A first model for the oxidized active form of the active site in galactose oxidase: a free-radical copper complex

 Copper(II) complexes derived from the tripodal ligand bis(3′-t–butyl-2′-hydroxybenzyl)(2-pyridylmethyl)amine (LH₂) have been studied in order to mimic the redox active site of the free radical-containing copper metalloenzyme galactose oxidase. In non-coordinating solvents such as dichloromethane, only an EPR-silent dimeric complex was obtained (L₂Cu₂). The crystal structure of L₂Cu₂ revealed a "butterfly" design of the [Cu(μOR)₂Cu] unit, which is not flattened and leads to a short Cu–Cu distance, the t–butyl groups being localized on the same side of the [Cu(μOR)₂Cu] unit. The dimeric structure was broken down by acetonitrile or by alcohols, leading quantitatively to a brown mononuclear copper(II) complex. UV-visible and EPR data indicated the coordination of the solvent in these mononuclear complexes. Electrochemical as well as chemical (silver acetate) one-electron oxidation of acetonitrile solutions of the monomeric complex led to a yellow-green solution. Based on EPR, UV-visible and resonance Raman spectroscopy, the one-electron oxidation product was identified as a cupric phenoxyl radical system. It slowly decomposes into a product where the ligand has been substituted (dimerization) in the para position of the hydroxyl group, for one of the phenolic groups. The data for the one-electron oxidized species provides strong evidence for a free-radical copper (II) complex. Received: 19 July 1996 / Accepted: 16 October 1996     

Mono- and dinuclear (o-thioetherphenolato)-copper(II) complexes. Structural models for galactose oxidase

Three new o-thioetherphenol ligands have been synthesized: 1,2-bis(3,5-di-tert-butyl-2-hydroxyphenylsulfanyl)ethane (H₂bse), 1,2-bis(3,5-di-tert-butyl-2-hydroxyphenylsulfanyl)benzene (H₂bsb), and 4,6-di-tert-butyl-2-phenylsulfanylphenol (Hpsp). Their complexes with copper(II) were prepared and investigated by UV-Vis-, EPR-spectroscopy; their electro- and magnetochemistry have also been studied: [Cu^II(psp)₂] (1), [Cu^II₂(bse)₂] (2), [Cu^II₂(bsb)₂] (3), [Cu^II(bsb)(py)₂] (4). The crystal structures of the ligands H₂bse, H₂bsb, Hpsp and of the complexes 1, 2, 3, 4 have been determined by X-ray crystallography.     

Studies on galactose oxidase active site model complexes: effects of ring substituents on Cu(II)-phenoxyl radical formation

Copper(II) complexes of new N₃O- and N₂O₂-donor tripodal ligands bearing one or two o-substituted phenol moieties have been synthesized as models for the galactose oxidase active site. The complexes of 2-[N-(1-methyl-2′-imidazolylmethyl)-N-(6″-methyl-2″-pyridylmethyl)-aminomethyl)]-4-methyl-6-methylthiophenol (MeSL), [Cu(MeSL)Cl], and N-(6-methyl-2-pyridylmethyl)-N,N-bis(2′-hydroxy-3′,5′-di-tert-butylbenzyl)amine (t-buL2mepy), [Cu(t-buL2mepy)(H₂O)], have been revealed by X-ray structural analysis to have a square-pyramidal structure with one and two phenolate oxygens in the basal plane, respectively. [Cu(MeSL)Cl] was converted into a Cu(II)-o-methylthiophenoxyl radical species by electrochemical or Ce(IV) oxidation. An o-methoxyphenoxyl radical in a similar complex was considerably more stable than the 2,4-di(tert-butyl)phenoxyl radical. While t-buL2mepy reacted with Cu(ClO₄)₂ to give [Cu(t-buL2mepy)(H₂O)] without disproportionation, an N2O2-donor ligand containing an o-methoxyphenol, a 2,4-di(tert-butyl)phenol, and an N-methylimidazole moiety gave a phenoxyl radical complex exhibiting the characteristic absorption peak at 478 nm as a reddish powder by the reaction with Cu(ClO₄)₂ as a result of spontaneous disproportionation. It exhibited a quasi-reversible redox wave at E_1/2=0.34 V (vs. Ag/AgCl) in CH₃CN, which is lower than the potentials of the copper complexes of various N₃O-donor ligands, and oxidized ethanol to acetaldehyde with a low turnover number.     

