thiyl radical species, with the reality probably lying between these two extremes. The reinterpretation of MCRox1 as a formal Ni(III) species rather than an Ni(I) species obviates the need to invoke a two-electron reduction of the F430 macrocyclic ligand on reductive activation of MCRox1 to yield MCRred1. [ABSTRACT FROM AUTHOR]

Methyl-coenzyme M reductase (MCR) catalyzes the reduction of methyl-coenzyme M (CH3–S–CoM) to methane. The enzyme contains as a prosthetic group the nickel porphinoid F430 which in the active enzyme is in the EPR-detectable Ni(I) oxidation state. Crystal structures of several inactive Ni(II) forms of the enzyme but not of the active Ni(I) form have been reported. To obtain structural information on the active enzyme–substrate complex we have now acquired X-ray absorption spectra of active MCR in the presence of either CH3–S–CoM or the substrate analog coenzyme M (HS–CoM). For both MCR complexes the results are indicative of the presence of a five-coordinate Ni(I), the five ligands assigned as four nitrogen ligands from F430 and one oxygen ligand. Analysis of the spectra did not require the presence of a sulfur ligand indicating that CH3–S–CoM and HS–CoM were not coordinated via their sulfur atom to nickel in detectable amounts. As a control, X-ray absorption spectra were evaluated of three enzymatically inactive MCR forms, MCR-silent, MCR-ox1-silent and MCR-ox1, in which the nickel is known to be six-coordinate. Comparison of the edge position of the X-ray absorption spectra revealed that the Ni(I) in the active enzyme is more reduced than the Ni in the two EPR-silent Ni(II) states. Surprisingly, the edge position of the EPR-active MCR-ox1 state was found to be the same as that of the two silent states indicating similar electron density on the nickel. Electronic supplementary material is available if you access this article at http://www.dx.doi.org/10.1007/s00775-002-0399-2. On that page (frame on the left side), a link takes you directly to the supplementary material. [ABSTRACT FROM AUTHOR]

Examines the synthesis and dimer cleavage reactions of monoderivatized N2S ligand based on the cyclic diamine diazacycloheptane. Role of nickel-thiolates in the function of cysteine-rich nickel enzymes; Functions of cyanide and alkylation agents in the protein modification reagent iodoacetamide; Electrochemical and spectral comparison among the nickel complexes.

Nickel enzymes are a relatively new class of metalloenzymes. The seven known nickel enzymes are urease, hydrogenase, CO-dehydrogenase, methyl-coenzyme M reductase, Ni-superoxide dismutase, glyoxalase I and cis-trans isomerase. The requirement for nickel implies the presence of a nickel-processing system, since free transition metals are harmful to the cell. A nickel-processing system involves the recognition and transport of nickel into the cell and the handling of the nickel once it enters the cell until it is inserted into the nickel enzyme. Several mechanisms for nickel transport have been identified and will be reviewed here. Accessory proteins required for the biosynthesis of the nickel active site have been identified. Accessory proteins bind the nickel when it enters the cell and are proposed to assist with the insertion of nickel into the enzyme. The function of the characterized nickel-processing proteins is described, and models for nickel insertion into the nickel enzymes are presented. [ABSTRACT FROM AUTHOR]

Examines the effect of nickel deficiency on lipid metabolism in liver and serum lipoproteins of rats. Triacylglycerol accumulation in liver as effect of nickel deficiency; Basis of nickel deficient classification; Determination of hematological measurements; Effects of iron deficiency in lipid metabolism.

From animal studies we know that oral administration of T-dependent antigens before sensitization effectively induces systemic immune unresponsiveness. Such 'oral tolerance' is persistent, dose dependent, antigen-specific and presumably T suppressor cell-mediated. Oral tolerance induction could be an effective way to prevent undesired T cell-mediated immune functions, such as playing a role in allograft reaction, autoimmune and allergic diseases. In the present study allergic contact hypersensitivity (ACH) to nickel, currently presenting the most frequent contact allergy in man, was chosen to establish the feasibility of oral prevention of undesired T cell-mediated immunity in man. Potentially tolerizing (oral nickel contacts via orthodontic braces) as well as sensitizing (ear piercing) events were studied retrospectively in 2176 patients attending nine European patch test clinics. Patients were interviewed by means of a confidential questionnaire. The results show that ear piercing strongly favoured development of nickel ACH, More importantly, patients having had oral contacts with nickel-releasing appliances (dental braces) at an early age, but only if prior to ear piercing, showed a reduced frequency of nickel hypersensitivity. Frequencies of other hypersensitivities, in particular to fragrance, were not affected. These results support our view that induction of specific systemic immunologic tolerance by timely oral administration of antigens is feasible in man. [ABSTRACT FROM AUTHOR]

