Ni-binding Sites Research Articles

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.

A De Novo-Designed Artificial Metallopeptide Hydrogenase: Insights into Photochemical Processes and the Role of Protonated Cys.

Hydrogenase enzymes produce H2 gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo-designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H2 photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Through combining these results, a fine balance is found between solution acidity and the electron transfer steps. This balance is critical to maximize the production of NiI -peptide and protonation of the NiII -H- intermediate (Ni-R) by a Cys (pKa ≈6.4) to produce H2 .

Redesign of a Copper Storage Protein into an Artificial Hydrogenase.

We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H+ reduction to H2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.

Investigating the mechanisms of Ni uptake and sub-lethal toxicity in the Atlantic killifish Fundulus heteroclitus in relation to salinity

The Atlantic killifish (Fundulus heteroclitus) is a resilient estuarine species that may be subjected to anthropogenic contamination of its natural habitat, by toxicants such as nickel (Ni). We investigated Ni accumulation and potential modes of Ni toxicity, in killifish, as a function of environmental salinity. Killifish were acclimated to 4 different salinities [0 freshwater (FW), 10, 30 and 100% seawater (SW)] and exposed to 5 mg/L of Ni for 96 h. Tissue Ni accumulation, whole body ions, critical swim speed and oxidative stress parameters were examined. SW was protective against Ni accumulation in the gills and kidney. Addition of Mg and Ca to FW protected against gill Ni accumulation, suggesting competition with Ni for uptake. Concentration-dependent Ni accumulation in the gill exhibited saturable relationships in both FW- and SW-acclimated fish. However SW fish displayed a lower Bmax (i.e. lower number of Ni binding sites) and a lower Km (i.e. higher affinity for Ni binding). No effect of Ni exposure was observed on critical swim speed (Ucrit) or maximum rate of oxygen consumption (MO2max). Markers of oxidative stress showed either no effect (e.g. protein carbonyl formation), or variable effects that appeared to depend more on salinity than on Ni exposure. These data indicate that the killifish is very tolerant to Ni toxicity, a characteristic that may facilitate the use of this species as a site-specific biomonitor of contaminated estuaries.

Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypBAT) with Ni ions. Transient association between HypA and HypBAT is controlled by the ATP hydrolysis cycle of HypBAT, which is accelerated by HypA. Only the ATP-bound form of HypBAT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypABAT complex has a nanomolar affinity (Kd = 7 nM), which is in contrast to the micromolar affinity (Kd = 4 µM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypBAT, affecting its association with HypA. These findings indicate that HypA and HypBAT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypBAT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.

CooC1 from Carboxydothermus hydrogenoformans Is a Nickel-Binding ATPase

The maturation of nickel-dependent enzymes requires the participation of several accessory proteins. Typically the hydrolysis of nucleotides is necessary for the final metal transfer steps. The ATPase CooC has been implicated in the insertion of nickel into the Ni,Fe cluster (C cluster) of the carbon monoxide dehydrogenase from Rhodospirillum rubrum. Analysis of the amino acid sequence of CooC suggests the presence of motifs typical for the MinD family of SIMIBI class NTPases, which contain a deviant Walker A motif. The genome of the carboxidotrophic hydrogenogenic bacterium Carboxydothermus hydrogenoformans contains three open reading frames with distinct sequence homology to CooC from R. rubrum. We overproduced, isolated, and studied CooC1 from C. hydrogenoformans. As-isolated CooC1 is monomeric in the absence of ligands but dimerizes in the presence of either nickel, ADP, or ATP. CooC1 shows ATPase activity, and the ADP- and ATP-bound dimeric states are distinguished by their stability. The K8A mutant of CooC1, in which alanine replaces the signature lysine typical for the deviant Walker A motif in the MinD family, is incapable of both ATP hydrolysis and ATP-dependent dimerization. This corroborates that CooC1 is indeed a member of the MinD family and suggests an analogous dynamic equilibrium between monomeric and dimeric states. CooC proteins are involved in the insertion of nickel into carbon monoxide dehydrogenases, and we found that one CooC1 dimer binds one Ni(II) ion with nanomolar affinity. Ni-induced dimerization and the Ni(II)-CooC1 stoichiometry suggest that the Ni-binding site of CooC1 occurs in the dimer interface.

Influence of pH, temperature, culture age and cations on adsorption and uptake of Ni by Chlorella vulgaris

Nickel accumulation by C. vulgaris was studied distinguishing adsorption and intracellular accumulation. The surface adsorption contributed maximally (<80%) to total Ni accumulation by the test alga. It was maximal and of equal magnitude at pH 3.5 and 5.5 accordingly suggesting the participation of strong and weak acidic functional groups of C. vulgaris with relatively low and high affinity for Ni adsorption. Nickel uptake was greatly reduced at acidic pH. The cultures in the decline phase of growth showed highest adsorption of Ni but the rate of Ni uptake was greatly reduced when cultures were in the decline or stationary phase of growth. A better exposure of Ni binding sites, or generation of new sites was perhaps responsible for greater Ni adsorption by old cultures of C. vulgaris. Sodium and K caused mixed inhibition whereas Ca and Mg caused noncompetitive inhibition of adsorption and uptake of Ni. Chromium was not able to competitively inhibit adsorption and uptake of Ni by the test alga. The competitive inhibition of Ni adsorption by Cu and Zn seems to be related to their similar ionic properties. Cu stimulated Ni uptake due possibly to increased permeability of the plasma membrane. The present study disagrees with the general conception that cations are competitive inhibitors of metal adsorption and uptake by algae.

