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Oxygen-activating copper proteins may possess His-X(aa)-His chelating sequences at their active sites and additionally exhibit imidiazole group δN vs εN tautomeric preferences. As shown here, such variations strongly affect copper ion's coordination geometry, redox behavior, and oxidative reactivity. Copper(I) complexes bound to either δ-HGH or ε-HGH tripeptides were synthesized and characterized. Structural investigations using X-ray absorption spectroscopy, density functional theory calculations, and solution conductivity measurements reveal that δ-HGH forms the Cu(I) dimer complex [{Cu(I)(δ-HGH)}2](2+) (1) while ε-HGH binds Cu(I) to give the monomeric complex [Cu(I)(ε-HGH)](+) (2). Only 2 exhibits any reactivity, forming a strong CO adduct, [Cu(I)(ε-HGH)(CO)](+), with properties closely matching those of the copper monooxygenase PHM. Also, 2 is reactive toward O2 or H2O2, giving a new type of O2-adduct or Cu(II)-OOH complex, respectively.

CuA is a dinuclear mixed-valence center located in subunit 2 of the ba3 type cytochrome oxidase from Thermus thermophilus. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of CuA formation from inorganic ions. The formulation of the CuA center as an electron-delocalized Cu1.5 – Cu1.5 system, implicates both Cu(II) and Cu(I) states in the metalation process. In earlier work we showed that selenomethionine (SeM) substitution of the coordinated M160 residue provided a ligand-directed probe for studying the copper coordination environment via the Se XAS signal, which was particularly useful for interrogating the Cu(I) states where other spectroscopic probes are absent. In the present study we have investigated the formation of mixed-valence CuA and its M160SeM derivative by stopped-flow UV-vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu(I) CuA has been studied by XAS alone. Our results establish the presence of previously undetected mononuclear intermediates, and show important differences from the metalation reactions of purple CuA azurin. XAS spectroscopy at Cu and Se edges has allowed us to extend mechanistic inferences to formation of the di-Cu(I) state which may be more relevant to biological CuA assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems, and that the dominant intermediate along the pathway to mixed-valence is a new green species with λmax = 460 nm. This intermediate has been isolated in a homogeneous state, and shown to be a mononuclear Cu(II)-(His)(Cys)2 species with no observable Cu(II)-(Met) interaction. Reduction with dithionite generates its Cu(I) homologue which is again mononuclear, but now shows a strong interaction with the Met160 thioether. The results are discussed within the framework of (i) the “coupled distortion” model for Cu(II) thiolates, and (ii) their relevance to biological metalation reactions of the CuA center.

Copper transfer to cuproproteins located in vesicular compartments of the secretory pathway depends on activity of the copper translocating ATPase (ATP7A or ATP7B) but the mechanism of transfer is largely unexplored. Copper-ATPase ATP7A is unique in having a sequence rich in histidine and methionine residues located on the lumenal side of the membrane. The corresponding fragment binds Cu(I) when expressed as a chimera with a scaffold protein, and mutations or deletions of His and/or Met residues in its sequence inhibit dephosphorylation of the ATPase, a catalytic step associated with copper release. Here we present evidence for a potential role of this lumenal region of ATP7A in copper transfer to cuproenzymes. Both Cu(II) and Cu(I) forms were investigated since the form in which copper is transferred to acceptor proteins is currently unknown. Analysis of Cu(II) using EPR demonstrated that at Cu:P ratios below 1:1, 15N-substituted protein had Cu(II) bound by 4 His residues, but this coordination changed as the Cu(II) to protein ratio increased towards 2:1. XAS confirmed this coordination via analysis of the intensity of outer-shell scattering from imidazole residues. The Cu(II) complexes could be reduced to their Cu(I) counterparts by ascorbate, but here again, as shown by EXAFS and XANES spectroscopy, the coordination was dependent on copper loading. At low copper Cu(I) was bound by a mixed ligand set of His + Met while at higher ratios His coordination predominated. The copper-loaded loop was able to transfer either Cu(II) or Cu(I) to peptidylglycine monooxygenase in the presence of chelating resin, generating catalytically active enzyme in a process that appeared to involve direct interaction between the two partners. The variation of coordination with copper loading suggests copper-dependent conformational change which in turn could act as a signal for regulating copper release by the ATPase pump.

