Nickel(II)-substituted azurin I from Alcaligenes xylosoxidans as characterized by resonance Raman spectroscopy at cryogenic temperature

Metal-substituted blue copper proteins (cupredoxins) have been successfully used to study the effect of metal-ion identity on their active-site properties, specifically the coordination geometry and metal–ligand bond strengths. In this work, low-temperature (77 K) resonance Raman (RR) spectra of the blue copper protein Alcaligenes xylosoxidans azurin I and its Ni(II) derivative are reported. A detailed analysis of all observed bands is presented and responsiveness to metal substitution is discussed in terms of structural and bonding changes. The native cupric site exhibits a RR spectrum characteristic of a primarily trigonal planar (type 1) coordination geometry, identified by the ν(Cu–S)_Cys markers at 373, 399, 409, and 430 cm⁻¹. Replacement of Cu(II) with Ni(II) results in optical and RR spectra that reveal (1) a large hypsochromic shift in the main (Cys)S → M(II) charge-transfer absorption from 622 to 440 nm, (2) greatly reduced metal–thiolate bonding interaction, indicated by substantially lower ν(Ni–S)_Cys stretching frequencies, (3) elevation of the cysteine ν(C _β –S) stretching, amide III, and ρ _s(C _β H₂) scissors vibrational modes, and (4) primarily four-coordinated, trigonally distorted tetrahedral geometry of the Ni(II) site that is marked by characteristic ν(Ni–S)_Cys stretching RR bands at 347, 364, and 391 cm⁻¹. Comparisons of the electronic and vibrational properties between A. xylosoxidans azurin I and its closely structurally related azurin from Pseudomonas aeruginosa are made and discussed. For cupric azurins, the intensity-weighted average M(II)–S(Cys) stretching frequencies are calculated to be ν(Cu–S)_iwa = 406.3 and 407.6 cm⁻¹, respectively. These values decreased to ν(Ni–S)_iwa = 359.3 and 365.5 cm⁻¹, respectively, after Ni(II) → Cu(II) exchange, suggesting that the metal–thiolate interactions are similar in the two native proteins but are much less alike in their Ni(II)-substituted forms.     

The binding of imidazole in an azurin-like blue-copper site

 Frozen solutions of the azurin mutant His117Gly in the presence of excess of methyl-substituted imidazoles have been investigated by electron spin-echo envelope modulation (ESEEM) spectroscopy at 9 GHz. The addition of imidazole is known to reconstitute a blue-copper site and variation of the non-protein bound ligand [N-methyl-, 2-methyl-, 4(5)-methylimidazole] has allowed the study of the copper-imidazole binding as a model for histidine binding in such sites. Quadrupole and hyperfine tensors of the remote nitrogen of the imidazoles have been determined. The quadrupole tensors indicate that the methyl-substituted imidazoles in the mutant adopt the same orientation relative to copper as the histidine-117 in the wild-type protein. Analysis of the hyperfine tensors in terms of spin densities reveals that the spin density on the remote nitrogen of the substituted imidazole has σ and a variable π character, depending on the position of the methyl group. For azurin the corresponding spin density is of virtually pure σ character. In conclusion, blue-copper sites show subtle variations as regards the histidine/imidazole centred part of the wavefunction of the unpaired electron. Received: 27 October 1998 / Accepted: 9 February 1999     

Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

Replacement of the cysteine at position 112 of Pseudomonas aeruginosa azurin with an aspartic acid residue results in a mutant (Cys112Asp) protein that retains a strong copper-binding site. Cu^II(Cys112Asp) azurin can be reduced by excess [Ru^II(NH₃)₆]²⁺, resulting in a Cu^I protein with an electronic absorption spectrum very similar to that of wild-type Cu^I azurin. Cys112Asp azurin exhibits reversible interprotein electron-transfer reactivity with P. aeruginosa cytochrome c ₅₅₁ (μ?=?0.1?M sodium phosphate (pH?7.0);E°(Cu^II/I)?=?180 mV vs NHE); this redox activity indicates that electrons can still enter and exit the protein through the partially solvent-exposed imidazole ring of His117. The structure of Cu^II(Cys112Asp) azurin at 2.4-Å resolution shows that the active-site copper is five coordinate: the pseudo-square base of the distorted square-pyramidal structure is defined by the imidazole N^δ atoms of His46 and His117 and the oxygen atoms of an asymmetrically-bound bidentate carboxylate group of Asp112; the apical position is occupied by the oxygen atom of the backbone carbonyl group of Gly45. The Cu^II–Asp112 interaction is distinguished by an approximately 1.2-Å displacement of the metal center from the plane defined by the Asp112 carboxylate group.     

