Role of the Surface-Exposed and Copper-Coordinating Histidine in Blue Copper Proteins: The Electron-Transfer and Redox-Coupled Ligand Binding Properties of His117Gly Azurin (original) (raw)
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JBIC Journal of Biological Inorganic Chemistry, 2007
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, p-p 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 p-p 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 elec-tronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell p-p interaction. We suggest that the p-p 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.
Inorganic Chemistry, 2014
His-tags are specific sequences containing six to nine subsequent histydyl residues, and they are used for purification of recombinant proteins by use of IMAC chromatography. Such polyhistydyl tags, often used in molecular biology, can be also found in nature. Proteins containing histidine-rich domains play a critical role in many life functions in both prokaryote and eukaryote organisms. Binding mode and the thermodynamic properties of the system depend on the specific metal ion and the histidine sequence. Despite the wide application of the His-tag for purification of proteins, little is known about the properties of metal-binding to such tag domains. This inspired us to undertake detailed studies on the coordination of Cu 2+ ion to hexa-His-tag. Experiments were performed using the potentiometric, UV−visible, CD, and EPR techniques. In addition, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were applied. The experimental studies have shown that the Cu 2+ ion binds most likely to two imidazoles and one, two, or three amide nitrogens, depending on the pH. The structures and stabilities of the complexes for the Cu 2+ -Ac-(His) 6 -NH 2 system using experimental and computational tools were established. Polymorphic binding states are suggested, with a possibility of the formation of α-helix structure induced by metal ion coordination. Metal ion is bound to various pairs of imidazole moieties derived from the tag with different efficiencies. The coordination sphere around the metal ion is completed by molecules of water. Finally, the Cu 2+ binding by Ac-(His) 6 -NH 2 is much more efficient compared to other multihistidine protein domains.
Journal of Inorganic Biochemistry, 2011
The systematic electrochemical studies of the copper complexes of various terminally protected tri-, tetra-, penta-and heptapeptides containing histidine in different location and number in the peptide chain and two histidine derivatives were carried out by cyclic voltammetry. The redox parameters of CuL and CuL 2 complexes coordinating exclusively through imidazole nitrogens were determined. For all studied Cu(II) complexes the characteristic redox reactions are quasi-reversible one electron reduction processes. The obtained formal reduction potential values fall into the 200-400 mV potential range supporting the former results that the CuL and CuL 2 complexes of these multihistidine peptides are not only structural but also good functional models of the Cu-Zn-superoxide dismutase (CuZnSOD) enzyme. These observations are confirmed by the results of SOD activity assay in a representative copper(II)-ligand system.
Central European Journal of Chemistry, 2012
An important function of many copper-containing proteins is activation of O2 and subsequent substrate oxidation. The Cu (III) oxidation state is generally considered to be less accessible because of the highly positive Cu (III)/Cu (II) redox potentials with typical amino acid ligands. Here, we employ density functional (DFT) calculations to explore to what extent copper (III) may be accessed in a biologically-relevant coordination environment around a mononuclear copper center, by breaking the oxygen-oxygen bond in a copper-(hydro) peroxide complex. In agreement with previous findings by Solomon and co-workers on copper models with related coordination patterns, the formally high-valent copper complex produced by O-O bond cleavage appears to harbor both oxidizing equivalents on the ligands. The potential energy surface for such a reaction reveals that with the three-histidine binding motif at the copper, O-O bond cleavage is not impossible, but rather disfavored thermodynamically.