Interactions of the axial ligand with its blue copper center are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of the blue copper center with a weak axial ligand to a green copper center containing a medium strength axial ligand has been demonstrated in cupredoxins, converting the blue copper center to a red copper center with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (R(L)) of absorption at 447 nm over that at 621 nm. Whereas no axial Cu-S(Cys121) interaction in Met121Cys was detectable by extended X-ray absorption fine structure (EXAFS) spectroscopy at pH 5, similar to what was observed in native azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 A is clearly visible at higher pH. Despite the higher R(L) and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A( parallel) = 54 x 10(-4) cm(-1) at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A(parallel) = 87 x 10(-4) cm(-1) at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with a nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (R(L)= 1.5, and A(parallel) = 180 x 10(-4) cm(-1)) and Cu-S(Cys) distance (2.22 A) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys or homocysteine resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 +/- 3 mV for Met121Cys azurin and 113 +/- 6 mV for Met121Hcy azurin at pH 7. The results strongly support the "coupled distortion" model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the wild-type (WT) protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/visible, Cu and S K-edge X-ray absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form, which is shown by extended X-ray absorption fine structure to be composed of a novel two-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49, H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans, which facilitates the formation of the highly stable two-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at approximately 2.24 A corresponding to the two thiolate-copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at approximately 2.16 A. S K-edge data give a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than that observed for nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, from which it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron-transfer reactivity.

Cytochrome oxidase is the terminal oxidase in both prokaryotic and eukaryotic cells and is responsible for the generation of cellular energy via the process known as oxidative phosphorylation. The enzyme contains two Fe and three Cu centers which together provide the redox machinery for the reduction of O2 to water. Recently, X-ray crystallography has provided the first three-dimensional description of the coordination spheres of the metal centers. However, the structures show the metal sites at low resolution, and in order to fully understand the mechanism of the reaction, it is desirable to determine the metrical details (bond lengths and angles) to much higher precision. X-ray absorption spectroscopy is unique in its ability to provide such detail, and we have applied the technique to determining the structure of the CuA center, a thiolate-bridged binuclear copper cluster in which the coppers are bridged by two cysteine ligands and have an extremely short Cu−Cu distance of ∼2.4 A. X-ray absorption spec...

In this study on model compounds for the iron−copper dinuclear center in heme-copper oxidases, we (i) detail the synthesis and reversible acid−base interconversion of μ-oxo and μ-hydroxo complexes [(F8-TPP)FeIII−(O2-)−CuII(TMPA)]+ (1) and [(F8-TPP)FeIII−(OH-)−CuII(TMPA)]2+ (2) [F8-TPP = tetrakis(2,6-difluorophenyl)porphyrinate(2−), TMPA = tris[(2-pyridylmethyl)amine]; (ii) compare their physical properties; (iii) establish the structure of 2 using XAS (X-ray absorption spectroscopy), a novel application of a three-body two-edge multiple-scattering (MS) analysis of ligand connectivity; and (iv) compare the XAS of 2 with those of 1 and an enzyme preparation. Complex 1 was prepared by reaction of [(TMPA)CuII(CH3CN)]2+ (3) and [(F8-TPP)FeIII−OH] (4) with triethylamine in acetonitrile (>70% yield). Salts 2-(ClO4)2 and 2-(CF3SO3)2 were synthesized (>60% yield) by addition of 3 with 4 in dichloroethane or by protonation of 1 with triflic acid. In a 1H-NMR spectroscopic titration (298 K) with triflic acid, the py...

The use of isocyanides as ligand-directed probes of Cu(I) coordination in proteins has been investigated. Reaction of 2,6-dimethylphenyl isocyanide (DIMPI) with reduced dopamine-β-monooxygenase (DβM) indicates the initial formation of monoisocyanide complexes at each of the two coppers (Cu A and Cu B ) with different frequencies (2148 and 2129 cm -1 ) indicative of inequivalent Cu(I) coordination at each copper. However, further addition of DIMPI leads to formation of a species containing multiple isocyanide ligands, believed to be a trisisocyanide adduct with a single IR band at 2160 cm -1 . This titration behavior can be interpreted by the active site model Cu A I (His) 2 X�"CuB I (His) 2 Y (X = His; Y = Met) where the first stage of the reaction with isocyanide is the formation of a mono-DIMPI four-coordinate complex at each Cu, giving rise to the two observed IR bands (2148 and 2129 cm -1 ) provided the protein ligands X and Y are different. The second stage is the displacement of protein-bound ligands by the isocyanide to form a protein-bound bis or tris complex (2160 cm -1 ). Closely analogous chemistry involving the reaction of DIMPI with deoxyHc is described, which illustrates the generality of isocyanides as probes of Cu(I) coordination in copper proteins. A model system [Cu I (MePY2)(DIMPI)]ClO4, II, is also described in which identical isocyanide-binding chemistry can be demonstrated, thus validating the conclusions on the protein systems. The crystal structure of II is described, and the clean conversion of II to a trisisocyanide complex is demonstrated by FTIR and FT Raman spectroscopy. © 1995, American Chemical Society. All rights reserved.

