Structure of the Reduced Copper Active Site in Pre-Processed Galactose Oxidase: Ligand Tuning for One-Electron O2 Activation in Cofactor Biogenesis

Galactose oxidase (GO) is a copper-dependent enzyme that accomplishes 2e- substrate oxidation by pairing a single copper with an unusual cysteinylated tyrosine (Cys-Tyr) redox cofactor. Previous studies have demonstrated that the post-translational biogenesis of Cys-Tyr is copper- and O2-dependent, resulting in a self-processing enzyme system. To investigate the mechanism of cofactor biogenesis in GO, the active-site structure of Cu(I)-loaded GO was determined using X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy, and density-functional theory (DFT) calculations were performed on this model. Our results show that the active-site tyrosine lowers the Cu potential to enable the thermodynamically unfavorable 1e- reduction of O2, and the resulting Cu(II)-O2¿- is activated toward H atom abstraction from cysteine. The final step of biogenesis is a concerted reaction involving coordinated Tyr ring deprotonation where Cu(II) coordination enables formation of the Cys-Tyr cross-link. These spectroscopic and computational results highlight the role of the Cu(I) in enabling O2 activation by 1e- and the role of the resulting Cu(II) in enabling substrate activation for biogenesis

Spectroscopic and Theoretical Study of CuI Binding to His111 in the Human Prion Protein Fragment 106-115

The ability of the cellular prion protein (PrPC) to bind copper in vivo points to a physiological role for PrPC in copper transport. Six copper binding sites have been identified in the nonstructured N-terminal region of human PrPC. Among these sites, the His111 site is unique in that it contains a MKHM motif that would confer interesting CuI and CuII binding properties. We have evaluated CuI coordination to the PrP(106-115) fragment of the human PrP protein, using NMR and X-ray absorption spectroscopies and electronic structure calculations. We find that Met109 and Met112 play an important role in anchoring this metal ion. CuI coordination to His111 is pH-dependent: at pH >8, 2N1O1S species are formed with one Met ligand; in the range of pH 5-8, both methionine (Met) residues bind to CuI, forming a 1N1O2S species, where N is from His111 and O is from a backbone carbonyl or a water molecule; at pH <5, only the two Met residues remain coordinated. Thus, even upon drastic changes in the chemical environment, such as those occurring during endocytosis of PrPC (decreased pH and a reducing potential), the two Met residues in the MKHM motif enable PrPC to maintain the bound CuI ions, consistent with a copper transport function for this protein. We also find that the physiologically relevant CuI-1N1O2S species activates dioxygen via an inner-sphere mechanism, likely involving the formation of a copper(II) superoxide complex. In this process, the Met residues are partially oxidized to sulfoxide; this ability to scavenge superoxide may play a role in the proposed antioxidant properties of PrPC. This study provides further insight into the CuI coordination properties of His111 in human PrPC and the molecular mechanism of oxygen activation by this site.Fil: Arcos López, Trinidad. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Qayyum, Munzarin. University of Stanford; Estados UnidosFil: Rivillas Acevedo, Lina. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Miotto, Marco César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Laboratory for Structural Biology; ArgentinaFil: Grande Aztatzi, Rafael. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Fernandez, Claudio Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario. Universidad Nacional de Rosario. Instituto de Investigaciones para el Descubrimiento de Fármacos de Rosario; Argentina. Max Planck Laboratory for Structural Biology; ArgentinaFil: Hedman, Britt. University of Stanford; Estados UnidosFil: Hodgson, Keith O.. University of Stanford; Estados UnidosFil: Vela, Alberto. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; MéxicoFil: Solomon, Edward I.. University of Stanford; Estados UnidosFil: Quintanar, Liliana. Instituto Politécnico Nacional. Centro de Investigación y de Estudios Avanzado; Méxic

Copper active sites in biology

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Copper active sites in biology

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Structure of the Reduced Copper Active Site in Pre-Processed Galactose Oxidase: Ligand Tuning for One-Electron O2 Activation in Cofactor Biogenesis