Model complexes of the active site of galactose oxidase. Effects of the metal ion binding sites

Model compounds of the active site of galactose oxidase have been developed by using new cofactor model ligands, L1H (2-methylthio-4-tert-butyl-6-[{bis(pyridin-2-ylmethyl)amino}methyl]phenol) and L2H (2-methylthio-4-tert-butyl-6-[{bis(6-methylpyridin-2-ylmethyl)amino}methyl]phenol). Treatment of the ligands with copper(II) and zinc(II) perchlorate in the presence of triethylamine followed by anion exchange reaction with NaPF₆ or NaBPh₄ provided the corresponding copper(II) and zinc(II) complexes, the crystal structures of which have been determined by X-ray crystallographic analysis. All the copper(II) and zinc(II) complexes have been isolated as a dimeric form in which the phenolate oxygen of each ligand acts as the bridging ligand to form a rhombic M₂(OAr)₂ core (M=Cu or Zn). The dimeric complexes can be converted into the corresponding monomer complexes by the treatment with exogenous ligand such as acetate ion. The redox potential and the spectroscopic features of the monomer complexes have also been examined. Furthermore, the copper(II)- and zinc(II)-complexes of the phenoxyl radical species of the ligands have been generated in situ by the oxidation of the phenolate complexes with (NH₄)₂[Ce^IV(NO₃)₆] (CAN) in CH₃CN, and their spectroscopic features have been explored. The structures and physicochemical properties of the phenolate and phenoxyl radical complexes of L1 and L2 have been compared to those of the previously reported copper(II) and zinc(II) complexes of L3 (2-methylthio-4-tert-butyl-6-[{bis(2-pyridin-2-ylethyl)amino}methyl]phenol) in order to get insights into the interaction between the metal ions and the organic cofactor moiety.     

Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495

 The catalytic mechanism of the copper-containing enzyme galactose oxidase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower k _cat / K _m) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism. Received: 17 December 1996 / Accepted: 12 March 1997     

A comparative study of galactose oxidase and active site analogs based on QM/MM Car-Parrinello simulations

A parallel study of the radical copper enzyme galactose oxidase (GOase) and a low molecular weight analog of the active site was performed with dynamical density functional and mixed quantum-classical calculations. This combined approach enables a direct comparison of the properties of the biomimetic and the natural systems throughout the course of the catalytic reaction. In both cases, five essential forms of the catalytic cycle have been investigated: the resting state in its semi-reduced (catalytically inactive) and its oxidized (catalytically active) form, A ^semi and A ^ox, respectively; a protonated intermediate B; the transition state for the rate-determining hydrogen abstraction step C, and its product D. For A and B the electronic properties of the biomimetic compound are qualitatively very similar to the ones of the natural target. However, in agreement with the experimentally observed difference in catalytic activity, the calculated activation energy for the hydrogen abstraction step is distinctly lower for GOase (16 kcal/mol) than for the mimetic compound (21 kcal/mol). The enzymatic transition state is stabilized by a delocalization of the unpaired spin density over the sulfur-modified equatorial tyrosine Tyr272, an effect that for geometric reasons is essentially absent in the biomimetic compound. Further differences between the mimic and its natural target concern the structure of the product of the abstraction step, which is characterized by a weakly coordinated aldehyde complex for the latter and a tightly bound linear complex for the former. Received 14 October 1999 · Accepted: 19 January 2000     

How useful are vibrational frequencies of isotopomeric O2 fragments for assessing local symmetry? Some simple systems and the vexing case of a galactose oxidase model

The tendency for mixed-isotope O₂ fragments to exhibit different stretching frequencies in asymmetric environments is examined with various levels of electronic structure theory for simple peroxides and peroxyl radicals, as well as for a variety of monocopper–O₂ complexes. The study of the monocopper species is motivated by their relevance to the active site of galactose oxidase. Extensive theoretical work with an experimental model characterized by Jazdzewski et al. (J. Biol. Inorg. Chem. 8:381–393, 2003) suggests that the failure to observe a splitting between ¹⁶O¹⁸O and ¹⁸O¹⁶O isotopomers cannot be taken as evidence against end-on O₂ coordination. Conformational analysis on an energetic basis, however, is complicated by biradical character inherent in all of the copper–O₂ singlet structures. Electronic Supplementary Material Supplementary material is available for this article at .     

