In vitro maturation of NiSOD reveals a role for cytoplasmic histidine in processing and metalation.

The importance of cellular low molecular weight ligands in metalloenzyme maturation is largely unexplored. Maturation of NiSOD requires post-translational N-terminal processing of the proenzyme, SodN, by its cognate protease, SodX. Here we provide evidence for the participation of L-histidine in the protease-dependent maturation of nickel-dependent superoxide dismutase (NiSOD) from Streptomyces coelicolor. In vitro studies using purified proteins cloned from S. coelicolor and overexpressed in E. coli support a model where a ternary complex formed between the substrate (SodN), the protease (SodX) and L-Histidine creates a novel Ni-binding site that is capable of the N-terminal processing of SodN and specifically incorporates Ni into the apo-NiSOD product. Thus, L-Histidine serves many of the functions associated with a metallochaperone or, conversely, eliminates the need for a metallochaperone in NiSOD maturation.

The structure of the high-affinity nickel-binding site in the Ni,Zn-HypA•UreE2 complex.

The maturation pathway for the nickel-dependent enzyme urease utilizes the protein UreE as a metallochaperone to supply Ni(II) ions. In Helicobacter pylori urease maturation also requires HypA and HypB, accessory proteins that are commonly associated with hydrogenase maturation. Herein we report on the characterization of a protein complex formed between HypA and the UreE2 dimer. Nuclear magnetic resonance (NMR) coupled with molecular modelling show that the protein complex apo, Zn-HypA•UreE2, forms between the rigorously conserved Met-His-Glu (MHE motif) Ni-binding N-terminal sequence of HypA and the two conserved His102A and His102B located at the dimer interface of UreE2. This complex forms in the absence of Ni(II) and is supported by extensive protein contacts that include the use of the C-terminal sequences of UreE2 to form additional strands of ß-sheet with the Ni-binding domain of HypA. The Ni-binding properties of apo, Zn-HypA•UreE2 and the component proteins were investigated by isothermal titration calorimetry using a global fitting strategy that included all of the relevant equilibria, and show that the Ni,Zn-HypA•UreE2 complex contains a single Ni(II)-binding site with a sub-nanomolar KD. The structural features of this novel Ni(II) site were elucidated using proteins produced with specifically deuterated amino acids, protein point mutations, and the analyses of X-ray absorption spectroscopy, hyperfine shifted NMR features, as well as molecular modeling coupled with quantum-mechanical calculations. The results show that the complex contains a six-coordinate, high-spin Ni(II) site with ligands provided by both component proteins.

Pro5 is not essential for the formation of 'Ni-hook' in nickel superoxide dismutase.

The N-terminus of nickel-dependent superoxide dismutase (NiSOD) forms a structural motif known as the "Ni-hook," where the peptide wraps around the metal to bring cysteine-2 and cysteine-6 into spatial proximity, allowing these residues to coordinate in a cis-geometry. A highly conserved proline-5 residue in the Ni-hook adopts a cis-conformation that is widely considered important for its formation. Herein, we investigate this role by point mutation of Pro5 to alanine. The results obtained show that the variant exhibits wild-type-like redox catalysis and features a Ni(III) center very similar to that found in enzyme. Structural analysis using X-ray absorption spectroscopy of the nickel sites in as-isolated P5A-NiSOD reveals changes in the variant and are consistent with a six-coordinate Ni site with (N/O)4S2 coordination. These changes are attributed to changes in the Ni(II) site structure. Nickel-binding studies using isothermal titration calorimetry reveal two binding events with Kd = 25(20) nM, and 250(60) nM. These events are attributed to i) Ni(II) binding to a preformed Ni-hook containing cis-Pro5 and ii) the combination of trans- to cis- isomerization upon Ni(II) binding, respectively. The higher-affinity binding event is absent in P5A-NiSOD, an observation attributed to the low abundance of the cis-Ala5 isomer in the apo-protein.

Nickel as a virulence factor in the Class I bacterial carcinogen, Helicobacter pylori.

Helicobacter pylori is a human bacterial pathogen that causes peptic ulcers and has been designated a Class I carcinogen by the International Agency for Research on Cancer (IARC). Its ability to survive in the acid environment of the stomach, to colonize the stomach mucosa, and to cause cancer, are linked to two enzymes that require nickel-urease and hydrogenase. Thus, nickel is an important virulence factor and the proteins involved in nickel trafficking are potential antibiotic targets. This review summarizes the nickel biochemistry of H. pylori with a focus on the roles of nickel in virulence, nickel homeostasis, maturation of urease and hydrogenase, and the unique nickel trafficking that occurs between the hydrogenase maturation pathway and urease nickel incorporation that is mediated by the metallochaperone HypA and its partner, HypB.