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.

The interactions of zinc, nickel, and cadmium with Xenopus transcription factor IIIA, assessed by equilibrium dialysis

Transcription factor IIIA (TFIIIA) was isolated from Xenopus ovary and treated with 1,10-phenanthroline to remove zinc. The interactions of apoTFIIIA with Zn 2+, Ni 2+, and Cd 2+ were studied by equilibrium dialysis under anaerobic conditions (pH 7.0, 25°C), using 65ZnCl 2, 62NiCl 2, and 109CdCl 2, as the radioligands. The data for binding of Zn 2+, Ni 2+, and Cd 2+ to apoTFIIIA were best-fitted by a model with two classes of binding sites. For Zn 2+, the apparent dissociation constants ( K dl Zn and K d 2 Zn) for the high- and low-affinity sites were 1.0 × 10 −8 and 2.6 × 10 −5 M; the apparent binding capacities of the two classes were 0.8 ± 0.5 and 9.6 ± 0.3 g-atoms of Zn/mol; the Hill coefficient was 1.18, consistent with positive cooperativity of Zn-binding sites. For Ni 2+, the apparent K dl Ni and K d 2 Ni values were 2.3 × 10 −5 and 5.2 × 10 −4 M; the apparent binding capacities were 2.3 ± 0.6 and 8.6 ± 0.6 g-atoms of Ni/mol; the Hill coefficient was 1.20 consistent with positive cooperativity of Ni-binding sites. For Cd 2+, the apparent K dl Cd and K d 2 Cd values were 2.8 × 10 −6 and 1.6 × 10 −4 M; the apparent binding capacities were 0.9 ± 0.3 and 2.4 ± 0.5 g-atoms of Cd/mol; the Hill coefficient was 0.53, consistent with negative cooperativity or heterogeneity of Cd-binding sites. This study has the following significance: First, it helps to resolve a controversy about the zinc content of purified TFIIIA. Second, it shows that the K dl Zn of apoTFIIIA is less than the reported K d Zn of thionein, consistent with the hypothesis that thionein modulates gene expression by competing with TFIIIA and other Zn-finger proteins for intracellular Zn 2+ stores. Third, it confirms previous indirect evidence that the affinity of apoTFIIIA for Zn 2+ is much greater than for Cd 2+, and that the affinity for Cd 2+ is greater than for Ni 2+.

Methanococcus voltae harbors four gene clusters potentially encoding two [NiFe] and two [NiFeSe] hydrogenases, each of the cofactor F420-reducing or F420-non-reducing types

Four gene clusters were identified in Methanococcus voltae which probably all encode hydrogenases of the [NiFe] type. One of these contains four genes, including those for the three subunits of the known [NiFeSe] hydrogenase capable of reducing the natural deazaflavin cofactor F420. In a second homologous cluster, the gene encoding the subunit corresponding to that which contains selenium in the known enzyme has a cysteine codon in the relevant position. In addition, two more gene clusters were detected which are very similar both in gene order and sequence to one which encodes a hydrogenase that reduces viologens in Methanobacterium thermoautotrophicum, but whose natural electron acceptor is as yet unknown. Again, in one of these clusters, one of the structural genes, which codes for a hydrogenase subunit containing the putative Ni-binding site, contains a selenocysteine codon. The homologous gene in the other clusters again shows a cysteine codon in the corresponding location. The four gene clusters are closely linked. Those encoding the two selenium-free enzymes are arranged in opposite polarities with a relatively short intergenic region. This arrangement is discussed in terms of a possible joint transcriptional regulation.

“Long” and “short” range electronic effects in the bonding of CO and H 2 on CuNi alloy

The thermal desorption data of CO from the CuNi alloys cannot be explained by a local binding site picture alone. The CO activation energy of desorption from a Ni binding site decreases linearly with increased surface Cu concentration indicating the presence of a ligand (long range electronic) effect. In contrast, the H 2 activation energy of desorption is constant with alloy composition.

Thermal desorption studies of CO and H 2 from the CuNi alloy

The desorption order and activation energies of CO and H 2 from a (110) oriented CuNi single crystal have been studied by thermal desorption spectroscopy. The surface composition of the alloy can be varied by annealing (after sputtering treatment which reduces the surface Cu concentration) in a temperature range where the surface composition is controlled by bulk diffusion. The surface composition is determined by the relative line intensities of the Auger M 1M 4,5M 4,5 Cu and Ni transitions at ∼-100 eV electron energy, and therefore represents an average concentration measurement within the electron escape length (approximately the first couple of atomic layers). The CO desorption spectra are mainly interpreted in terms of the ensemble effect, which relates the heat of chemisorption to the number and the species of the surface atoms in contact with the CO molecule. The CO desorption kinetics is first order. Pour desorption states are resolved, two of which are readily identified as desorption from a pure Cu or pure Ni ensemble (i.e., pure Cu or pure Ni binding site). The pure Ni binding state activation energy decreases linearly with increasing surface Cu concentration, indicating that a ligand (long range) effect is also present in the bonding. The H 2 desorption kinetics is second order. Two binding states are resolved, and both of them are shown to have a pure Ni origin. The activation energy of the high temperature bonding state remains constant with changing surface composition.