Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly.

The Klebsiella aerogenes ureE gene product was previously shown to facilitate assembly of the urease metallocenter (Lee, M.H., et al., 1992, J. Bacteriol. 174, 4324-4330). UreE protein has now been purified and characterized. Although it behaves as a soluble protein, UreE is predicted to possess an amphipathic beta-strand and exhibits unusually tight binding to phenyl-Sepharose resin. Immunogold electron microscopic studies confirm that UreE is a cytoplasmic protein. Each dimeric UreE molecule (M(r) = 35,000) binds 6.05 + 0.25 nickel ions (Kd of 9.6 +/- 1.3 microM) with high specificity according to equilibrium dialysis measurements. The nickel site in UreE was probed by X-ray absorption and variable-temperature magnetic circular dichroism spectroscopies. The data are most consistent with the presence of Ni(II) in pseudo-octahedral geometry with 3-5 histidyl imidazole ligands. The remaining ligands are nitrogen or oxygen donors. UreE apoprotein has been crystallized and analyzed by X-ray diffraction methods. Addition of nickel ion to apoprotein crystals leads to the development of fractures, consistent with a conformational change upon binding nickel ion. We hypothesize that UreE binds intracellular nickel ion and functions as a nickel donor during metallocenter assembly into the urease apoprotein.     

Purification and activation properties of UreD-UreF-urease apoprotein complexes.

In vivo assembly of the Klebsiella aerogenes urease nickel metallocenter requires the presence of UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. Prior studies had shown that urease apoprotein exists in an uncomplexed form as well as in a series of UreD-urease (I.-S. Park, M.B. Carr, and R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994) and UreD-UreF-UreG-urease (I.-S. Park and R.P. Hausinger, J. Bacteriol. 177:1947-1951, 1995) apoprotein complexes. This study demonstrates the existence of a distinct series of complexes consisting of UreD, UreF, and urease apoprotein. These novel complexes exhibited activation properties that were distinct from urease and UreD-urease apoprotein complexes. Unlike the previously described species, the UreD-UreF-urease apoprotein complexes were resistant to inactivation by NiCl2. The bicarbonate concentration dependence for UreD-UreF-urease apoenzyme activation was significantly decreased compared with that of the urease and UreD-urease apoproteins. Western blot (immunoblot) analyses with polyclonal anti-urease and anti-UreD antibodies indicated that UreD is masked in the UreD-UreF-urease complexes, presumably by UreF. We propose that the binding of UreF modulates the UreD-urease apoprotein activation properties by excluding nickel ions from binding to the active site until after formation of the carbamylated lysine metallocenter ligand.     

Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation

The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a UreD-UreF-UreG complex bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. This study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein-protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in copurification of UreD and urease apoprotein, whereas UreG bound to only a subset of the species. Notably, weakened interaction with UreG correlated with the low-activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD-UreF-UreG-urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the noncognate metal Zn.     

UreE stimulation of GTP-dependent urease activation in the UreD-UreF-UreG-urease apoprotein complex

The activation of metal-containing enzymes often requires the participation of accessory proteins whose roles are poorly understood. In the case of Klebsiella aerogenes urease, a nickel-containing enzyme, metallocenter assembly requires UreD, UreF, and UreG acting as a protein chaperone complex and UreE serving as a nickel metallochaperone. Urease apoprotein within the UreD-UreF-UreG-urease apoprotein complex is activated to wild-type enzyme activity levels under physiologically relevant conditions (100 microM bicarbonate and 20 microM Ni2+) in a process that requires GTP and UreE. The GTP concentration needed for optimal activation is greatly reduced in the presence of UreE compared to that required in its absence. The amount of UreE provided is critical, with maximal activation observed at a concentration equal to that of Ni2+. On the basis of its ability to facilitate urease activation in the presence of chelators, UreE is proposed to play an active role in transferring Ni2+ to urease apoprotein. Studies involving site-directed variants of UreE provide evidence that His96 has a direct role in metal transfer. The results presented here parallel those obtained from previous in vivo studies, demonstrating the relevance of this in vitro system to the cellular metallocenter assembly process.     