Complexation of the nickel and cobalt transcriptional regulator RcnR with DNA.

RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. Escherichia coli RcnR was crystallized with DNA that encompasses the DNA-binding site. X-ray diffraction data were collected to 2.9 Šresolution. The crystal belonged to space group P6122 or P6522, with unit-cell parameters a = b = 73.59, c = 157.66 Å, α = ß = 90, γ = 120°.

Structural, Photophysical, and Photochemical Characterization of Zinc Protoporphyrin IX in a Dimeric Variant of an Iron Storage Protein: Insights into the Mechanism of Photosensitized H2 Generation.

Some of us have previously reported the preparation of a dimeric form of the iron storage protein, bacterioferritin (Bfr), in which the native heme b is substituted with the photosensitizer, Zn(II)-protoporphyrin IX (ZnPP-Bfr dimer). We further showed that the ZnPP-Bfr dimer can serve as a photosensitizer for platinum-catalyzed H2 generation in aqueous solution without the usually added electron relay between photosensitizer and platinum ( Clark , E. R. , Inorg. Chem. 2017 , 56 , 4584 - 4593 ). We proposed reductive or oxidative quenching pathways involving the ZnPP anion radical (ZnPP•-) or the ZnPP cation radical, (ZnPP•+), respectively. The present report describes structural, photophysical, and photochemical properties of the ZnPP in the ZnPP-Bfr dimer. X-ray absorption spectroscopic studies at 10 K showed a mixture of five- and six-coordinated Zn centers with axial coordination by one long Zn-SγMet distance of ∼2.8 Å and ∼40% having an additional shorter Zn-S distance of ∼2.4 Å, in addition to the expected 4 nitrogen atom coordination from the porphyrin. The ZnPP in ZnPP-Bfr dimer was prone to photosensitized oxidation to ZnPP•+. The ZnPP•+ was rapidly reduced by ascorbic acid, which we previously determined was essential for photosensitized H2 production in this system. These results are consistent with an oxidative quenching pathway involving electron transfer from 3ZnPP* to platinum, which may be assisted by a flexible ZnPP axial coordination sphere. However, the low quantum yield for H2 production (∼1%) in this system could make reductive quenching difficult to detect, and can, therefore, not be completely ruled out. The ZnPP-Bfr dimer provides a simple but versatile framework for mechanistic assessment and optimization of porphyrin-photosensitized H2 generation without an electron relay between porphyrin and the platinum catalyst.

Ni(II) Sensing by RcnR Does Not Require an FrmR-Like Intersubunit Linkage.

E. coli RcnR (resistance to cobalt and nickel regulator) is a homotetrameric DNA binding protein that regulates the expression of a Ni(II) and Co(II) exporter (RcnAB) by derepressing expression of rcnA and rcnB in response to binding Co(II) or Ni(II). Prior studies have shown that the cognate metal ions, Ni(II) and Co(II), bind in six-coordinate sites at subunit interfaces and are distinguished from noncognate metals (Cu(I), Cu(II), and Zn(II)) by coordination number and ligand selection. In analogy with FrmR, a formaldehyde-responsive transcriptional regulator in the RcnR/CsoR family, the interfacial site allows the metal ions to "cross-link" the N-terminal domain of one subunit with the invariant Cys35 residue in another, which has been deemed to be key to mediating the allosteric response of the tetrameric protein to metal binding. Through the use of mutagenesis to disconnect one subunit from the metal-mediated cross-link, X-ray absorption spectroscopy (XAS) as a structural probe, LacZ reporter assays, and metal binding studies using isothermal titration calorimetry (ITC), the work presented here shows that neither the interfacial binding site nor the coordination number of Ni(II) is important to the allosteric response to binding of this cognate metal ion. The opposite is found for the other cognate metal ion, Co(II), with respect to the interfacial binding site, suggesting that the molecular mechanisms for transcriptional regulation by the two ions are distinct. The metal binding studies reveal that tight metal binding is maintained in the variant. XAS is further used to demonstrate that His33 is not a ligand for Co(II), Ni(II), or Zn(II) in WT-RcnR. The results are discussed in the context of the overall understanding of the molecular mechanisms of metallosensors.

