Jake W. Ginsbach

Copper Active Sites in Biology

Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2. Figure 1 Copper active sites in biology. Figure 2 Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6. This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond. Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

Copper Dioxygen (Bio)Inorganic Chemistry

Cu/O2 intermediates in biological, homogeneous, and heterogeneous catalysts exhibit unique spectral features that reflect novel geometric and electronic structures that make significant contributions to reactivity. This review considers how the respective intermediate electronic structures overcome the spin-forbidden nature of O2 binding, activate O2 for electrophilic aromatic attack and H-atom abstraction, catalyze the 4 e- reduction of O2 to H2O, and discusses the role of exchange coupling between Cu ions in determining reactivity.

Amine Oxidative N-Dealkylation via Cupric Hydroperoxide Cu-OOH Homolytic Cleavage Followed by Site-Specific Fenton Chemistry

Copper(II) hydroperoxide species are significant intermediates in processes such as fuel cells and (bio)chemical oxidations, all involving stepwise reduction of molecular oxygen. We previously reported a Cu(II)-OOH species that performs oxidative N-dealkylation on a dibenzylamino group that is appended to the 6-position of a pyridyl donor of a tripodal tetradentate ligand. To obtain insights into the mechanism of this process, reaction kinetics and products were determined employing ligand substrates with various para-substituent dibenzyl pairs (-H,-H; -H,-Cl; -H,-OMe, and -Cl,-OMe), or with partially or fully deuterated dibenzyl N-(CH2Ph)2 moieties. A series of ligand-copper(II) bis-perchlorate complexes were synthesized, characterized, and the X-ray structures of the -H,-OMe analogue were determined. The corresponding metastable Cu(II)-OOH species were generated by addition of H2O2/base in acetone at -90 °C. These convert (t1/2 ≈ 53 s) to oxidatively N-dealkylated products, producing para-substituted benzaldehydes. Based on the experimental observations and supporting DFT calculations, a reaction mechanism involving dibenzylamine H-atom abstraction or electron-transfer oxidation by the Cu(II)-OOH entity could be ruled out. It is concluded that the chemistry proceeds by rate limiting Cu-O homolytic cleavage of the Cu(II)-(OOH) species, followed by site-specific copper Fenton chemistry. As a process of broad interest in copper as well as iron oxidative (bio)chemistries, a detailed computational analysis was performed, indicating that a Cu(I)OOH species undergoes O-O homolytic cleavage to yield a hydroxyl radical and Cu(II)OH rather than heterolytic cleavage to yield water and a Cu(II)-O(•-) species.

A N3S(thioether)-Ligated CuII-Superoxo with Enhanced Reactivity

Previous efforts to synthesize a cupric superoxide complex possessing a thioether donor have resulted in the formation of an end-on trans-peroxo-dicopper(II) species, [{(Ligand)Cu(II)}2(μ-1,2-O2(2-))](2+). Redesign/modification of previous N3S tetradentate ligands has now allowed for the stabilization of the monomeric, superoxide product possessing a S(thioether) ligation, [((DMA)N3S)Cu(II)(O2(•-))](+) (2(S)), as characterized by UV-vis and resonance Raman spectroscopies. This complex mimics the putative Cu(II)(O2(•-)) active species of the copper monooxygenase PHM and exhibits enhanced reactivity toward both O-H and C-H substrates in comparison to close analogues [(L)Cu(II)(O2(•-))](+), where L contains only nitrogen donor atoms. Also, comparisons of [(L)Cu(II/I)](n+) compound reduction potentials (L = various N4 vs (DMA)N3S ligands) provide evidence that (DMA)N3S is a weaker donor to copper ion than is found for any N4 ligand-complex.

Correlation of the electronic and geometric structures in mononuclear copper(II) superoxide complexes.

The geometry of mononuclear copper(II) superoxide complexes has been shown to determine their ground state where side-on bonding leads to a singlet ground state and end-on complexes have triplet ground states. In an apparent contrast to this trend, the recently synthesized (HIPT3tren)Cu(II)O2(•-) (1) was proposed to have an end-on geometry and a singlet ground state. However, reexamination of 1 with resonance Raman, magnetic circular dichroism, and (2)H NMR spectroscopies indicate that 1 is, in fact, an end-on superoxide species with a triplet ground state that results from the single Cu(II)O2(•-) bonding interaction being weaker than the spin-pairing energy.

Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF

The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxygenase that converts o-aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosinase, is able to mono-oxygenate monophenols (monophenolase activity) but not o-aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxy-tyrosinase indicates that oxy-NspF contains a Cu2O2 core where peroxide is coordinated in a μ-η2∶ η2 mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent μ-η2∶η2 peroxy bridged coupled binuclear copper active sites.