Effect of nickel(II) substitution on the resonance Raman and NMR spectra of Alcaligenes xylosoxidans azurin II: implications for axial-ligand bonding interactions in cupredoxin active sites

Assignment of the resonance Raman (RR) spectrum of Ni(II)-substituted azurin II from Alcaligenes xylosoxidans (NCIMB 11015) using Ni isotope substitution reveals an anomalously low Ni-S(Cys) stretching frequency of 349?cm^–1, suggesting the presence of significant axial-ligand bonding interactions. The X-ray crystal structure of Ni(II)-substituted azurin from Pseudomonas aeruginosa shows that there are two potential axial ligands to the Ni ion: a peptide carbonyl O at a distance of 2.46?Å, together with a long-range interaction from a methionine sulfur (S′) at a distance of 3.30?Å. Comparison of the RR properties of Ni(II)-substituted azurin II with stellacyanin (which contains an axial carbonyl ligand, but no methionine) suggests that the interaction from the carbonyl oxygen ligand alone is not sufficient to account for the weak Ni azurin metal-thiolate bond. Instead, it appears that a Ni-methionine bonding interaction is also required to explain the low Ni-S(Cys) stretching frequency in Ni(II)-substituted azurin II. This hypothesis is supported by NMR studies which show a large paramagnetic shift for the protons of the methionine side-chain. Thus, it appears that Ni-substituted azurin II is best described as five-coordinate, and that significant Ni(II)-methionine bonding interactions can occur at a distance of 3.3?Å.     

Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

Low-frequency (90–435 cm⁻¹) NIR-excitation (875–900 nm) resonance Raman (RR) studies are reported for the H(M202)G cavity mutant of bacterial photosynthetic reaction centers (RCs) from Rb. sphaeroides that was first described by Goldsmith et al. [(1996) Biochemistry 35: 2421–2428]. In this mutant, the His residue that axially ligates the Mg ion of the M-side bacteriochlorophyll (BChl) of the special pair primary donor (P) is replaced by a non-ligating Gly residue. Regardless, the Mg ion of P_M in the H(M202)G RCs remains pentacoordinates and is presumably ligated by a water molecule, although this axial ligand has not been definitively identified. The low-frequency RR studies of the H(M202)G RCs are accompanied by studies of RCs exchanged with D₂O and incubated with imidazole (Im). The RR studies of the cavity mutant RCs reveal the following: (1) The structure of P_M in the H(M202)G RCs is different from that of the wild-type, consistent with an altered BChl core. (2) A water ligand for P_M in the H(M202)G RCs is generally consistent with the low-frequency RR spectra. The Mg-OH₂ stretching vibration is tentatively assigned to a band at 318 cm⁻¹, a frequency higher than that of the Mg-His stretch of the native pigment (∼ ∼235 cm⁻¹). (3) The BChl core structure of P_M in the cavity mutant is rendered similar (but not identical) to that of the wild-type when the adventitious water axial ligand is replaced by Im. (4) Exchange with D₂O results in more global structural changes, likely involving the protein, which in turn affect the structure of the BChls in P. (5) Assignment of the low-frequency vibrational spectrum of P is generally more complex than originally suggested.     