The Role of Mixed Amine/Amide Ligation in Nickel Superoxide Dismutase.

Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel-dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (∼300 mV vs. NHE). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue was added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with density functional theory (DFT) geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and time-dependent DFT computations are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide, and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis-sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.

Chloride Supports O2 Activation in the D201G Facial Triad Variant of Factor-Inhibiting Hypoxia Inducible Factor, an α-Ketoglutarate Dependent Oxygenase.

α-Ketoglutarate (αKG) dependent oxygenases comprise a large superfamily of enzymes that activate O2 for varied reactions. While most of these enzymes contain a nonheme Fe bound by a His2(Asp/Glu) facial triad, a small number of αKG-dependent halogenases require only the two His ligands to bind Fe and activate O2. The enzyme "factor inhibiting HIF" (FIH) contains a His2Asp facial triad and selectively hydroxylates polypeptides; however, removal of the Asp ligand in the Asp201→Gly variant leads to a highly active enzyme, seemingly without a complete facial triad. Herein, we report on the formation of an Fe-Cl cofactor structure for the Asp201→Gly FIH variant using X-ray absorption spectroscopy (XAS), which provides insight into the structure of the His2Cl facial triad found in halogenases. The Asp201→Gly variant supports anion dependent peptide hydroxylation, demonstrating the requirement for a complete His2X facial triad to support O2 reactivity. Our results indicated that exogenous ligand binding to form a complete His2X facial triad was essential for O2 activation and provides a structural model for the His2Cl-bound nonheme Fe found in halogenases.

The Helicobacter pylori HypA·UreE2 Complex Contains a Novel High-Affinity Ni(II)-Binding Site.

Helicobacter pylori is a human pathogen that colonizes the stomach, is the major cause of ulcers, and has been associated with stomach cancers. To survive in the acidic environment of the stomach, H. pylori uses urease, a nickel-dependent enzyme, to produce ammonia for maintenance of cellular pH. The bacteria produce apo-urease in large quantities and activate it by incorporating nickel under acid shock conditions. Urease nickel incorporation requires the urease-specific metallochaperone UreE and the (UreFGH)2 maturation complex. In addition, the H. pylori nickel urease maturation pathway recruits accessory proteins from the [NiFe] hydrogenase maturation pathway, namely, HypA and HypB. HypA and UreE dimers (UreE2) are known to form a protein complex, the role of which in urease maturation is largely unknown. Herein, we examine the nickel-binding properties and protein-protein interactions of HypA and UreE2 using isothermal titration calorimetry and fluorometric methods under neutral and acidic pH conditions to gain insight into the roles played by HypA in urease maturation. The results reveal that HypA and UreE2 form a stable complex with micromolar affinity that protects UreE from hydrolytic degradation. The HypA·UreE2 complex contains a unique high-affinity (nanomolar) Ni2+-binding site that is maintained under conditions designed to mimic acid shock (pH 6.3). The data are interpreted in terms of a proposed mechanism wherein HypA and UreE2 act as co-metallochaperones that target the delivery of Ni2+ to apo-urease with high fidelity.

Selenium versus sulfur: Reversibility of chemical reactions and resistance to permanent oxidation in proteins and nucleic acids.

This review highlights the contributions of Jean Chaudière to the field of selenium biochemistry. Chaudière was the first to recognize that one of the main reasons that selenium in the form of selenocysteine is used in proteins is due to the fact that it strongly resists permanent oxidation. The foundations for this important concept was laid down by Al Tappel in the 1960's and even before by others. The concept of oxygen tolerance first recognized in the study of glutathione peroxidase was further advanced and refined by those studying [NiFeSe]-hydrogenases, selenosubtilisin, and thioredoxin reductase. After 200 years of selenium research, work by Marcus Conrad and coworkers studying glutathione peroxidase-4 has provided definitive evidence for Chaudière's original hypothesis (Ingold et al., 2018) [36]. While the reaction of selenium with oxygen is readily reversible, there are many other examples of this phenomenon of reversibility. Many reactions of selenium can be described as "easy in - easy out". This is due to the strong nucleophilic character of selenium to attack electrophiles, but then this reaction can be reversed due to the strong electrophilic character of selenium and the weakness of the selenium-carbon bond. Several examples of this are described.

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae.

Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M-1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed.

Co(II) and Ni(II) binding of the Escherichia coli transcriptional repressor RcnR orders its N terminus, alters helix dynamics, and reduces DNA affinity.