Characterization of UreG, identification of a UreD-UreF-UreG complex, and evidence suggesting that a nucleotide-binding site in UreG is required for in vivo metallocenter assembly of Klebsiella aerogenes urease.

In vivo urease metallocenter assembly in Klebsiella aerogenes requires the presence of several accessory proteins (UreD, UreF, and UreG) and is further facilitated by UreE. In this study, UreG was isolated and shown to be a monomer with an Mr of 21,814 +/- 20 based on gel filtration chromatography and mass spectrometric results. Although it contains a P-loop motif typically found in nucleotide-binding proteins, UreG did not bind or hydrolyze ATP or GTP, and it exhibited no affinity for ATP- and GTP-linked agarose resins. Site-directed mutagenesis of ureG allowed the substitution of Ala for Lys-20 or Thr-21 in the P-loop motif and resulted in the production of inactive urease in cells grown in the presence of nickel; hence, an intact P-loop may be essential for UreG to function in vivo. These mutant cells were unable to synthesize the UreD-UreF-UreG-urease apoprotein species that are thought to be the key urease activation complexes in the cell. An insoluble protein species containing UreD, UreF, and UreG (termed the DFG complex) was detected in cells carrying deletions in ureE and the urease structural genes. The DFG complex was solubilized in 0.5% Triton X-100 detergent, shown to bind to an ATP-linked agarose resin, and found to elute from the resin in the presence of Mg-ATP. In cells containing a UreG P-loop variant, the DFG complex was formed but did not bind to the nucleotide-linked resin. These results suggest that the UreG P-loop motif may be essential for nucleotide binding by the DFG complex and support the hypothesis that nucleotide hydrolysis is required for in vivo urease metallocenter assembly.     

Effect of the urease accessory genes on activation of the Helicobacter pylori urease apoprotein

The roles that accessory gene products play in activating the Helicobacter pylori urease apoprotein were examined. The activity of the urease apoprotein increased in the following order when it was expressed with the accessory genes: ureG相似文献   

Thermodynamic Modeling of Poorly Complexing Metals in Concentrated Electrolyte Solutions: An X-Ray Absorption and UV-Vis Spectroscopic Study of Ni(II) in the NiCl2-MgCl2-H2O System

Knowledge of the structure and speciation of aqueous Ni(II)-chloride complexes is important for understanding Ni behavior in hydrometallurgical extraction. The effect of concentration on the first-shell structure of Ni(II) in aqueous NiCl₂ and NiCl₂-MgCl₂ solutions was investigated by Ni K edge X-ray absorption (XAS) and UV-Vis spectroscopy at ambient conditions. Both techniques show that no large structural change (e.g., transition from octahedral to tetrahedral-like configuration) occurs. Both methods confirm that the Ni(II) aqua ion (with six coordinated water molecules at R _Ni-O = 2.07(2) Å) is the dominant species over the whole NiCl₂ concentration range. However, XANES, EXAFS and UV-Vis data show subtle changes at high salinity (> 2 mol∙kg^-1 NiCl₂), which are consistent with the formation of small amounts of the NiCl⁺ complex (up to 0.44(23) Cl at a Ni-Cl distance of 2.35(2) Å in 5.05 mol∙kg^-1 NiCl₂) in the pure NiCl₂ solutions. At high Cl:Ni ratio in the NiCl₂-MgCl₂-H₂O solutions, small amounts of [NiCl₂]⁰ are also present. We developed a speciation-based mixed-solvent electrolyte (MSE) model to describe activity-composition relationships in NiCl₂-MgCl₂-H₂O solutions, and at the same time predict Ni(II) speciation that is consistent with our XAS and UV-Vis data and with existing literature data up to the solubility limit, resolving a long-standing uncertainty about the role of chloride complexing in this system.     

Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein

 The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to function in Ni(II) delivery to the urease apoprotein. Wild-type UreE contains a histidine-rich region at its carboxyl terminus and binds 5–6 Ni per dimer, whereas the functionally active but truncated H144*UreE lacks the histidine-rich motif and binds only two Ni per dimer [Brayman TG, Hausinger RP (1996) J Bacteriol 178 : 5410-5416]. For both proteins, Cu(II), Co(II), and Zn(II) ions compete for the Ni-binding sites. In order to characterize the coordination environments of bound metals, especially features that are unique to Ni, the Ni-, Cu-, and Co-bound forms of H144*UreE were studied by a combination of EPR, ESEEM, hyperfine-shifted ¹H-NMR, XAS, and RR spectroscopic methods. For each metal ion, the two binding sites per homodimer were spectroscopically distinguishable. For example, the two Ni-binding sites each have pseudo-octahedral geometry in an N/O coordination environment, but differ in their number of histidine donors. The two Cu-binding sites have tetragonal geometry with two histidine donors each; however, the second Cu ion is bound by at least one cysteine donor in addition to the N/O-type donors found for the first Cu ion. Two Co ions are bound to H144*UreE in pseudo-octahedral geometry with N/O coordination, but the sites differ in the number of histidine donors that can be observed by NMR. The differences in coordination for each type of metal ion are relevant to the proposed function of UreE to selectively facilitate Ni insertion into urease in vivo. Received: 8 October 1997 / Accepted: 30 December 1997     

Ni Uptake and Limitation in Marine Synechococcus Strains

Ni accumulation and utilization were studied in two strains of marine Synechococcus, isolated from both coastal (CC9311; clade I) and open-ocean (WH8102; clade III) environments, for which complete genome sequences are available. Both strains have genes encoding an Ni-containing urease and when grown on urea without Ni become Ni-N colimited. The Ni requirements of these strains also depend upon the genomic complement of genes encoding superoxide dismutase (SOD). WH8102, with a gene encoding only an Ni-SOD, has a novel obligate requirement for Ni, regardless of the N source. Reduced SOD activity in Ni-depleted cultures of WH8102 supports the link of this strain's Ni requirement to Ni-SOD. The genome of CC9311 contains a gene for a Cu/Zn-SOD in addition to a predicted pair of Ni-SODs, yet this strain cannot grow without Ni on NO₃⁻ and can grow only slowly on NH₄⁺ without Ni, implying that the Cu/Zn-SOD cannot completely replace Ni-SOD in marine cyanobacteria. CC9311 does have a greater tolerance for Ni starvation. Both strains increase their Ni uptake capabilities and actively bioconcentrate Ni in response to decreasing extracellular and intracellular Ni. The changes in Ni uptake rates were more pronounced in WH8102 than in CC9311 and for growth on urea or nitrate than for growth on ammonia. These results, combined with an analysis of fully sequenced marine cyanobacterial genomes, suggest that the growth of many marine Synechococcus and all Prochlorococcus strains is dependent upon Ni.     

X-ray absorption spectroscopic analysis of Fe(II) and Cu(II) forms of a herbicide-degrading α-ketoglutarate dioxygenase

 The first step in the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by Ralstonia eutropha JMP134 is catalyzed by the α-ketoglutarate (α-KG)-dependent dioxygenase TfdA. Previously, EPR and ESEEM studies on inactive Cu(II)-substituted TfdA suggested a mixture of nitrogen/oxygen coordination with two imidazole-like ligands. Differences between the spectra for Cu TfdA and α-KG- and 2,4-D-treated samples were interpreted as a rearrangement of the g–tensor principal axis system. Herein, we report the use of X-ray absorption spectroscopy (XAS) to further characterize the metal coordination environment of Cu TfdA as well as that in the active, wild-type Fe(II) enzyme. The EXAFS data are interpreted in terms of four N/O ligands (two imidazole-like) in the Cu TfdA sample and six N/O ligands (one or two imidazole-like) in the Fe TfdA sample. Addition of α-KG results in no significant structural change in coordination for Cu or Fe TfdA. However, addition of 2,4-D results in a decrease in the number of imidazole ligands in both Cu and Fe TfdA. Since this change is seen both in the Fe and Cu EXAFS, loss of one histidine ligand upon 2,4-D addition best describes the phenomenon. These XAS data clearly demonstrate that changes occur in the atomic environment of the metallocenter upon substrate binding. Received: 3 July 1998 / Accepted: 13 October 1998     

Function of UreB in Klebsiella aerogenes urease

Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreBΔ1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreBΔ1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.     

Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate

The properties of the tight and specific binding of 2-C-carboxy-d-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO₂ and Mg²⁺, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of [¹⁴C]CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported (S Johal, BE Partridge, R Chollet [1985] J Biol Chem 260: 9894-9904). The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg²⁺. Addition of [¹⁴C]CABP and EDTA stopped binding of Mg²⁺ and allowed tight binding of the radiolabel only to sites which were CO₂/Mg²⁺-activated at that moment. This approach estimated the amount of CO₂/Mg²⁺-activated sites in the presence of inactive sites and carbamylated sites lacking Mg²⁺. The rate of CO₂ fixation was proportional to the CO₂/Mg²⁺-activated sites. During light-dependent CO₂ fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg²⁺ and CO₂. Lysis of chloroplasts in media with [¹⁴C]CABP plus EDTA estimated those carbamylated sites having Mg²⁺. The loss of Rubisco activation during illumination was partially due to the lack of Mg²⁺ to stabilize the carbamylated sites.     

XAS investigation of the structure and function of Ni in acireductone dioxygenase

Acireductone dioxygenases (ARDs) are enzymes involved in the methionine recycle pathway, which regulates aspects of the cell cycle. Klebsiella pneumoniae produces two enzymes that share a common polypeptide sequence and differ only in the metal ion present. Reaction of acireductone (1,2-dihydroxy-3-keto-5-methylthiopentene) with Fe-ARD and dioxygen produces formate and 2-keto-4-methylthiobutanoic acid, the alpha-ketoacid precursor of methionine. Ni-ARD reacts with acireductone and dioxygen to produce methylthiopropionate, CO, and formate and does not lie on the methionine recycle pathway. An X-ray absorption spectroscopy (XAS) study of the structure of the catalytic Ni center in resting Ni-ARD enzyme and the enzyme-substrate complex is reported. This study establishes the structure of the Ni site in resting Ni-ARD as containing a six coordinate Ni site composed of O/N-donor ligands including 3-4 histidine residues, demonstrates that the substrate binds to the Ni center in a bidentate fashion by displacing two ligands, at least one of which is a histidine ligand, and provides insight into the mechanism of catalysis employed by a Ni-containing dioxygenase. Efficiently relaxed and hyperfine-shifted resonances are observed in the (1)H nuclear magnetic resonance spectrum of Ni-ARD that can be attributed to the His imidazoles ligating the paramagnetic Ni ion and are consistent with the XAS results regarding His ligation. These resonances show significant perturbation in the presence of substrate, confirming that the metal ion interacts directly with the substrate.     

The reactivity of tetracarbonylnickel encapsulated in zeolite X. A case history of intrazeolite coordination chemistry.

Zeolite X shows a high capacity for tetracarbonylnickel (up to 28 weight percent) such that complete pore filling with ‘liquid like’ material takes place. The adsorbed material may be removed simply by evacuation at room temperature. Partial decomposition of the Ni(CO)₄ occurs on standing at room temperature under N₂. The resultant orange species is highly reactive and has spectroscopic properties consistent with a coordinatively unsaturated ‘Ni(CO)₃’. Complete and irreversible decomposition by heating to 200 °C in vacuo gives a black zeolite, with an undefined metal phase, which is unreactive towards carbon monoxide. Reaction of the zeolite supported Ni(CO)₄ with various phosphorus ligands is highly dependent on the original loading level as well as the physical size of the ligands involved. At low loadings two kinds of reactivity are observed: 1) With ligands too large to gain access to the zeolite crystal interior, reaction occurs only in solution and so drags the Ni(CO)₄ from the zeolite: 2) With smaller ligands, reaction takes place inside the zeolite cages leading to well-defined, encapsulated, ship-in-bottle complexes which have a stoichiometry dictated by the available space in the cages. At high loading levels, pore blocking phenomena lead to inhomogeneous distributions of encapsulated complexes wherein a complete shell of phosphorous ligand substituted nickel carbonyl species forms at the crystal surface layers and prevents further reaction deeper inside the crystal. The reactivity with large phosphines has been used to study the diffusion of Ni(CO)₄ from the zeolite. Monitoring the appearance of the Ni(CO)₃L (where L = phosphine) by 31-P NMR of the supernatant solution shows that Ni(CO)₄ leaves the zeolite with a first order rate constant of at least 2 × 10^?2 sec^?1 at 298 K.     