Effect of nickel(II) substitution on the resonance Raman and NMR spectra of Alcaligenes xylosoxidans azurin II: implications for axial-ligand bonding interactions in cupredoxin active sites

Assignment of the resonance Raman (RR) spectrum of Ni(II)-substituted azurin II from Alcaligenes xylosoxidans (NCIMB 11015) using Ni isotope substitution reveals an anomalously low Ni-S(Cys) stretching frequency of 349?cm^–1, suggesting the presence of significant axial-ligand bonding interactions. The X-ray crystal structure of Ni(II)-substituted azurin from Pseudomonas aeruginosa shows that there are two potential axial ligands to the Ni ion: a peptide carbonyl O at a distance of 2.46?Å, together with a long-range interaction from a methionine sulfur (S′) at a distance of 3.30?Å. Comparison of the RR properties of Ni(II)-substituted azurin II with stellacyanin (which contains an axial carbonyl ligand, but no methionine) suggests that the interaction from the carbonyl oxygen ligand alone is not sufficient to account for the weak Ni azurin metal-thiolate bond. Instead, it appears that a Ni-methionine bonding interaction is also required to explain the low Ni-S(Cys) stretching frequency in Ni(II)-substituted azurin II. This hypothesis is supported by NMR studies which show a large paramagnetic shift for the protons of the methionine side-chain. Thus, it appears that Ni-substituted azurin II is best described as five-coordinate, and that significant Ni(II)-methionine bonding interactions can occur at a distance of 3.3?Å.     

Systematic development of computational models for the catalytic site in galactose oxidase: impact of outer-sphere residues on the geometric and electronic structures

A systematic in silico approach has been employed to generate sound, experimentally validated active-site models for galactose oxidase (GO) using a hybrid density functional, B(38HF)P86. GO displays three distinct oxidation states: oxidized [Cu(II)-Y*]; semireduced [Cu(II)-Y]; and reduced [Cu(I)-Y]. Only the [Cu(II)-Y*] and the [Cu(I)-Y] states are assumed to be involved in the catalytic cycle, but their structures have not yet been determined. We have developed several models (1-7) for the [Cu(II)-Y*] state that were evaluated by comparison of our computational results with experimental data. An extended model system (6) that includes solvent molecules and second coordination sphere residues (R330, Y405, and W290) is essential to obtain an experimentally correct electronic structure of the active site. The optimized structure of 6 resulted in a five-coordinate Cu site with a protein radical centered on the Tyr-Cys cofactor. We further validated our converged model with the largest model (7) that included additional outer-sphere residues (Q406, H334, Y329, G513, and T580) and water molecules. Adding these residues did not affect significantly the active site's electronic and geometric structures. Using both 6 and 7, we explored the redox dependence of the active-site structure. We obtained four- and three-coordinate Cu sites for [Cu(II)-Y] and [Cu(I)-Y] states, respectively, that corroborate well with the experimental data. The relative energies of these states were validated by a comparison with experimental redox potentials. Collectively, our computational GO models well reproduce the physicochemical characteristics of the individual states, including their redox behaviors.     