Galactose oxidase (GO) is a copper-dependent enzyme that accomplishes 2e- substrate oxidation by pairing a single copper with an unusual cysteinylated tyrosine (Cys-Tyr) redox cofactor. Previous studies have demonstrated that the post-translational biogenesis of Cys-Tyr is copper- and O2-dependent, resulting in a self-processing enzyme system. To investigate the mechanism of cofactor biogenesis in GO, the active-site structure of Cu(I)-loaded GO was determined using X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy, and density-functional theory (DFT) calculations were performed on this model. Our results show that the active-site tyrosine lowers the Cu potential to enable the thermodynamically unfavorable 1e- reduction of O2, and the resulting Cu(II)-O2¿- is activated toward H atom abstraction from cysteine. The final step of biogenesis is a concerted reaction involving coordinated Tyr ring deprotonation where Cu(II) coordination enables formation of the Cys-Tyr cross-link. These spectroscopic and computational results highlight the role of the Cu(I) in enabling O2 activation by 1e- and the role of the resulting Cu(II) in enabling substrate activation for biogenesis

Spectroscopic and Crystallographic Characterization of "Alternative Resting" and "Resting Oxidized" Enzyme Forms of Bilirubin Oxidase: Implications for Activity and Electrochemical Behavior of Multicopper Oxidases

4 pagesInternational audienceWhile there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu−Cu distance, while the alternative resting form shows unusually small A∥ and high g∥ EPR features, lack of 330 nm absorption intensity, and a long T3 Cu−Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis

Geometric and Electronic Structure of [{Cu(MeAN)}₂(μ-η²:η²(O₂^2–))]²⁺ with an Unusually Long O–O Bond: O–O Bond Weakening vs Activation for Reductive Cleavage

Certain side-on peroxo-dicopper­(II) species with particularly low ν_O–O (710–730 cm^–1) have been found in equilibrium with their bis-μ-oxo-dicopper­(III) isomer. An issue is whether such side-on peroxo bridges are further activated for O–O cleavage. In a previous study (Liang, H.-C., et al. <i>J. Am. Chem. Soc.</i> <b>2002</b>, <i>124</i>, 4170), we showed that oxygenation of the three-coordinate complex [Cu^I(MeAN)]⁺ (MeAN = <i>N</i>-methyl-<i>N</i>,<i>N</i>-bis­[3-(dimethylamino)­propyl]­amine) leads to a low-temperature stable [{Cu^II(MeAN)}₂(μ-η²:η²-O₂^2–)]²⁺ peroxo species with low ν_O–O (721 cm^–1), as characterized by UV–vis absorption and resonance Raman (rR) spectroscopies. Here, this complex has been crystallized as its SbF₆^– salt, and an X-ray structure indicates the presence of an unusually long O–O bond (1.540(5) Å) consistent with the low ν_O–O. Extended X-ray absorption fine structure and rR spectroscopic and reactivity studies indicate the exclusive formation of [{Cu^II(MeAN)}₂(μ-η²:η²-O₂^2–)]²⁺ without any bis-μ-oxo-dicopper­(III) isomer present. This is the first structure of a side-on peroxo-dicopper­(II) species with a significantly long and weak O–O bond. DFT calculations show that the weak O–O bond results from strong σ donation from the MeAN ligand to Cu that is compensated by a decrease in the extent of peroxo to Cu charge transfer. Importantly, the weak O–O bond does not reflect an increase in backbonding into the σ* orbital of the peroxide. Thus, although the O–O bond is unusually weak, this structure is not further activated for reductive cleavage to form a reactive bis-μ-oxo dicopper­(III) species. These results highlight the necessity of understanding electronic structure changes associated with spectral changes for correlations to reactivity

L‑Edge X‑ray Absorption Spectroscopy and DFT Calculations on Cu₂O₂ Species: Direct Electrophilic Aromatic Attack by Side-on Peroxo Bridged Dicopper(II) Complexes