Probing copper ligands in denatured <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> azurin: unfolding His117Gly and His46Gly mutants

Azurin is a single-domain beta-barrel protein with a redox-active copper cofactor. Upon Pseudomonas aeruginosa azurin unfolding, the cofactor remains bound to the polypeptide, coordinating three ligands: cysteine-112, one histidine imidazole, and a third, unknown ligand. In order to identify which histidine (histidine-117 and histidine-46 both coordinate copper in native azurin) is involved in copper coordination in denatured azurin, two single-site (histidine to glycine) mutants, His117Gly and His46Gly azurin, are investigated here. Equilibrium denaturation experiments of His46Gly azurin loaded with copper demonstrate that copper remains bound to this mutant in high urea concentrations where the protein's secondary structure is lost. In contrast, for copper-loaded His117Gly azurin, copper does not stay coordinated upon polypeptide unfolding. The copper absorption at 370 nm in denatured His46Gly azurin agrees with that for copper in complex with a peptide corresponding to residues 111-123 in azurin, suggesting similar metal coordination. We conclude that histidine-117 (and not histidine-46) is the histidine copper ligand in denatured azurin. This is also in accord with the proximity of histidine-117 to cysteine-112 in the primary sequence.     

Ligand replacement study at the His120 site of purple CuA azurin

The CuA center is a dinuclear Cu2S2(Cys) electron transfer center found in cytochrome c oxidase and nitrous oxide reductase. In a previous investigation of the equatorial histidine ligands' effect on the reduction potential, electron transfer and spectroscopic properties of the CuA center, His120 in the engineered CuA azurin was mutated to Asn, Asp, and Ala. The identical absorption and EPR spectra of these mutants indicate that a common ligand is bound to the copper center. To identify this replacement ligand, the His120Gly CuA azurin mutant was constructed and purified. Absorption and X-band EPR spectra show that His120Gly is similar to the other His120X (X = Asn, Asp, Ala) mutant proteins. Titrations with chloride, imidazole, and azide suggest that the replacement ligand is not exchangeable with exogenous ligands. The possibility of an internal amino acid acting as the replacement ligand for His120 in the His120X mutant proteins was investigated by analyzing the CuA azurin crystal structure and then converting the likely internal ligand, Asn 119, to Asp, Ser, or Ala in the His120Gly mutant. The double mutants H120G/Asn 119X (X = Asp, Ser, or Ala) displayed UV-Vis absorption and EPR spectra that are identical to His120Gly and the other His120X mutants, indicating that Asn119 is not the internal ligand replacing His120 in the His120X mutant proteins. These results demonstrate the remarkable stability of the dinuclear His120 mutants of CuA azurin.     

Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

 Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k ₁ = 1.4×10² M^–1s^–1) compared with both native-glycosylated and recombinant forms of HRPC (k ₁ = 1.7×10⁷ M^–1s^–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k ₁ = 1.1×10⁴ M^–1s^–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k ₂ = 142 s^–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly, stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38 explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases. Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are involved in the modulation of substrate affinity. Received: 21 July 1995 / Accepted: 27 November 1995     

Azide adducts of stearoyl-ACP desaturase: a model for μ-1,2 bridging by dioxygen in the binuclear iron active site

 The stearoyl-acyl carrier protein Δ⁹ desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O₂–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O₂ to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (k _app≈3.5 s^–1 in 0.7 M NaN₃, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η¹–terminal structure at pH 7.8 (¹⁴N₃ ^– asymmetric stretch at 2073 cm^–1, resolved into two bands with ¹⁵N¹⁴N₂ ^–) and a μ-1,3 bridging structure at pH<7 (¹⁴N₃ ^– asymmetric stretch at 2100 cm^–1, shifted as a single band with ¹⁵N¹⁴N₂ ^–). Both adducts also exhibit an Fe–N₃ stretching mode at ≈380 cm^–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries. Received: 23 August 1996 / Accepted: 4 October 1996     

The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [Cu_M(His)₂(OH₂)₂–Cu_H(His)₃(OH₂)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [Cu_M(His)₂S(Met)–Cu_H(His)₃] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the Cu_M Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the Cu_M site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the Cu_M center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM.     