RcnR, a transcriptional regulator in Escherichia coli, derepresses the expression of the export proteins RcnAB upon binding Ni(II) or Co(II). Lack of structural information has precluded elucidation of the allosteric basis for the decreased DNA affinity in RcnR's metal-bound states. Here, using hydrogen-deuterium exchange coupled with MS (HDX-MS), we probed the RcnR structure in the presence of DNA, the cognate metal ions Ni(II) and Co(II), or the noncognate metal ion Zn(II). We found that cognate metal binding altered flexibility from the N terminus through helix 1 and modulated the RcnR-DNA interaction. Apo-RcnR and RcnR-DNA complexes and the Zn(II)-RcnR complex exhibited similar 2H uptake kinetics, with fast-exchanging segments located in the N terminus, in helix 1 (residues 14-24), and at the C terminus. The largest difference in 2H incorporation between apo- and Ni(II)- and Co(II)-bound RcnR was observed in helix 1, which contains the N terminus and His-3, and has been associated with cognate metal binding. 2H uptake in helix 1 was suppressed in the Ni(II)- and Co(II)-bound RcnR complexes, in particular in the peptide corresponding to residues 14-24, containing Arg-14 and Lys-17. Substitution of these two residues drastically affected DNA-binding affinity, resulting in rcnA expression in the absence of metal. Our results suggest that cognate metal binding to RcnR orders its N terminus, decreases helix 1 flexibility, and induces conformational changes that restrict DNA interactions with the positively charged residues Arg-14 and Lys-17. These metal-induced alterations decrease RcnR-DNA binding affinity, leading to rcnAB expression.

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori.

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

An XAS investigation of the nickel site structure in the transcriptional regulator InrS.

InrS (Internal nickel-responsive Sensor) is a transcriptional repressor of the nickel exporter NrsD and de-represses expression of the exporter upon binding Ni(II) ions. Although a crystal structure of apo-InrS has been reported, no structure of the protein with metal ions bound is available. Herein we report the results of metal site structural investigations of Ni(II) and Cu(II) complexes of InrS using X-ray absorption spectroscopy (XAS) that are complementary to data available from the apo-InrS crystal structure, and are consistent with a planar four-coordinate [Ni(His)2(Cys)2] structure, where the ligands are derived from the side chains of His21, Cys53, His78, and Cys82. Coordination of Cu(II) to InrS forms a nearly identical planar four-coordinate complex that is consistent with a simple replacement of the Ni(II) center by Cu(II).

Glutamate Ligation in the Ni(II)- and Co(II)-Responsive Escherichia coli Transcriptional Regulator, RcnR.

Escherichia coli RcnR (resistance to cobalt and nickel regulator, EcRcnR) is a metal-responsive repressor of the genes encoding the Ni(II) and Co(II) exporter proteins RcnAB by binding to PRcnAB. The DNA binding affinity is weakened when the cognate ions Ni(II) and Co(II) bind to EcRcnR in a six-coordinate site that features a (N/O)5S ligand donor-atom set in distinct sites: while both metal ions are bound by the N terminus, Cys35, and His64, Co(II) is additionally bound by His3. On the other hand, the noncognate Zn(II) and Cu(I) ions feature a lower coordination number, have a solvent-accessible binding site, and coordinate protein ligands that do not include the N-terminal amine. A molecular model of apo-EcRcnR suggested potential roles for Glu34 and Glu63 in binding Ni(II) and Co(II) to EcRcnR. The roles of Glu34 and Glu63 in metal binding, metal selectivity, and function were therefore investigated using a structure/function approach. X-ray absorption spectroscopy was used to assess the structural changes in the Ni(II), Co(II), and Zn(II) binding sites of Glu → Ala and Glu → Cys variants at both positions. The effect of these structural alterations on the regulation of PrcnA by EcRcnR in response to metal binding was explored using LacZ reporter assays. These combined studies indicate that while Glu63 is a ligand for both metal ions, Glu34 is a ligand for Co(II) but possibly not for Ni(II). The Glu34 variants affect the structure of the cognate metal sites, but they have no effect on the transcriptional response. In contrast, the Glu63 variants affect both the structure and transcriptional response, although they do not completely abolish the function of EcRcnR. The structure of the Zn(II) site is not significantly perturbed by any of the glutamic acid variations. The spectroscopic and functional data obtained on the mutants were used to calculate models of the metal-site structures of EcRcnR bound to Ni(II), Co(II), and Zn(II). The results are interpreted in terms of a switch mechanism, in which a subset of the metal-binding ligands is responsible for the allosteric response required for DNA release.