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

 Reactions (25  °C) of galactose oxidase, GOase_ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH₂OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O₂. The GOase_ox state has a tyrosyl radical coordinated at a square-pyramidal Cu^II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase_ox] (0.8–10 μM), RCH₂OH (1.0–6.0 mM), and O₂ (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O₂. Each trace was fitted to the competing reactions GOase_ox+RCH₂ OH → GOase_redH₂+RCHO (k ₁), and GOase_redH₂+O₂→ GOase_ox+H₂O₂ (k ₂), with GOase_redH₂ written as the doubly protonated two-electron-reduced Cu^I product. It was necessary to avoid auto-redox interconversion of GOase_ox and GOase_semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10^–4 k ₁ / M^–1 s^–1 for the reactions of GOase_ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k ₁ values decreased by factors of up to a half, and acid dissociation pK _as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOase_ox gave an independently determined pK _a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK _a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k ₂ (1.01×10⁷ M^–1 s^–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H⁺ (or H-atoms) for the O₂ → H₂O₂ change are provided by the active site of GOase_red . The Cu^I of GOase_red is less extensively complexed, and a coordination number of three is likely. Received: 4 February 1997 / Accepted: 16 May 1997     

A new dinuclear unsymmetric copper(II) complex as model for the active site of catechol oxidase

The crystal structure, magnetic, redox and spectroscopic properties of a novel unsymmetrical dinuclear copper(II) complex, prepared by the reaction between copper(II) perchlorate, sodium acetate and the unsymmetrical, binucleating ligand HTPPNOL, where HTPPNOL is N,N,N′-tris-(2-pyridylmethyl)-1,3-diaminopropan-2-ol, is reported. HTPPNOL (1 equiv.) reacted with 1 equiv. of copper(II) ion, in methanol, and produced the mononuclear copper complex [Cu(TPPNOL)](ClO₄)(BPh₄) (1). On the other hand, the reaction of 1 equiv. of HTPPNOL with 2 equiv. each of copper (II) ion and acetate, in methanol, produced the dinuclear complex [Cu₂(TPPNOL)(OOCCH₃)](ClO₄)₂ (2), whose structure has been determined by X-ray diffraction. In complex 2, as a result of the inherent asymmetry of the ligand HTPPNOL, one copper ion is five-coordinated (distorted trigonal-bipyramidal) while the other copper is four-coordinated (distorted square-planar). Then, as a result of the presence of distinct geometries for the metal centres, complex 2 exhibits a ferromagnetic coupling (J=+25.41 cm⁻¹). Titration experiments carried out on the dinuclear complex suggest a pK_a=8.0, which was related to the aquo/hydroxo equilibrium. Complex 2 is able to oxidise 3,5-di-tert-butylcatechol to the respective o-quinone. The oxidation reaction was studied by following the appearance of the quinone spectrophotometrically, at pH 8.0 and 25 °C.     

Trimethylsilyloxo complexes of oxomolybdenum(V)

The complexes LMo^VIO₂X [L?=?hydrotris(3,5-dimethylpyrazol-1-yl)borate; X?=?Cl, Br, NCS, OPh, SPh, SCH₂Ph] are converted to air-stable complexes LMo^VO(OSiMe₃)X by one-electron coupled electron-electrophile transfer (CEET) reactions involving cobaltocene and the electrophilic reagent Me₃SiCl. These complexes may also be obtained from LMo^VO(OH)X by reaction with Me₃SiCl in the presence of base. LMo^VO(OSiMe₃)(SCH₂Ph) crystallises in space group P2₁/n, with a?=?8.526 (1) Å, b?=?23.141 (3) Å, c?=?16.499 (2) Å, β?=?103.75 (12)° and Z?=?4. The complex exhibits a distorted octahedral structure with a facially tridentate L ligand and mutually cis oxo [Mo=O?=?1.675 (4) Å], silyloxo [Mo–O?=?1.932 (4) Å] and thiolato [Mo–S?=?2.398 (2) Å] ligands. The detailed redox properties of LMo^VO(OR)X (R?=?SiMe₃, alkyl, aryl) differ from those of LMo^VO(OH)X. Centres [Mo^VO(OR)] are candidates for the stable "inhibited" forms of certain molybdenum enzymes formed under conditions which apparently disfavour the catalytically active [Mo^VO(OH)] centres. In the coordinating solvent pyridine (py), both LMo^VIO₂(SPh) and LMo^VO(OSiMe₃)(SPh) are reduced in one-electron steps to stable LMo^IVO(py)(SPh). LMo^IVO(py)(SR) complexes are also obtained from LMo^VIO₂(SR) (R?=?Ph, CH₂Ph, CHMe₂) via a two-electron oxygen atom transfer reaction with tertiary phosphines in pyridine. Consequently, the Mo(IV) product is accessible via a concerted two-electron step or via two one-electron steps.     