The relationship between HLA antigens (A, B and DR) and nickel contact sensitivity was examined in a series of 26 female patients with unequivocal positive patch-test reactions to nickel. Acomparison of patient antigen frequency versus Yorkshire Region HLA antigen figures showed an increased incidence of HLA-B35 in our patient populations, (P<0.01). The relative risk (RR) for the development of nickel sensitivity was reflected in the increased values for HLA-B35 and BW22 in our patients when compared with Yorkshire frequencies. We found no significant association between DR antigens and RR in our patients. The aetiological fraction (EF) showed no significant association between HLA antigens and nickel contract sensitivity. [ABSTRACT FROM AUTHOR]

The large subunits of nickel-containing hydrogenases are synthesised in a precursor form which, after nickel incorporation, is processed by proteolytic cleavage at the C-terminal end. The protease involved in processing of HycE. the large subunit of hydrogenase 3 from Escherichia coli, was purified by three chromatographic steps to apparent homogeneity. Its gene was identified by using a hybridisation probe generated by PCR with oligonucleotide primers the sequence of which was derived from the N-terminal and internal amino acid sequences. Determination of tile nucleotide sequence showed that the gene is located distally and as a hitherto uncharacterised gene within the hyc operon, coding for hydrogenase 3 components. It was designated hycI. The HycI protease has a molecular mass of 17 k Da and is a monomer. Its cleavage reaction is not inhibited by conventional inhibitors of serine and metalloproteases, which correlates with the fact that the sequence does not contain signal are motifs characteristic of serine-, metallo-, cysteine- or acid proteases. Homologous genes are present in other transcriptional units coding for hydrogenases. [ABSTRACT FROM AUTHOR]

The five known nickel-dependent enzymes include urease, hydrogenase, carbon monoxide dehydrogenase (and CO dehydrogenase/acetyl-coenzyme A synthase), methyl- S–coenzyme M reductase, and one class of superoxide dismutase. Consistent with their disparate functions, these Ni enzymes have distinct metallocenter structures that vary in Ni coordination geometry, number and types of metals, and the presence of additional components. Sophisticated cellular Ni processing systems have been devised to allow for specific and functional incorporation of Ni into these proteins. This review highlights several themes that are common to the enzyme activation processes and summarizes current concepts related to the enzyme-specific Ni assembly pathways. [ABSTRACT FROM AUTHOR]

CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) is one of the four known nickel enzymes. It is a bifunctional protein that catalyzes the oxidation of CO to CO at a nickel iron-sulfur cluster (Cluster C) and a remarkable condensation reaction between a methyl group (donated from a methylated corrinoid iron-sulfur protein), carbon monoxide, and coenzyme A to form acetyl-CoA at a separate nickel iron-sulfur cluster (Cluster A). This review focuses on the current understanding of the structure and function of Cluster A and on related model chemistry. It describes studies that uncovered the first example of a biological organometallic reaction sequence. The mechanism of acetyl-CoA synthesis includes enzymebound methylnickel, iron-carbonyl, and acylmetal intermediates. Discovery of the methylnickel species constituted the first example of an alkylnickel species in biology and unveiled a new biological role for nickel. [ABSTRACT FROM AUTHOR]

A new mononuclear low-spin nickel(II) dithiolato complex, [NiL2] (1), reacts with copper iodide to form the heterooctanuclear cluster [{Ni(L)2}2(CuI)6] (2) with four trigonal-planar CuI2S and two tetrahedral CuI2S2 sites; anagostic interactions between the nickel(II) ions and aromatic protons have been demonstrated by variable-temperature NMR studies to pertain in solution. [ABSTRACT FROM AUTHOR]

The advancements of quantum chemical methods and computer power allow detailed mechanistic investigations of metalloenzymes. In particular, both quantum chemical cluster and combined QM/MM approaches have been used, which have been proven to successfully complement experimental studies. This review starts with a brief introduction of nickel-dependent enzymes and then summarizes theoretical studies on the reaction mechanisms of these enzymes, including NiFe hydrogenase, methyl-coenzyme M reductase, nickel CO dehydrogenase, acetyl CoA synthase, acireductone dioxygenase, quercetin 2,4-dioxygenase, urease, lactate racemase, and superoxide dismutase. [ABSTRACT FROM AUTHOR]