Tuning of the Copper–Thioether Bond in Tetradentate N3S(thioether) Ligands; O–O Bond Reductive Cleavage via a [CuII2(μ-1,2-peroxo)]2+/[CuIII2(μ-oxo)2]2+ Equilibrium

Current interest in copper/dioxygen reactivity includes the influence of thioether sulfur ligation, as it concerns the formation, structures, and properties of derived copper-dioxygen complexes. Here, we report on the chemistry of {L-CuI}2-(O2) species L = DMMESE, DMMESP, and DMMESDP, which are N3S(thioether)-based ligands varied in the nature of a substituent on the S atom, along with a related N3O(ether) (EOE) ligand. CuI and CuII complexes have been synthesized and crystallographically characterized. Copper(I) complexes are dimeric in the solid state, [{L-CuI}2](B(C6F5)4)2, however are shown by diffusion-ordered NMR spectroscopy to be mononuclear in solution. Copper(II) complexes with a general formulation [L-CuII(X)]n+ {X = ClO4–, n = 1, or X = H2O, n = 2} exhibit distorted square pyramidal coordination geometries and progressively weaker axial thioether ligation across the series. Oxygenation (−130 °C) of {(DMMESE)CuI}+ results in the formation of a trans-μ-1,2-peroxodicopper(II) species [{(DMMESE)CuII}2(μ-1,2-O22–)]2+ (1P). Weakening the Cu–S bond via a change to the thioether donor found in DMMESP leads to the initial formation of [{(DMMESP)CuII}2(μ-1,2-O22–)]2+ (2P) that subsequently isomerizes to a bis-μ-oxodicopper(III) complex, [{(DMMESP)CuIII}2(μ-O2–)2]2+ (2O), with 2P and 2O in equilibrium (Keq = [2O]/[2P] = 2.6 at −130 °C). Formulations for these Cu/O2 adducts were confirmed by resonance Raman (rR) spectroscopy. This solution mixture is sensitive to the addition of methylsulfonate, which shifts the equilibrium toward the bis-μ-oxo isomer. Further weakening of the Cu–S bond in DMMESDP or substitution with an ether donor in DMMEOE leads to only a bis-μ-oxo species (3O and 4O, respectively). Reactivity studies indicate that the bis-μ-oxodicopper(III) species (2O, 3O) and not the trans-peroxo isomers (1P and 2P) are responsible for the observed ligand sulfoxidation. Our findings concerning the existence of the 2P/2O equilibrium contrast with previously established ligand-CuI/O2 reactivity and possible implications are discussed.

Peroxo and Superoxo Moieties Bound to Copper Ion: Electron-Transfer Equilibrium with a Small Reorganization Energy

Oxygenation of [Cu2(UN-O(-))(DMF)](2+) (1), a structurally characterized dicopper Robin-Day class I mixed-valent Cu(II)Cu(I) complex, with UN-O(-) as a binucleating ligand and where dimethylformamide (DMF) binds to the Cu(II) ion, leads to a superoxo-dicopper(II) species [Cu(II)2(UN-O(-))(O2(•-))](2+) (2). The formation kinetics provide that kon = 9 × 10(-2) M(-1) s(-1) (-80 °C), ΔH(‡) = 31.1 kJ mol(-1) and ΔS(‡) = -99.4 J K(-1) mol(-1) (from -60 to -90 °C data). Complex 2 can be reversibly reduced to the peroxide species [Cu(II)2(UN-O(-))(O2(2-))](+) (3), using varying outer-sphere ferrocene or ferrocenium redox reagents. A Nernstian analysis could be performed by utilizing a monodiphenylamine substituted ferrocenium salt to oxidize 3, leading to an equilibrium mixture with Ket = 5.3 (-80 °C); a standard reduction potential for the superoxo-peroxo pair is calculated to be E° = +130 mV vs SCE. A literature survey shows that this value falls into the range of biologically relevant redox reagents, e.g., cytochrome c and an organic solvent solubilized ascorbate anion. Using mixed-isotope resonance Raman (rRaman) spectroscopic characterization, accompanied by DFT calculations, it is shown that the superoxo complex consists of a mixture of μ-1,2- (2(1,2)) and μ-1,1- (2(1,1)) isomers, which are in rapid equilibrium. The electron transfer process involves only the μ-1,2-superoxo complex [Cu(II)2(UN-O(-))(μ-1,2-O2(•-))](2+) (2(1,2)) and μ-1,2-peroxo structures [Cu(II)2(UN-O(-))(O2(2-))](+) (3) having a small bond reorganization energy of 0.4 eV (λin). A stopped-flow kinetic study results reveal an outer-sphere electron transfer process with a total reorganization energy (λ) of 1.1 eV between 2(1,2) and 3 calculated in the context of Marcus theory.

CCDC 1430274: Experimental Crystal Structure Determination

Related Article: Rui Cao, Claudio Saracini, Jake W. Ginsbach, Matthew T. Kieber-Emmons, Maxime A. Siegler, Edward I. Solomon, Shunichi Fukuzumi, and Kenneth D. Karlin|2016|J.Am.Chem.Soc.|138|7055|doi:10.1021/jacs.6b02404