Nickel Ligation of the N-Terminal Amine of HypA Is Required for Urease Maturation in Helicobacter pylori.

The human pathogen Helicobacter pylori requires nickel for colonization of the acidic environment of the stomach. HypA, a Ni metallochaperone that is typically associated with hydrogenase maturation, is also required for urease maturation and acid survival of H. pylori. There are two proposed Ni site structures for HypA; one is a paramagnetic six-coordinate site characterized by X-ray absorption spectroscopy (XAS) in unmodified HypA, while another is a diamagnetic four-coordinate planar site characterized by solution nuclear magnetic resonance in an N-terminally modified HypA construct. To determine the role of the N-terminal amine in Ni binding of HypA, an N-terminal extension variant, L2*-HypA, in which a leucine residue was inserted into the second position of the amino acid sequence in the proposed Ni-binding motif, was characterized in vitro and in vivo. Structural characterization of the Ni site using XAS showed a coordination change from six-coordinate in wild-type HypA (WT-HypA) to five-coordinate pyramidal in L2*-HypA, which was accompanied by the loss of two N/O donor protein ligands and the addition of an exogenous bromide ligand from the buffer. The magnetic properties of the Ni sites in WT-HypA compared to those of the Ni sites in L2*-HypA confirmed that a spin-state change from high to low spin accompanied this change in structure. The L2*-HypA H. pylori strain was shown to be acid sensitive and deficient in urease activity in vivo. In vitro characterization showed that L2*-HypA did not disrupt the HypA-UreE interaction that is essential for urease maturation but was at least 20-fold weaker in Ni binding than WT-HypA. Characterization of the L2*-HypA variant clearly demonstrates that the N-terminal amine of HypA is involved in proper Ni coordination and is necessary for urease activity and acid survival.

Non-thiolate ligation of nickel by nucleotide-free UreG of Klebsiella aerogenes.

Nickel-dependent ureases are activated by a multiprotein complex that includes the GTPase UreG. Prior studies showed that nucleotide-free UreG from Klebsiella aerogenes is monomeric and binds one nickel or zinc ion with near-equivalent affinity using an undefined binding site, whereas nucleotide-free UreG from Helicobacter pylori selectively binds one zinc ion per dimer via a universally conserved Cys-Pro-His motif in each protomer. Iodoacetamide-treated K. aerogenes UreG was nearly unaffected in nickel binding compared to non-treated sample, suggesting the absence of thiolate ligands to the metal. X-ray absorption spectroscopy of nickel-bound UreG showed the metal possessed four-coordinate geometry with all O/N donor ligands including one imidazole, thus confirming the absence of thiolate ligation. The nickel site in Strep-tag II-modified protein possessed six-coordinate geometry, again with all O/N donor ligands, but now including two or three imidazoles. An identical site was noted for the Strep-tag II-modified H74A variant, substituted in the Cys-Pro-His motif, ruling out coordination by this His residue. These results are consistent with metal binding to both His6 and a His residue of the fusion peptide in Strep-tagged K. aerogenes UreG. We conclude that the nickel- and zinc-binding site in nucleotide-free K. aerogenes UreG is distinct from that of nucleotide-free H. pylori UreG and does not involve the Cys-Pro-His motif. Further, we show the Strep-tag II can perturb metal coordination of this protein.

The mechanism of a formaldehyde-sensing transcriptional regulator.

Most organisms are exposed to the genotoxic chemical formaldehyde, either from endogenous or environmental sources. Therefore, biology has evolved systems to perceive and detoxify formaldehyde. The frmRA(B) operon that is present in many bacteria represents one such system. The FrmR protein is a transcriptional repressor that is specifically inactivated in the presence of formaldehyde, permitting expression of the formaldehyde detoxification machinery (FrmA and FrmB, when the latter is present). The X-ray structure of the formaldehyde-treated Escherichia coli FrmR (EcFrmR) protein reveals the formation of methylene bridges that link adjacent Pro2 and Cys35 residues in the EcFrmR tetramer. Methylene bridge formation has profound effects on the pattern of surface charge of EcFrmR and combined with biochemical/biophysical data suggests a mechanistic model for formaldehyde-sensing and derepression of frmRA(B) expression in numerous bacterial species.