Multifrequency EPR Studies of [Cu^(1.5)Cu^(1.5)]+ for Cu_2(μ-NR_2)_2 and Cu_2(μ-PR_2)_2 Diamond Cores

Multifrequency electron paramagnetic resonance (EPR) spectroscopy is used to explore the electronic structures of a series of dicopper complexes of the type {(LXL)Cu}_2^+. These complexes contain two four-coordinate copper centers of highly distorted tetrahedral geometries linked by two [LXL]^− ligands featuring bridging amido or phosphido ligands and associated thioether or phosphine chelate donors. Specific chelating [LXL]^− ligands examined in this study include bis(2-tert-butylsulfanylphenyl)amide (SNS), bis(2-di-iso-butylphosphinophenyl)amide (PNP), and bis(2-di-iso-propylphosphinophenyl)phosphide (PPP). To better map the electronic coupling to copper, nitrogen, and phosphorus in these complexes, X-, S-, and Q-band EPR spectra have been obtained for each complex. The resulting EPR parameters implied by computer simulation are unusual for typical dicopper complexes and are largely consistent with previously published X-ray absorption spectroscopy and density functional theory data, where a highly covalent {Cu_2(μ-XR_2)_2}^+ diamond core has been assigned in which removal of an electron from the neutral {Cu_2(μ-XR_2)_2} can be viewed as ligand-centered to a substantial degree. To our knowledge, this is the first family of dicopper diamond core model complexes for which the compendium of X-, S-, and Q-band EPR spectra have been collected for comparison to Cu_A

A Cu\u3csub\u3e4\u3c/sub\u3eS Model for the Nitrous Oxide Reductase Active Sites Supported Only by Nitrogen Ligands

To model the (His)7Cu4Sn (n = 1 or 2) active sites of nitrous oxide reductase, the first Cu4(μ4-S) cluster supported only by nitrogen donors has been prepared using amidinate supporting ligands. Structural, magnetic, spectroscopic, and computational characterization is reported. Electrochemical data indicates that the 2-hole model complex can be reduced reversibly to the 1-hole state and irreversibly to the fully reduced state

A One-Hole Cu\u3csub\u3e4\u3c/sub\u3eS Cluster with N\u3csub\u3e2\u3c/sub\u3eO Reductase Activity: A Structural and Functional Model for Cu\u3csub\u3eZ\u3c/sub\u3e

During bacterial denitrification, two-electron reduction of N2O occurs at a [Cu4(μ4-S)] catalytic site (CuZ*) embedded within the nitrous oxide reductase (N2OR) enzyme. In this Communication, an amidinate-supported [Cu4(μ4-S)] model cluster in its one-hole (S = 1/2) redox state is thoroughly characterized. Along with its two-hole redox partner and fully reduced clusters reported previously, the new species completes the two-electron redox series of [Cu4(μ4-S)] model complexes with catalytically relevant oxidation states for the first time. More importantly, N2O is reduced by the one-hole cluster to produce N2 and the two-hole cluster, thereby completing a closed cycle for N2O reduction. Not only is the title complex thus the best structural model for CuZ* to date, but it also serves as a functional CuZ* mimic

Damage to Mitochondrial Complex I During Cardiac Ischemia Reperfusion Injury is Reduced Indirectly by Anti-anginal Drug Ranolazine

Ranolazine, an anti-anginal drug, is a late Na+ channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if ranolazine\u27s protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH Ubiquinone oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with ranolazine just before 30 min ischemia and then isolated cardiac mitochondria at the end of 30 min ischemia and/or 30 min ischemia followed by 10 min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, Western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe–S clusters, enzyme linked immuno sorbent assay (ELISA) for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ranolazine treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ranolazine treatment also led to more normalized electron transfer via Fe–S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with ranolazine were associated with improved cardiac function after IR. However, these protective effects of ranolazine are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I

EPR of Cu\u3csup\u3e2+\u3c/sup\u3e Prion Protein Constructs at 2 GHz Using the \u3cem\u3eg\u3c/em\u3e\u3csub\u3e⊥\u3c/sub\u3e Region to Characterize Nitrogen Ligation

A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu2+ complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g⊥ region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu2+. In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the MI = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric Ax and Ay hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the g⊥ part of the spectrum is sensitive to the rhombic distortion parameters Ax and Ay. Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains ∼1 mL of frozen sample

Structurally Distinct Active Sites in the Copper(II)-Substituted Aminopeptidases from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e and \u3cem\u3eEscherichia coli\u3c/em\u3e

