Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.

Superoxide dismutases rely on protein structural elements to adjust the redox potential of the metallocenter to an optimum value near 300 mV (vs. NHE), to provide a source of protons for catalysis, and to control the access of anions to the active site. These aspects of the catalytic mechanism are examined herein for recombinant preparations of the nickel-dependent SOD (NiSOD) from Streptomyces coelicolor, and for a series of mutants that affect a key tyrosine residue, Tyr9 (Y9F-, Y62F-, Y9FY62F- and D3A-NiSOD). Structural aspects of the nickel sites are examined by a combination of EPR and x-ray absorption spectroscopies, and by single crystal x-ray diffraction at ~ 1.9 Å resolution in the case of Y9F- and D3A-NiSODs. The functional effects of the mutations are examined by kinetic studies employing pulse radiolytic generation of O2− and by redox titrations. These studies reveal that although the structure of the nickel center in NiSOD is unique, the ligand environment is designed to optimize the redox potential at 290 mV and results in the oxidation of 50% of the nickel centers in the oxidized hexamer. Kinetic investigations show that all of the mutant proteins have considerable activity. In the case of Y9F-NiSOD, the enzyme shows saturation behavior that is not observed in WT-NiSOD and suggests that release of peroxide is inhibited. The crystal structure of Y9F-NiSOD reveals an anion binding site that is occupied by either Cl− or Br− and is located close to, but not within bonding distance of the nickel center. The structure of D3A-NiSOD reveals that in addition to affecting the interaction between subunits, this mutation repositions Y9 and leads to altered chemistry with peroxide. Comparisons with Mn(SOD) and Fe(SOD) reveal that although different strategies are employed to adjust the redox potential and supply of protons, NiSOD has evolved a similar strategy to control the access of anions to the active site.

Helicobacter pylori, a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic, and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys) 4 site to a Zn(His) 2 (Cys) 2 site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the β-CH 2 protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys) 4 ) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH. KeywordsHelicobacter pylori; XAS; HypA; metallochaperone; zinc; nickel; ITC; NMR mmaroney@chemistry.umass.edu. Supporting Information Available: Figures of CD spectra for zinc-site cysteine mutants of HypA, Thermal melts of WT-and zinc-site cysteine and histidine mutants, molecular weight determinations by size-exclusion chromatography, ITC thermograms for zinc-site cysteine and histidine mutants, raw ITC titration data, zinc K-edge XANES and EXAFS data and fits for Cys → Asp and His95A mutations, nickel K-edge XANES and EXAFS data and fits for Cys → Asp zinc-site mutations, and UV-vis spectra of HypA with nickel bound. Tables of mutagenic primers, best EXAFS fits to Zn K-edge data for Cys → Asp mutations, best EXAFS fits to Ni Kedge data for zinc-site Cys → Asp mutations, alternate fits for zinc and nickel K-edge EXAFS (39 pages). This information is available free of charge via the Internet at

Hypoxia sensing is the generic term for pO 2 -sensing in humans and other higher organisms. These cellular responses to pO 2 are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the Factor Inhibiting HIF (FIH-1), which couples O 2 -activation to the hydroxylation of the Hypoxia Inducible Factor α (HIFα). Uncoupled O 2 -activation by human FIH-1 was studied by exposing the resting form of FIH-1, (αKG+Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ max = 583 nm) arising from enzyme auto-hydroxylation of ; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue, (oxo)Met 275 . The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however product yield was saturable with increasing [αKG] and required excess Fe (II). Yellow FIH-1 was formed from (succinate+Fe)FIH-1, or by glycerol addition to (αKG+Fe) FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1, (αKG+Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O 2 -activation by loss of an H 2 O ligand from the metal center.