Biochemistry, 1996
A pH-dependent X-ray absorption fine structure (XAFS) study has been undertaken to provide a structural interpretation of the spectroscopic properties of the Met121Glu mutant of azurin from Pseudomonas aeruginosa (Az p). Ligand binding studies have been carried out to investigate the effect of the cavity formed at the Cu site as a result of the mutation. The optical spectrum at pH 4 exhibits an intense band at ∼600 nm and a weaker band at ∼450 nm, typical for the blue copper proteins. As the pH is increased, these bands decrease in intensity and shift to 570 and 413 nm, respectively, with the latter becoming the more intense of the two [Karlsson, B. G., et al. (1991) Protein Eng. 4 (3), 343-349]. These changes are accompanied by a change in the EPR spectrum from a rhombic type 1 Cu spectrum at pH 4 to a spectrum with the rhombic splitting decreasing to zero and the hyperfine coupling increasing from 25 to 83 G. X-ray absorption at the Cu K-edge shows that this change results from the lengthening of the Cu-His (by 0.07 Å) and Cu-Cys (by 0.06 Å) bonds and the coordination of one of the oxygen atoms of the glutamate ligand at pH 8, at a distance as close as 1.90 Å. The copper site thus changes from a normal type 1 copper center with three strong bonds at pH 4 to a copper site with four strong bonds at pH 8, with Cu-His distances significantly longer than known distances for type 1 copper centres measured using the XAFS technique. The XAFS of the azide derivative measured at pH 8 shows a similar Cu coordination, with azide replacing glutamate as the fourth ligand. Azide binding at pH 8 is accompanied by a further increase in the EPR hyperfine coupling to 110 G. This structural information when taken together with recent structural sudies on copper proteins points toward the need for a reexamination of the basis on which copper proteins are classified.
European Journal of Biochemistry, 1993
The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5 -8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-+Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK, 9-10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR gll value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation. Fax: +31 71 274537. Abbreviations. Ches, 2-(N-cyclohexylamino)ethmesulfonic acid; Cu(1) azurin, reduced azurin; Cu(I1) azurin, oxidized azurin; Em, midpoint potential ; ESE, electron self-exchange ; ESEEM, electron spin-echo envelope modulation; M44K, Met44+Lys ; RR, resonance Raman; wt, wild type; FT, Fourier transform. due His117 is located in the centre of this patch. The replacement of Met44 by the protonated lysine residue hardly affects the spectroscopic properties of the Cu site, but causes a considerable decrease of the k,,, value of azurin. At pH >8, deprotonation of Lys44 produces a new type-] Cu site, whereas the magnitude of the k,,, value of the protein is largely restored. At pH 5 and pH 8, the midpoint potential, Em, of Met44+Lys (M44K) azurin is higher than the wildtype (wt) azurin Em by 60 mV. At pH > 8, deprotonation of Lys44 in M44K azurin reduces the En, difference from 60 mV to 40 mV. The pH dependence of the wt and M44K azurin En, values is analyzed in terms of the titration of His35, His83 and Lys44. In addition, the effects of the M44K mutation and the Cu-ion oxidation state (I or TI) on the pK, values of the titratable His35 and His83 of azurin are reported and analyzed. The structural and functional implications of the findings are discussed.
Journal of Inorganic Biochemistry, 2007
The systematic investigation of the copper(II) complexes of tripeptides Xaa-Xaa-His, Xaa-His-Xaa and His-Xaa-Xaa, where Xaa = Gly or Ala was performed by combined pH-metry, spectrophotometry, CD and in part EPR spectroscopy. The matrix rank analysis of the spectral data revealed the number of the coloured and optically active species as a basis for the solution speciation. A critical evaluation on the speciation and solution structure of the complexes formed is presented on the basis of their d-d band optical activity. The replacement of a Gly residue with the chiral Ala amino acid allowed us to gain decisive information on the solution structure of the complexes by CD spectroscopy. It was shown that the tripeptides with histidine in the third position formed CuH À2 L species with (NH 2 , 2N À , ImN-where Im stands for imidazole) coordination sphere as a major species, and only the macrochelated CuL complexes as minor species around pH 5.0. In copper(II)-Xaa-His-Xaa tripeptide systems the CuH À1 L (NH 2 , N À , ImN) is the most stable species at physiological pH, but the vacant fourth site around copper(II)ions is offered for further deprotonation, most probably resulting in mixed hydroxo species at low (<5 • 10 À4 M) metal ion concentrations, while a tetrameric complex is dominant when the copper concentration exceeds 3 • 10 À3 M. The histamine type coordination mode in CuL and CuL 2 complexes of His-Xaa-Xaa ligands predominates at low pH. The structural consequences drawn from the CD spectra for the mono and bis parent complexes were supported by theoretical calculations. CD spectra strongly suggest the participation of the imidazole nitrogen both in the Cu 2 H À2 L 2 and CuH À2 L complexes.