The aminopeptidase from Aeromonas proteolytica (AAP) was titrated with copper, which bound sequentially at two distinct sites. Both the mono- and disubstituted forms of AAP exhibited catalytic hyperactivity relative to the native dizinc enzyme. Monosubstituted AAP exhibited an axial Cu(II) EPR spectrum with slight pH dependence:  at pH 6.0 g|| = 2.249, g⊥ = 2.055, and A||(63/65Cu) = 1.77 × 10-2 cm-1, whereas at pH 9.65 g|| = 2.245, g⊥ = 2.056, and A||(63/65Cu) = 1.77 × 10-2 cm-1. These data indicate oxygen and nitrogen ligation of Cu. AAP further substituted with copper exhibited a complex signal with features around g ∼ 2 and 4. The features at g ∼ 4 were relatively weak in the B0 ⊥ B1 (perpendicular) mode EPR spectrum but were intense in the B0 || B1 (parallel) mode spectrum. The g ∼ 2 region of the perpendicular mode spectrum exhibited two components, one corresponding to mononuclear Cu(II) with g|| = 2.218, g⊥ = 2.023, and A||(63/65Cu) = 1.55 × 10-2 cm-1 and likely due to adventitious binding of Cu(II) to a site distant from the active site. Excellent simulations were obtained for the second component of the spectrum assuming that two Cu(II) ions experience dipolar coupling corresponding to an inter-copper distance of 5 Å with the two Cu(II) gz directions parallel to each other and at an angle of ∼17° to the inter-copper vector (ℋ = βB·gCuA·SCuA + βB·gCuB·SCuB + [S·A·I]CuA + [S·A·I]CuB + [SCuA·J·SCuB]; g||(CuA,CuB) = 2.218, g⊥(CuA,CuB) = 2.060; A||(CuA,CuB)(63/65Cu) = 1.59 × 10-2 cm-1, Jisotropic = 50 cm-1, rCu-Cu = 4.93 Å, and χ = 17°). The exchange coupling between the two copper ions was found to be ferromagnetic as the signals exhibited Curie law temperature dependence. The Cu−Cu distance of ∼5 Å indicated by EPR was significantly higher than the inter-zinc distance of 3.5 Å in the native enzyme, and the dicopper species therefore represents a novel dinuclear site capable of catalysis of hydrolysis. In contrast to AAP, the related methionyl aminopeptidase from Escherichia coli (EcMetAP) was found to bind only one Cu(II) ion despite possessing a dinuclear binding site motif. A further difference was the marked pH dependence of the signal in EcMetAP, suggestive of a change in ligation. The structural motifs of these two Cu(II)-substituted aminopeptidases provide important insight into the observed catalytic activity

Three-Coordinate Copper(I) Amido and Aminyl Radical Complexes

Electron transfer (ET) through proteins often utilizes copper-containing active sites as efficient one-electron relays. The type-1 active sites of the blue copper proteins are prominent examples. It is generally thought that high ET rates through type-1 redox sites occur because the protein environments enforce unusual trigonally distorted coordination spheres to allow for minimal structural reorganization during ET. Though large Cu^II/Cu^I self-exchange ET rate constants (kS) in the range observed for type-1 sites have been achieved in certain synthetic monocopper systems using geometries distinct from trigonal environments, ET studies have yet to be conducted in a synthetic system featuring isolated, trigonally disposed copper centers. The simplest such systems would contain a trigonal planar geometry. Here we report the structural characterization of a trigonal planar system featuring formally Cu^II and Cu^I amido complexes related by a reversible one-electron redox event. We find in this system that ET is extremely rapid and is accompanied by a small degree of structural reorganization during redox. We propose that this structural rigidity in the absence of secondary coordination sphere effects results from significant covalency of the copper-amide linkages. In fact, a CuI-aminyl radical description of the formally Cu^II-amide complex may be most appropriate

Di- and Trinuclear Mixed-Valence Copper Amidinate Complexes from Reduction of Iodine

Molecular examples of mixed-valence copper complexes through chemical oxidation are rare but invoked in the mechanism of substrate activation, especially oxygen, in copper-containing enzymes. To examine the cooperative chemistry between two metals in close proximity to each other we began studying the reactivity of a dinuclear Cu(I) amidinate complex. The reaction of [(2,6-Me2C6H3N)2C(H)]2Cu2, 1, with I2 in tetrahydrofuran (THF), CH3CN, and toluene affords three new mixed-valence copper complexes [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I3)(THF)2, 2, [(2,6-Me2C6H3N)2C(H)]2Cu2(μ2-I) (NCMe)2, 3, and [(2,6-Me2C6H3N)2C(H)]3Cu3(μ3-I)2, 4, respectively. The first two compounds were characterized by UV-vis and electron paramagnetic resonance spectroscopies, and their molecular structure was determined by X-ray crystallography. Both di- and trinuclear mixed-valence intermediates were characterized for the reaction of compound 1 to compound 4, and the molecular structure of 4 was determined by X-ray crystallography. The electronic structure of each of these complexes was also investigated using density functional theory

The Octarepeat Domain of the Prion Protein Binds Cu(II) with Three Distinct Coordination Modes at pH 7.4

The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copper−copper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4