An EXAFS study of jack bean urease,a nickel metalloenzyme

EXAFS and XANES spectra have been recorded above the nickel K edge of urease and three model compounds. Preliminary results indicate that the local environment of the nickel ions in urease resemble most closely that of the nickel ions in the model compound [Ni(L)₂(L)₁] (ClO₄)₁, where L is 1-n-propyl-2-α-hydroxybenzyl benzimidazole and L is the deprotonated form.     

Nickel and copper complexes with few amide-based macrocyclic and open-chain ligands

The present work shows three new amide-based ligands H₂L¹, H₂L² and H₂L³ and their nickel and copper complexes. The X-ray structural analysis substantiate that the ligands constitute a square-based basal plane around the metal center. The crystal structures also show interesting solid state packing due to hydrogen-bonding and various weak C?H interactions. The solution-based spectral studies support the solid-state geometry observed for these complexes. The electrochemical results show that the Ni^3+/2+ and Cu^3+/2+ redox couple primarily depends on the N₄ donors composed of N_amide and N_amine atoms. It was observed that the ligands H₂L¹ and H₂L² are better suited to stabilize the Cu(III) species whereas ligand H₂L³ is ideal for the stabilization of Ni(III) species. On the basis of electrochemical findings, transient Ni³⁺ species were generated and characterized by the absorption spectroscopy.     

Inhibition of urease by bismuth(III): Implications for the mechanism of action of bismuth drugs

Bismuth compounds are widely used for the treatment of peptic ulcers and Helicobacter pylori infections. It has been suggested that enzyme inhibition plays an important role in the antibacterial activity of bismuth towards this bacterium. Urease, an enzyme that converts urea into ammonia and carbonic acid, is crucial for colonization of the acidic environment of the stomach by H. pylori. Here, we show that three bismuth complexes exhibit distinct mechanisms of urease inhibition, with some differences dependent on the source of the enzyme. Bi(EDTA) and Bi(Cys)₃ are competitive inhibitors of jack bean urease with K _i values of 1.74 ± 0.14 and 1.84 ± 0.15 mM, while the anti-ulcer drug, ranitidine bismuth citrate (RBC) is a non-competitive inhibitor with a K _i value of 1.17 ± 0.09 mM. A ¹³C NMR study showed that Bi(Cys)₃ reacts with jack bean urease during a 30 min incubation, releasing free cysteines from the metal complex. Upon incubation with Bi(EDTA) and RBC, the number of accessible cysteine residues in the homohexameric plant enzyme decreased by 5.80 ± 0.17 and 11.94 ± 0.13, respectively, after 3 h of reaction with dithiobis(2-nitrobenzoic acid). Kinetic analysis showed that Bi(EDTA) is both a competitive inhibitor and a time-dependent inactivator of the recombinant Klebsiella aerogenes urease. The active C319A mutant of the bacterial enzyme displays a significantly reduced sensitivity toward inactivation by Bi(EDTA) compared with the wild-type enzyme, consistent with binding of Bi³⁺ to the active site cysteine (Cys³¹⁹) as the mechanism of enzyme inactivation.     

Evidence for the presence of urease apoprotein complexes containing UreD, UreF, and UreG in cells that are competent for in vivo enzyme activation.

In vivo activation of Klebsiella aerogenes urease, a nickel-containing enzyme, requires the presence of functional UreD, UreF, and UreG accessory proteins and is further facilitated by UreE. These accessory proteins are proposed to be involved in metallocenter assembly (M. H. Lee, S. B. Mulrooney, M. J. Renner, Y. Markowicz, and R. P. Hausinger, J. Bacteriol. 174:4324-4330, 1992). A series of three UreD-urease apoprotein complexes are present in cells that express ureD at high levels, and these complexes are thought to be essential for in vivo activation of the enzyme (I.-S. Park, M. B. Carr, and R. P. Hausinger, Proc. Natl. Acad. Sci. USA 91:3233-3237, 1994). In this study, we describe the effect of accessory gene deletions on urease complex formation. The ureE, ureF, and ureG gene products were found not to be required for formation of the UreD-urease complexes; however, the complexes from the ureF deletion mutant exhibited delayed elution during size exclusion chromatography. Because these last complexes were of typical UreD-urease sizes according to native gel electrophoretic analysis, we propose that UreF alters the conformation of the UreD-urease complexes. The same studies revealed the presence of an additional series of urease apoprotein complexes present only in cells containing ureD, ureF, and ureG, along with the urease subunit genes. These new complexes were shown to contain urease, UreD, UreF, and UreG. We propose that the UreD-UreF-UreG-urease apoprotein complexes represent the activation-competent form of urease apoprotein in the cell.     