Electron relaxation and solvent accessibility of the metal site in wild-type and mutated azurins as determined from nuclear magnetic relaxation dispersion experiments

The interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different degrees of solvent accessibility are found. The low relaxivity of wild-type azurin agrees with a solvent-protected copper site in solution, the closest water being found at a distance of more than 5?Å from the copper. This low relaxivity contrasts with the relatively large relaxivity of the His46Gly and His117Gly azurin mutants, which shows clear evidence of copper-coordinated water. The data on the latter mutants are best analyzed in terms of one and two water molecules coordinated to the copper in His46Gly and His117Gly, respectively. The Met121His azurin mutant shows an intermediate behavior. The data are analyzed in terms of an increased solvent accessibility with respect to the wild-type azurin, resulting in semi-coordination of water at low pH. These different modes of coordination lead to different geometries, ranging from the trigonal type 1 site of wild-type azurin to the tetragonal type 2 copper sites of the His117Gly and His46Gly azurin mutants through a so-called type 1.5 site of the Met121His mutant. A correlation is found between the relaxation time (τ_s) of the unpaired electron of copper(II) and the geometry of the metal site: as the tetragonal character decreases the relaxation becomes significantly faster. τ_s values of ≤1?ns are found for the tetrahedrally distorted type 1 and type 1.5 sites and of 5–15?ns for the tetragonal type 2 sites.     

Electrochemistry of the CuA domain of Thermus thermophilus cytochrome ba 3

 The electrochemistry of a water-soluble fragment from the Cu_A domain of Thermus thermophilus cytochrome ba ₃ has been investigated. At 25  °C, Cu_A exhibits a reversible reduction at a pyridine-4-aldehydesemicarbazone-modified gold electrode (0.1 M Tris, pH 8) with E° = 0.24 V vs NHE. Thermodynamic parameters for the [Cu(Cys)₂Cu]^+/0 electrode reaction were determined by variable-temperature electrochemistry (ΔS°_rc = –5.4(12) eu, ΔS° = –21.0(12) eu, ΔH° = –11.9(4) kcal/mol;ΔG° = –5.6 (11) kcal/mol). The relatively small reaction entropy is consistent with a low reorganization energy for [Cu(Cys)₂Cu]^+/0 electron transfer. An irreversible oxidation of [Cu(Cys)₂Cu]⁺ at 1 V vs NHE confirms that the Cu^II:Cu^II state of Cu_A is significantly destabilized relative to the Cu^II state of analogous blue-copper proteins. Received: 3 June 1996 / Accepted: 26 August 1996     

Arctic cyanobacteria and limnological properties of their environment: Bylot Island, Northwest Territories, Canada (73°N, 80°W)

Cyanobacteria were a major constituent of phototrophic communities in the lakes, ponds and streams of Bylot Island, in the Canadian high Arctic. The waters spanned a range of temperatures (1.8–16.8°C in late July), pH regimes (6.2–9.2) and conductivities (1.5–1700 μS cm⁻¹) but nutrient concentrations were consistently low (< 1 μg dissolved reactive P l⁻¹ at all sites; < 10 μg NO₃-N l⁻¹ at most sites). Picoplanktonic species (Synechococcus spp.) were often the numerical dominants in the plankton, and periphytic filamentous species (Oscillatoriaceae) commonly formed thick (5–50 mm) benthic mats. Bloom-forming species of cyanobacteria were either absent or poorly represented even in Chla-rich ponds. The total community biomass ranged from 0.1 to 29.8 μg Chla l⁻¹ in the plankton and from 1.1 to 34.8 μg Chla cm⁻² in the benthos. The in vivo absorbance characteristics of isolates from these environments indicated a genetically diverse range of species in each group of Arctic cyanobacteria. Growth versus irradiance relationships were determined for each of the isolates and similarly revealed large genetic differences (maximum growth rates from 0.17 to 0.61 day⁻¹), even between morphologically identical taxa. A comparison of nutrients, pigment concentrations and species composition underscores the strong similarities between freshwater ecosystems in the north and south polar zones. Received: 3 June 1996 / Accepted: 3 November 1996     

Catalytic efficiency of HAP phytases is determined by a key residue in close proximity to the active site

