Noncoupled Copper Research Articles

Investigation of the Hydroxylation Mechanism of Noncoupled Copper Oxygenases by Ab Initio Molecular Dynamics Simulations

In Nature, the family of copper monooxygenases comprised of peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM) is known to perform dioxygen-dependent hydroxylation of aliphatic C-H bonds by using two uncoupled metal sites. In spite of many investigations, including biochemical, chemical, and computational, details of the C-H bond oxygenation mechanism remain elusive. Herein we report an investigation of the mechanism of hydroxylation by PHM by using hybrid quantum/classical potentials (i.e., QM/MM). Although previous investigations using hybrid QM/MM techniques were restricted to geometry optimizations, we have carried out ab initio molecular dynamics simulations in order to include the intrinsic flexibility of the active sites in the modeling protocol. The major finding of this study is an extremely fast rebound step after the initial hydrogen-abstraction step promoted by the cupric-superoxide adduct. The hydrogen-abstraction/rebound sequence leads to the formation of an alkyl hydroperoxide intermediate. Long-range electron transfer from the remote copper site subsequently triggers its reduction to the hydroxylated substrate. We finally show two reactivity consequences inherent in the new mechanistic proposal, the investigation of which would provide a means to check its validity by experimental means.

Evidence for Substrate Preorganization in the Peptidylglycine α-Amidating Monooxygenase Reaction Describing the Contribution of Ground State Structure to Hydrogen Tunneling

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme which catalyzes the post-translational modification of inactive C-terminal glycine-extended peptide precursors to the corresponding bioactive α-amidated peptide hormone. This conversion involves two sequential reactions both of which are catalyzed by the separate catalytic domains of PAM. The first step, the copper-, ascorbate-, and O(2)-dependent stereospecific hydroxylation at the α-carbon of the C-terminal glycine, is catalyzed by peptidylglycine α-hydroxylating monooxygenase (PHM). The second step, the zinc-dependent dealkylation of the carbinolamide intermediate, is catalyzed by peptidylglycine amidoglycolate lyase. Quantum mechanical tunneling dominates PHM-dependent C(α)-H bond activation. This study probes the substrate structure dependence of this chemistry using a set of N-acylglycine substrates of varying hydrophobicity. Primary deuterium kinetic isotope effects (KIEs), molecular mechanical docking, alchemical free energy perturbation, and equilibrium molecular dynamics were used to study the role played by ground-state substrate structure on PHM catalysis. Our data show that all Ν-acylglycines bind sequentially to PHM in an equilibrium-ordered fashion. The primary deuterium KIE displays a linear decrease with respect to acyl chain length for straight-chain N-acylglycine substrates. Docking orientation of these substrates displayed increased dissociation energy proportional to hydrophobic pocket interaction. The decrease in KIE with hydrophobicity was attributed to a preorganization event which decreased reorganization energy by decreasing the conformational sampling associated with ground state substrate binding. This is the first example of preorganization in the family of noncoupled copper monooxygenases.

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 [CuM(His)2(OH2)2–CuH(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 [CuM(His)2S(Met)–CuH(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 CuM 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 CuM 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 CuM 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00775-010-0677-3) contains supplementary material, which is available to authorized users.

Dioxygen Activation by Mononuclear Copper Enzymes: Insights from a Tripodal Ligand Mimicking Their CuM Coordination Sphere

A mononuclear cuprous complex is proposed as a novel in silico model for the Cu(M) active site of noncoupled copper monooxygenases. To the best of our knowledge, it is one of the first biomimicking models that allows one to recover the intimate structural features of the enzymatic oxygenated adducts and to gain clear-cut insights relevant to dioxygen activation by these enzymes.

Models of noncoupled dinuclear copper centers in azurin

The successful modeling of metalloproteins is an important step in understanding their structure and function. Toward this goal, models of the noncoupled copper centers found in the enzymes peptidyl alpha-hydroxylating monooxygenase (PHM), dopamine beta-monooxygenase (DBM), and nitrite reductase (NiR) were designed into the small soluble protein azurin. The models are significant because they maintain the existing type 1 (T1) copper, electron transfer site of azurin while including the second designed type 2 (T2) copper center that mimics the T2 catalytic sites in the target enzymes. UV-vis absorption and EPR spectroscopy data of the model sites are consistent with T2 centers and establish copper binding at the sites, thus modeling those found in PHM/DBM and NiR. Importantly the models' approximate 11-13 A separation between the T1 and T2 copper sites is comparable with the separations in the native systems. This, along with the power to tune the T1 site redox potential in azurin, allows for the future evaluation of relevant activity assays in these models.

A copper(II) azide compound of pyrazinic acid containing a new dinuclear complex anion [Cu 2(N 3) 6] 2−. Synthesis, spectral and study of KCu 2(pyrazinato) (N 3) 4

A new complex of copper(II) azide with pyrazinic acid, of general formula KCu 2(pyrazinato)(N 3) 4 has been synthesized and characterized by spectroscopic and crystallographic methods. The structure of the complex consists of a new dinuclear complex anion [Cu 3(N 3) 6] 2−, potassium cations and neutral polymeric complex [Cu(pyrazinato)(N 3)]. Each distorted square-planar copper centre [Cu(1)] in the complex anion is coordinated to four nitrogen atoms from two terminal and two μ-(1,1) bridging azido ligands [CuN distances 1.940(6)–2.011(5) Å]. In the polymeric complex, each copper atom, Cu(2), is coordinated to N(41) of a terminal azido group and N(61), N(62) and O(31) from pyrazinato anions to form infinite, “neutral” chains of polyhedra with orientations along [010]. The potassium cations are located in broad channels which are a consequence of stacking of the copper complexes. The IR spectrum confirms the presence of different asymmetric azido ligands. The absorption frequencies of diagnostic values are given and discussed. The electronic spectrum displays three absorption bands associated with N 3 −Cu II charge transfer transitions. The EPR spectrum of the solid complex suggests an axial structure with non-coupled copper(II) atoms.

E.F.R and Spectroscopic Studies of Cu(3-Piooline)2(N3)2Crystal

Abstract EPR, UV-Vie, and IR measurements were performed to determine the molecular structure of Cu(3-picoline)2(N3)2 in solid state. Single crystal EPR spectra indicate non-coupled copper (I1) complexes with gn = 2.265 and g⊥ = 2.056. A binuclear complex structure is suggested with end-to-end azido copper (I1) bibridges which are not optimiaed for exchange coupling transmission.