Manganese Oxide Modified Nickel Catalysts for Photothermal CO Hydrogenation to Light Olefins

Ni‐based catalysts are traditionally considered unsuitable for the Fischer–Tropsch syntheses of olefins, due to the very strong hydrogenation ability of metallic Ni. Herein, this paradigm is challenged. A series of MnO supports nickel catalysts (denoted herein as Ni‐x) are fabricated by H₂ reduction of a nickel‐manganese mixed metal oxide at temperatures (x) ranging from 250 to 600 °C. The Ni‐500 catalyst displays unprecedented performance for photothermal CO hydrogenation to olefins, with an olefin selectivity of 33.0% under ultraviolet–visible irradiation. High‐resolution transmission electron microscopy, X‐ray absorption spectroscopy (XAS), and X‐ray diffraction analyses reveal that the Ni‐x catalysts contain metallic Ni nanoparticles supported by MnO. X‐ray photoelectron spectroscopy and XAS establish that electron transfer from MnO to the Ni⁰ nanoparticles is responsible for modifying the electronic structure of nickel (creating Ni^δ? states), thereby shifting the CO hydrogenation selectivity toward light olefins. Further, density functional theory calculations show that this electron transfer lowers the adsorption energies of olefins on Ni surfaces, thus minimizing the undesirable deep hydrogenation reactions to higher alkanes. This study conclusively demonstrates that MnO‐modified Ni‐based catalyst systems can be highly selective for CO hydrogenation to light olefins.     

Structural variations in dinuclear model hydrolases and hydroxamate inhibitor models: synthetic, spectroscopic and structural studies

Reactions of the structural model hydrolases [M₂(OAc)₄(H₂O)(Im)₄]; M=Mn (E^′); M=Co (D^′); M=Ni (B^′) and [M₂(OPiv)₄(H₂O)(tmen)₂]; M=Mn (E″); M=Co (D″); M=Ni (B″) with a number of hydroxamic acids, RHA (aceto- (R=CH₃), benzo- (R = C₆H₅) and N-phenylacetohydroxamic acid (NPhAHA)) gave a series of hydroxamate dibridged complexes [M₂(OAc)(RA)₂(Im)₄][OTf] and [M₂(OPiv)(RA)₂(tmen)₂][OTf]; M=Co, Ni, in which the bridging hydroxamates exhibit a novel bonding mode in which the deprotonated hydroxamate hydroxyl bridges the two metal centres only. The formation of this type of structure by NPhAHA is the first example involving a secondary hydroxamic acid. These complexes are good structural models of the acetohydroxamate-inhibited C³¹⁹A variant of Klebsiella aerogenes urease (KAU) and their structures are close to those previously reported for complexes containing tmen capping ligands. Reaction with glutarodihydroxamic acid leads to hydroxylamine elimination and formation of a dimer containing deprotonated N-hydroxyglutarimide as bridging ligand but in this case the structure contains pentacoordinated Co(II) and only one bridging acetate in contrast to the tmen-based series where the analogous complex contains hexacoordinated Co(II) and two bridging acetates. Reaction of [Mn₂(OAc)₂(μ-OAc)₂(μ-H₂O)(tmen)₂] with acetohydroxamic acid (AHA) gave the first structurally characterized manganese hydroxamate, [Mn₂(OAc)₃(AA)(tmen)₂] with the same bridging/chelating mode of hydroxamate bonding as in the analogous cobalt and nickel complexes, although only one bridging hydroxamate occurs in the manganese complex in contrast to the two bridging hydroxamates in the cobalt and nickel complexes. The isolation of the dimanganese hydroxamate bridged complex suggests that hydroxamic acids may also inhibit the dimanganese based metallohydrolase, arginase.