The maximum activity of Yersinia enterocolitica phytase (YeAPPA) occurs at pH 5.0 and 45 °C, and notably, its specific activity (3.28 ± 0.24 U mg⁻¹) is 800-fold less than that of its Yersinia kristeensenii homolog (YkAPPA; 88% amino acid sequence identity). Sequence alignment and molecular modeling show that the arginine at position 79 (Arg79) in YeAPPA corresponding to Gly in YkAPPA as well as other histidine acid phosphatase (HAP) phytases is the only non-conserved residue near the catalytic site. To characterize the effects of the corresponding residue on the specific activities of HAP phytases, Escherichia coli EcAPPA, a well-characterized phytase with a known crystal structure, was selected for mutagenesis—its Gly73 was replaced with Arg, Asp, Glu, Ser, Thr, Leu, or Tyr. The results show that the specific activities of all of the corresponding EcAPPA mutants (17–2,400 U mg⁻¹) were less than that of the wild-type phytase (3,524 U mg⁻¹), and the activity levels were approximately proportional to the molecular volumes of the substituted residues’ side chains. Site-directed replacement of Arg79 in YeAPPA (corresponding to Gly73 of EcAPPA) with Ser, Leu, and Gly largely increased the specific activity, which further verified the key role of the residue at position 79 for determining phytase activity. Thus, a new determinant that influences the catalytic efficiency of HAP phytases has been identified.     

The photomechanic infrared receptor for the detection of forest fires in the beetle Melanophila acuminata (Coleoptera: Buprestidae)

We recorded from single units of individual sensilla of the thoracic infrared (IR) pit organs of Melanophila acuminata. When the organ was stimulated with a thermal radiator whose emission spectrum was similar to that of a typical forest fire, units responded phasically with up to seven spikes within 30–40 ms at a radiation power of 24 mW cm⁻². In the experiments all wavelengths shorter than 1.6 μm were excluded by a longpass IR filter. Response latencies were about 4 ms and initial impulse frequencies were up to 250 impulses per second (ips). A single spike could be generated even when stimulus duration was only 2 ms. Reduction of total radiation power from 24 mW cm⁻² to 5 mW cm⁻² resulted in increased response latencies of 5–6 ms and the occurrence of only two to three spikes. Initial impulse frequencies decreased to 125 ips. According to our physiological results and calculations, Melanophila should be able to detect a 10-hectare fire from a distance of 12 km. Mechanical stimuli also evoked responses of the IR sensilla. All present morphological and physiological findings lead to the conclusion that the IR receptors of Melanophila must function by means of a hitherto undescribed photomechanic mechanism. Accepted: 1 November 1997     

Crystal structure of yeast Sco1

The Sco family of proteins are involved in the assembly of the dinuclear Cu_A site in cytochrome c oxidase (COX), the terminal enzyme in aerobic respiration. These proteins, which are found in both eukaryotes and prokaryotes, are characterized by a conserved CXXXC sequence motif that binds copper ions and that has also been proposed to perform a thiol:disulfide oxidoreductase function. The crystal structures of Saccharomyces cerevisiae apo Sco1 (apo-ySco1) and Sco1 in the presence of copper ions (Cu–ySco1) were determined to 1.8- and 2.3-Å resolutions, respectively. Yeast Sco1 exhibits a thioredoxin-like fold, similar to that observed for human Sco1 and a homolog from Bacillus subtilis. The Cu–ySco1 structure, obtained by soaking apo-ySco1 crystals in copper ions, reveals an unexpected copper-binding site involving Cys181 and Cys216, cysteine residues present in ySco1 but not in other homologs. The conserved CXXXC cysteines, Cys148 and Cys152, can undergo redox chemistry in the crystal. An essential histidine residue, His239, is located on a highly flexible loop, denoted the Sco loop, and can adopt positions proximal to both pairs of cysteines. Interactions between ySco1 and its partner proteins yeast Cox17 and yeast COX2 are likely to occur via complementary electrostatic surfaces. This high-resolution model of a eukaryotic Sco protein provides new insight into Sco copper binding and function.     