Progress has been made in determining the individual coordination of each of the copper sites (Cu A and Cu B ) which comprise the active center in dopamine-β-hydroxylase. Previous studies have determined the average ligand environment per copper in the fully metalated enzyme as two to three histidines and one to two O/N donors in the Cu(II) form changing to 2-3 histidines and 0.5 sulfur donors upon reduction to the Cu(I) form. Derivatives of the Cu(I) form of DBH have been made in which CuA has been selectively removed, allowing Cu B , the O 2 -binding center to be studied by EXAFS and FTIR

Studies of copper complexes with the 1,2-dimethylimidazole (Me[sub 2]im) system have provided insights into the factors which control dioxygen (O[sub 2]) binding and activation in imidazole (histidine) ligated copper complexes and proteins. A two-coordinate complex [Cu(Me[sub 2]im)[sub 2]](PF[sub 6]) (1(PF[sub 6])) is formed by the reaction of 1,2-dimethylimidazole with [Cu(CH[sub 3]CN)[sub 4]](PF[sub 6]). Although 1 is unreactive toward O[sub 2] or CO, reaction with one additional molar equivalent of Me[sub 2]im yields a three-coordinate complex [Cu(Me[sub 2]im)[sub 3]](PF[sub 6])(2(PF[sub 6])) which reacts with O[sub 2](Cu/O[sub 2] = 2:1, manometry), producing the EPR silent dioxygen adduct, formulated as [Cu[sub 2](Me[sub 2]im)[sub 6](O[sub 2])][sup 2+] (3). The structure of 1 has been studied by X-ray crystallography; it crystallizes in the monoclinic space group C2/c with Z = 4, a = 14.877 (2) [angstrom], b = 15.950 (4) [angstrom], c = 6.931 (4) [angstrom], and [beta] = 108.54 (2)[degrees]. The linear two-coordinate Cu(I) structure is typical and contains crystallographically equivalent Cu-N(imid) distances of 1.865 [angstrom]. The structures of 2 and 3 have been studied by X-ray absorption spectroscopy, using imidazole group-fitting and full curved-wave multiple scattering analysis. Complex 2 is best fit by a T-shaped structure involving two short (1.89 [angstrom]) and onemore » longer (2.08 [angstrom]) Cu-N(imid) distances. Absorption edge data confirm that the dioxygen complex 3 should be formulated as a Cu(II)-peroxo species. The EXAFS of 3 can be fit by either of two models, A and B. 50 refs., 9 figs., 3 tabs.« less

Sco1 is a mitochondrial membrane protein involved in the assembly of the CuA site of cytochrome c oxidase. The Bacillus subtilis genome contains a homologue of yeast Sco1, YpmQ (hereafter termed BSco), deletion of which leads to a phenotype lacking in caa3 (CuA-containing) oxidase activity but expressing normal levels of aa3 (quinol) oxidase activity. Here, we report the characterization of the metal binding site of BSco in its Cu(I)-, Cu(II)-, Zn(II)-, and Ni(II)-bound forms. Apo BSco was found to bind Cu(II), Zn(II), and Ni(II) at a 1:1 protein/metal ratio. The Cu(I) protein could be prepared by either dithionite reduction of the Cu(II) derivative or by reconstitution of the apo protein with Cu(I). X-ray absorption (XAS) spectroscopy showed that Cu(I) was coordinated by two cysteines at 2.22 +/- 0.01 A and by a weakly bound low-Z scatterer at 1.95 +/- 0.03 A. The Cu(II) derivative was reddish-orange and exhibited a strong type-2 thiolate to Cu(II) transition around 350 nm. Multifrequency electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and electron spin-echo envelope modulation (ESEEM) studies on the Cu(II) derivative provided evidence of one strongly coupled histidine residue, at least one strongly coupled cysteine, and coupling to an exchangeable proton. XAS spectroscopy indicated two cysteine ligands at 2.21 A and two O/N donor ligands at 1.95 A, at least one of which is derived from a coordinated histidine. The Zn(II) and Ni(II) derivatives were 4-coordinate with MS2N(His)X coordination. These results provide evidence that a copper chaperone can engage in redox chemistry at the metal center and may suggest interesting redox-based mechanisms for metalation of the mixed-valence CuA center of cytochrome c oxidase.