Inorganica Chimica Acta, 2005
The macrocycles L 1 -L 3 having N 2 S 2 O-, N 2 S 2 -, and N 2 S 3 -donor sets, respectively, and incorporating the 1,10-phenanthroline unit interact in EtOH and MeCN solutions with Cu II to give 1:1 [M(L)] 2+ complex species. The compounds [Cu(L 1 )(ClO 4 )]ClO 4 (1), [Cu(L 2 )(ClO 4 )]ClO 4 AE 1 2 H 2 O (2) and [Cu(L 3 )](ClO 4 ) 2 (3) were isolated at the solid state and the first two also characterised by Xray diffraction studies. The conformation adopted by L 1 and L 2 in the cation complexes reveals the aliphatic portion of the rings folded over the plane containing the heteroaromatic moiety with the ligands encapsulating the metal centre within their cavity by imposing, respectively, a square-based pyramidal and a square planar geometry. In both complexes, the metal ion completes its coordination sphere by interacting with a ClO 4 À ligand. The compound [Cu(L 3 ) 2 ](PF 6 ) 2 (4) containing a 1:2 cation complex was also isolated at the solid state: EPR spectroscopy measurements suggest the presence of a CuN 4 chromophore in this complex. The EPR and electronic spectral features of 1-4 have been studied and their redox properties examined in comparison with those observed for Type-1 blue copper proteins.
Tetrahedron: Asymmetry, 1999
The ligand piperazine-1,4-bis[4-(N-(1-acetoxy-3-(1-methyl-1H-imidazol-4-yl))-2-propyl)-N-(1-methyl-1Himidazol-2-ylmethyl)aminobutyl] (PHI) was synthesized by a multistep procedure starting from N τ -methyl-Lhistidine, piperazine-1,4-bis[4-(4-oxo-4-butanoic) acid] and 1-methyl-1H-imidazole-2-carbaldehyde. This ligand has two potential tridentate, aminobis(imidazole) (A sites), and one bidentate, piperazine (B site), binding sites for metal ions and was employed for the synthesis of the binuclear [Cu 2 PHI] 4+ and the trinuclear [Cu 3 PHI] 6+ complexes, the latter of which features a coordination environment mimicking that present in the trinuclear clusters of the blue copper oxidases. For comparison purposes, the mononucleating ligand L-N α -(1-methyl-1Himidazol-2-ylmethyl)-N τ -methylhistidine methyl ester (IH) and its complex [CuIH] 2+ have been also prepared. These copper(II) model complexes are the first reported which are directly derived from chiral L-histidine residues. A detailed analysis of the UV-vis, CD and EPR spectra of the complexes has established that the Cu(II) centers bound to PHI A sites are square-pyramidal in solution, with the amino and one imidazole donor in the equatorial plane and the additional imidazole group bound axially. This arrangement implies the adoption of an unusual conformation of λ chirality by the L-histidine residue and is determined by the attempts to minimize steric interference between the substituents at the tertiary amine donor group and the histidine residue bearing the C-α substituent acetoxymethylene group of the bound PHI ligand. For the less sterically crowded secondary amine group of the bound IH ligand, the histidine C-α substituent can occupy a pseudoaxial position, so that in the complex [CuIH] 2+ the 'normal' arrangement with three equatorial nitrogen donors and δ chirality in the L-histidine chelate ring occurs.