Short-Term Exposure to Nickel Alters the Adult Rat Brain Antioxidant Status and the Activities of Crucial Membrane-Bound Enzymes: Neuroprotection by L-Cysteine

Nickel (Ni) is an environmental pollutant towards which human exposure can be both occupational (mainly through inhalation) and dietary (through water and food chain-induced bioaccumulation). The aim of this study was to investigate the effects of short-term Ni-administration (as NiCl₂, 13 mg/kg) on the adult rat whole brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), Na⁺,K⁺-ATPase, and Mg²⁺-ATPase; in addition, the potential effect of the co-administration of the antioxidant L-cysteine (Cys, 7 mg/kg) on the above parameters was studied. Twenty-eight male Wistar rats were divided into four groups: A (saline-treated control), B (Ni), C (Cys), and D (Ni and Cys). All rats were treated once daily with intraperitoneal injections of the tested compounds, for 1-week. Rats were sacrificed by decapitation and the above-mentioned parameters were measured spectrophotometrically. Rats treated with Ni exhibited a significant reduction in brain TAS (-47%, p < 0.001, BvsA) that was efficiently limited by the co-administration of Cys (-4%, p > 0.05, DvsA; +83%, p < 0.001, DvsB), while Cys (group C) had no effect on TAS. The rat brain AChE activity was found significantly increased by both Ni (+30%, p < 0.001, BvsA) and Cys (+62%, p < 0.001, CvsA), while it tended to adjust to control levels by the co-administration of Ni and Cys (+13%, p < 0.001, DvsA; −13%, p < 0.001, DvsB). The activity of rat brain Na⁺,K⁺-ATPase was significantly decreased by Ni-administration (−49%, p < 0.001, BvsA), while Cys supplementation could not reverse this decrease (-44%, p < 0.001, DvsA). The activity of Mg²⁺-ATPase was not affected by Ni-administration (−3%, p > 0.05, BvsA), but was significantly reduced when combined with Cys administration (−17%, p < 0.001, DvsA). The above findings suggest that Ni short-term in vivo administration causes a statistically significant decrease in the rat brain TAS and an increase in AChE activity. Both effects can be, partially or totally, reversed to control levels by Cys co-administration; Cys could thus be considered (for future applications) as a potential neuroprotective agent against chronic exposure to Ni. The activity of Na⁺,K⁺-ATPase that was inhibited by Ni, could not be reversed by Cys co-administration. The matter requires further investigation in order to fully elucidate the spectrum of the neurotoxic effects of Ni.     

<Emphasis Type="Italic">π</Emphasis>–<Emphasis Type="Italic">π</Emphasis> interaction between aromatic ring and copper-coordinated His81 imidazole regulates the blue copper active-site structure

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, π–π interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the π–π interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell π–π interaction. We suggest that the π–π interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and (15)N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His-->Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS*L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)(3) donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS*N donor set deriving from His46, Cys112 and Met121. The Cu-S(delta)(Met) distance is reduced from about 3.2 to 2.3 A. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the beta-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.     

The heme environment of mouse neuroglobin: histidine imidazole plane orientations obtained from solution NMR and EPR spectroscopy as compared with X-ray crystallography

The ¹H NMR chemical shifts of the heme methyl groups of the ferriheme complex of metneuroglobin (Du et al. in J. Am. Chem. Soc. 125:8080–8081, 2003) predict orientations of the axial histidine ligands (Shokhirev and Walker in J. Biol. Inorg. Chem. 3:581–594, 1998) that are not consistent with the X-ray data (Vallone et al. in Proteins Struct. Funct. Bioinf. 56:85–94, 2004), and the EPR spectrum (Vinck et al. in J. Am. Chem. Soc. 126:4516–4517, 2004) is only marginally consistent with these data. The reasons for these inconsistencies appear to be rooted in the high degree of aqueous solution exposure of the heme group and the fact that there are no strong hydrogen-bond acceptors for the histidine imidazole N–H protons provided by the protein. Similar inconsistencies may exist for other water-soluble heme proteins, and ¹H NMR spectroscopy provides a simple means to verify whether the solution structure of the heme center is the same as or different from that in the crystalline state.