The hydroxylation of aromatic substrates catalyzed by coupled binuclear copper enzymes has been observed with side-on-peroxo-dicopper­(II) (<b>P</b>) and bis-μ-oxo-dicopper­(III) (<b>O</b>) model complexes. The substrate-bound-<b>O</b> intermediate in [Cu­(II)₂(DBED)₂(O)₂]²⁺ (DBED = <i>N</i>,<i>N</i>′-di-<i>tert</i>-butyl-ethylenediamine) was shown to perform aromatic hydroxylation. For the [Cu­(II)₂(NO₂-XYL)­(O₂)]²⁺ complex, only a <b>P</b> species was spectroscopically observed. However, it was not clear whether this O–O bond cleaves to proceed through an <b>O</b>-type structure along the reaction coordinate for hydroxylation of the aromatic xylyl linker. Accurate evaluation of these reaction coordinates requires reasonable quantitative descriptions of the electronic structures of the <b>P</b> and <b>O</b> species. We have performed Cu L-edge XAS on two well-characterized <b>P</b> and <b>O</b> species to experimentally quantify the Cu 3d character in their ground state wave functions. The lower per-hole Cu character (40 ± 6%) corresponding to higher covalency in the <b>O</b> species compared to the <b>P</b> species (52 ± 4%) reflects a stronger bonding interaction of the bis-μ-oxo core with the Cu­(III) centers. DFT calculations show that 10–20% Hartree–Fock (HF) mixing for <b>P</b> and ∼38% for <b>O</b> species are required to reproduce the Cu–O bonding; for the <b>P</b> species this HF mixing is also required for an antiferromagnetically coupled description of the two Cu­(II) centers. B3LYP (with 20% HF) was, therefore, used to calculate the hydroxylation reaction coordinate of <b>P</b> in [Cu­(II)₂(NO₂-XYL)­(O₂)]²⁺. These experimentally calibrated calculations indicate that the electrophilic attack on the aromatic ring does not involve formation of a Cu­(III)₂(O^2–)₂ species. Rather, there is direct electron donation from the aromatic ring into the peroxo σ* orbital of the Cu­(II)₂(O₂^2–) species, leading to concerted C–O bond formation with O–O bond cleavage. Thus, species <b>P</b> is capable of direct hydroxylation of aromatic substrates without the intermediacy of an <b>O</b>-type species

Geometric and Electronic Structure of [{Cu(MeAN)}₂(μ-η²:η²(O₂^2–))]²⁺ with an Unusually Long O–O Bond: O–O Bond Weakening vs Activation for Reductive Cleavage

Certain side-on peroxo-dicopper­(II) species with particularly low ν_O–O (710–730 cm^–1) have been found in equilibrium with their bis-μ-oxo-dicopper­(III) isomer. An issue is whether such side-on peroxo bridges are further activated for O–O cleavage. In a previous study (Liang, H.-C., et al. <i>J. Am. Chem. Soc.</i> <b>2002</b>, <i>124</i>, 4170), we showed that oxygenation of the three-coordinate complex [Cu^I(MeAN)]⁺ (MeAN = <i>N</i>-methyl-<i>N</i>,<i>N</i>-bis­[3-(dimethylamino)­propyl]­amine) leads to a low-temperature stable [{Cu^II(MeAN)}₂(μ-η²:η²-O₂^2–)]²⁺ peroxo species with low ν_O–O (721 cm^–1), as characterized by UV–vis absorption and resonance Raman (rR) spectroscopies. Here, this complex has been crystallized as its SbF₆^– salt, and an X-ray structure indicates the presence of an unusually long O–O bond (1.540(5) Å) consistent with the low ν_O–O. Extended X-ray absorption fine structure and rR spectroscopic and reactivity studies indicate the exclusive formation of [{Cu^II(MeAN)}₂(μ-η²:η²-O₂^2–)]²⁺ without any bis-μ-oxo-dicopper­(III) isomer present. This is the first structure of a side-on peroxo-dicopper­(II) species with a significantly long and weak O–O bond. DFT calculations show that the weak O–O bond results from strong σ donation from the MeAN ligand to Cu that is compensated by a decrease in the extent of peroxo to Cu charge transfer. Importantly, the weak O–O bond does not reflect an increase in backbonding into the σ* orbital of the peroxide. Thus, although the O–O bond is unusually weak, this structure is not further activated for reductive cleavage to form a reactive bis-μ-oxo dicopper­(III) species. These results highlight the necessity of understanding electronic structure changes associated with spectral changes for correlations to reactivity