Azurin is a small electron-transfer protein belonging to the cupredoxin family. The Cu atom is located within a trigonal plane coordinated by two histidines (His46 and His117) and a cysteine (Cys112) with two more distant ligands (Gly45 and Met121) providing axial interactions. A Cys112SeCys derivative has been prepared by expressed protein ligation, and detailed UV/vis, EPR and EXAFS studies at the Cu and Se K-edges have been carried out. Marked changes are observed between the EPR parameters of the Cys112SeCys and WT azurin derivatives, which include a 2-fold increase in A(||), a decrease in g-values, and a large increase in rhombicity of the g-tensor. The Cu-Se and Se-Cu bond lengths obtained from analysis of the Cu and Se K-EXAFS of the oxidized protein were found to be 2.30 and 2.31 A, respectively, 0.14 A longer than the Cu-S distance of the WT protein. Unexpectedly, the Cu-Se bond lengths were found to undergo only minor changes during reduction, suggesting a very similar structure in both redox states and extending the "rack" hypothesis to the Se-substituted protein.

Expressed protein ligation was used to replace the axial methionine of the blue copper protein azurin from Pseudomonas aeruginosa with unnatural amino acids. The highly conserved methionine121 residue was replaced with the isostructural amino acids norleucine (Nle) and selenomethionine (SeM). The UV-visible absorption, X- and Q-band EPR, and Cu EXAFS spectra of the variants are slightly perturbed from WT. All variants have a predominant S(Cys) to Cu(II) charge transfer band around 625 nm and narrow EPR hyperfine splittings. The Se EXAFS of the M121SeM variant is also reported. In contrast to the small spectral changes, the reduction potentials of M121SeM, M121Leu, and M121Nle are 25, 135, and 140 mV, respectively, higher than that of WT azurin. The use of unnatural amino acids allowed deconvolution of different factors affecting the reduction potentials of the blue copper center. A careful analysis of the WT azurin and its variants obtained in this work showed the large reduction potential variation was linearly correlated with the hydrophobicity of the axial ligand side chains. Therefore, hydrophobicity is the dominant factor in tuning the reduction potentials of blue copper centers by axial ligands.

Modified His−His dipeptides have been reacted with copper(I) salts to model active-site Cu ions bound by contiguous His residues in certain oxygen-activating copper proteins, as well as amyloid β-peptide. Chelation of copper(I) by these ligands affords linear, two-coordinate complexes as studied structurally by X-ray absorption spectroscopy. The complexes are robust toward oxidation, showing limited to no reactivity with O2, and they bind CO weakly. Reaction with a third ligand (N-methylimidazole) affords complexes with a markedly different structure (distorted T-shaped) and reactivity, binding CO and oxidizing rapidly upon exposure to dioxygen.

The temperature dependence of the primary and secondary intrinsic isotope effects for the C-H bond cleavage catalyzed by peptidylglycine alpha-hydroxylating monooxygenase has been determined. Analysis of the magnitude and Arrhenius behavior of the intrinsic isotope effects provides strong evidence for the use of tunneling as a primary catalytic strategy for this enzyme. Modeling of the isotope effect data allows for a comparison to the hydrogen transfer catalyzed by soybean lipoxygenase in terms of environmental reorganization energy and frequency of the protein vibration that controls the hydrogen transfer.

Despite its prevalence in transition metal chemistry in general, to our knowledge the doubly-bridged M{sub 2}({mu}-SR){sub 2} motif is unknown for M = Cu(II), presumably because of the tendency for operation of eq 2Cu(II) + 2SR{sup -1} {yields} 2Cu(I) + RSSR, for which the appropriate stoichiometry would be provided in attempted assembly reactions. Herein we report the use of a new, sterically hindered ligand for the isolation of the first example of such a complex, which has been subjected to structural investigations oriented toward relating its properties to those of the CcO and N{sub 2}OR electron transfer sites. 21 refs., 2 figs.

Preparation et proprietes du complexe [Cu 2 (XYL-O-)(OOH)] 2+ structure cristalline de [Cu 2 (XYL-OH) (PPh 3 ) 2 ] (ClO 4 ) 2