HypA is an accessory protein and putative metallochaperone that is critical for supplying nickel to the active site of NiFe hydrogenases. In addition to binding Ni(II), HypA is known to contain a Zn site that has been suggested to play a structural role. X-ray absorption spectroscopy has been used to show that the Zn site changes structure upon binding nickel, from a S 3 (O/N)-donor ligand environment to an S 4 -donor ligand environment. This provides a potential mechanism for discriminating Ni(II) from other divalent metal ions. The Ni(II) site is shown to be a six-coordinate complex composed of O/N-donors including two histidines. As such, it resembles the nickel site in UreE, a nickel metallochaperone involved in nickel incorporation into urease.The use of reactive and potentially toxic transition metals in enzyme active sites depends on proteins that can acquire metals from the environment (e.g., permeases), transport them inside cells and incorporate them into apoenzymes (e.g., metallochaperones), and control their intracellular concentration (e.g. transcriptional regulators), often with great specificity for the cognate metal. 1 How proteins achieve this specificity is largely unknown. However, competition for ligands between metals and changes in ligand environments would provide a mechanism for discriminating different metals. Several accessory proteins have been implicated in the highly choreographed incorporation of nickel in the bimetallic active site of NiFe hydrogenases, 2 including HypA, 2-4 HypB, 3, 5, 6 and SlyD. 7 HypA is critical for supplying Ni to the active site of NiFe hydrogenases. 2, 3 In addition to binding Ni(II), HypA is known to contain a Zn site that has been suggested to play a structural role in E. coli 4 as has its homolog, HybF. 8 Herein we show that the Zn site of H. pylori HypA (HpHypA) undergoes a structural change in response to Ni binding, suggesting that it has a role in the specific binding of nickel.H. pylori hypA was cloned, expressed and purified as described in the supporting information. The purification procedure was a modification of that previously described by Mehta et al. 3 Most notably, 1 mM DTT was included in all stages of the purification, but removed before the addition of nickel. The protein eluted from gel-filtration at a molecular weight consistent with a monomer. Metal content was determined by ICP-AE after treating metallated samples with Chelex and buffer exchanging ten times to remove any trace metal that was not tightly bound. Urea denaturation experiments indicated the protein unfolded cooperatively (see supporting information). The addition of stoichiometric Ni(II) had only a slight effect on the stability. Nickel titration of the nickel-free protein monitored by Tyr fluorescence showed a decrease in signal that reached a minimum at 1:1 Ni(II):HypA stoichiometry. XAS data were collected on frozen protein solutions in Tris-buffer (20 mM Tris with 100 mM NaCl, pH 7.2, no DTT) held at ~50 K using a He displex cryostat. Protein solutions were...

The adsorption and intercalation of the cationic luminescence probe, tris(2,2′-bipyridine)ruthenium(II) complex ([Ru(bpy) 3 ] 2+ or Rubpy), into hectorite and Laponite host clay films were investigated. Because the photophysical properties of Rubpy are strongly influenced by the lamellar nanospace of the smectite host, Rubpy serves as a unique photoprobe of host-guest and guest-guest interfaces within inorganic-organic nanocomposites. The stacking patterns of the host tactoids influence guest luminescence through direct mediation of ion clustering and self-quenching phenomena. Spectral red shift of emission wavelengths and decreased lifetimes were observed with increased guest loading. The extent of red shift in the Rubpy/Laponite films indicated a more fluid guest microenvironment. Rubpy/Laponite films exhibit enhanced potential for photonic and sensor applications with increased optical transparency, intense luminescence, and longer luminescence lifetimes. Cointercalation of the cationic surfactant, trimethylcetylammonium cation, promotes two-dimensional tiling of Laponite tactoids and may afford selective tuning of fluorophore packing.

A study of the step-wise oxidation of a Ni(II) diaminodithiolate complex through the formation of sulfate, the ultimate sulfur oxygenate, is reported. Controlled oxygenations or peroxidations of a neutral, planar, tetracoordinate, low-spin Ni(II) complex of a N(2)S(2)-donor ligand, (N,N'-dimethyl-N-N'-bis(2-mecaptoethyl)-1,3-propanediaminato) nickel(ii) (1), led to a series of sulfur oxygenates that have been isolated and characterized by ESI-MS and single-crystal X-ray diffraction. A monosulfenate complex (2) was detected by ESI-MS as a product of oxidation with one equivalent of H(2)O(2). However, this complex proved too unstable to isolate. Reaction of the dithiolate (1) with two equivalents of H(2)O(2) or one O(2) molecule leads to the formation of a monosulfinate complex (3), which was isolated and fully characterized by crystallography. The oxidation product of the monosulfinate (3) produced with either O(2) or H(2)O(2) is an interesting dimeric complex containing both sulfonate and thiolate ligands (4), this complex was fully characterized by crystallography, details of which were reported earlier by us. A disulfonate complex (7) is produced by reaction of 1 in the presence of O(2) or by reaction with exactly six equivalents of H(2)O(2). This complex was isolated and also fully characterized by crystallography. Possible intermediates in the conversion of the monosulfinate complex (3) to the disulfonate complex (7) include complexes with mixed sulfonate/sulfenate (5) or sulfonate/sulfinate (6) ligands. Complex 5, a four-oxygen adduct of 1, was not detected, but the sulfonate/sulfinate complex (6) was isolated and characterized. The oxidation chemistry of 1 is very different from that reported for other planar cis-N(2)S(2) Ni(ii) complexes including N,N'-dimethyl-N-N'-bis(2-mecaptoethyl)-1,3-ethylenediaminato) nickel(II), (8), and N,N'-bis(mercaptoethyl)-1,5-diazacyclooctane nickel(II). To address the structural aspects of the reactivity differences, the crystal structure of 8 was also determined. A comparison of the structures of planar Ni(II) complexes containing cis-dithiolate ligands, strongly suggests that the differences in reactivity are determined in part by the degree of flexibility that is allowed by the NN' chelate ring.

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