Properties of the Trp290His variant of Fusarium NRRL 2903 galactose oxidase: interactions of the GOasesemi state with different buffers, its redox activity and ability to bind azide

The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short ～8?Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400?mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25??°C, I=0.100?M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the non-radical GOase_semi (Cu^II) form of the Trp290His variant. Equilibrium constants K _eq/M^–1 from absorbance changes at 635?nm are for 1?:?1 interactions with the OH-containing buffers H₂PO₄ ^– (231), Hepes (43) and Tris (202), concentrations 0–60?mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at ～635?nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOase_ox/GOase_semi couple is 730?mV, which compares with 400?mV for the WT GOase couple. Consistent with the 730?mV value the GOase_semi form is not oxidised with [Fe(CN)₆]^3– (410?mV) or [W(CN)₈]^3– (530?mV), and much stronger oxidants such as [Mo(CN)₈]^3– (800?mV) and [IrCl₆]^2– (890?mV) are required. The GOase_ox product is unstable and decays within 20?min with re-formation of GOase_semi. From changes in UV-Vis spectra with pH, Trp290His GOase_semi gives a pK _a of 6.9, and rate constants for the oxidation of GOase_semi with [Mo(CN)₈]^3– are dependent on this same pK _a. The latter compares with 7.9 for WT GOase_semi, and is assigned here also as protonation of Tyr-495. The 1?:?1 binding of azide at the substrate-binding (H₂O) site of Trp290His GOase_semi was studied and gives a formation constant 330?M^–1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOase_semi. The trends observed indicate less affinity of Trp290His GOase_semi for the ionic reactants H⁺ and N₃ ^–.     

Copper(II) PMDTA and copper(II) TMEDA complexes: precursors for the synthesis of dinuclear dicationic copper(II) complexes

Copper(II) cations coordinated with PMDTA (pentamethyldiethylenetriamine) and TMEDA (tetramethylethylenediamine) possess a high synthetic potential. The synthesis of these cations was carried out by metathesis reactions with silver salts. The cationic copper(II) complexes, [Cu(PMDTA)(Me₂CO)Cl]⁺, [Cu(PMDTA)(H₂O)Cl]⁺, [Cu(PMDTA)(DMF)]⁺, [Cu(PMDTA)Cl]⁺, [Cu(PMDTA)OAc]⁺, [Cu(PMDTA)(MeCN)₂]²⁺, [Cu₂(TMEDA)₂Cl₃]⁺ and [Cu(TMEDA)(MeCN)₃]²⁺ were synthesised as PF₆ salts, crystallised and characterised by single-crystal X-ray diffraction.     

Synthesis and structural analysis of copper(II) cysteine complexes

This report describes the synthesis and structural analysis of stable copper(II) cysteine complexes. Pale pink copper(II) cysteine complexes were synthesized in mole ratios of 1:2, 1:4, and 1:6 of copper(II):cysteine in ethanol. Infrared spectroscopy and X-ray absorption spectroscopy confirmed that copper(II) binding occurred via the thiol ligand of cysteine. XANES analysis showed that the oxidation state of copper remained as copper(II) and the local atomic geometry was similar in all of the cysteine complexes. The EXAFS data indicate that the copper(II) cysteine complexes are forming ring type structures with sulfur ligands from the cysteines acting as bridging ligands. X-ray diffraction revealed that the copper(II) cysteine complexes formed monoclinic cells with maximum crystallinity found in the 1:4 copper(II):cysteine complex.     

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase

It is known that germin, which is a marker of the onset of growth in germinating wheat, is an oxalate oxidase, and also that germins possess sequence similarity with legumin and vicilin seed storage proteins. These two pieces of information have been combined in order to generate a 3D model of germin based on the structure of vicilin and to examine the model with regard to a potential oxalate oxidase active site. A cluster of three histidine residues has been located within the conserved β-barrel structure. While there is a relatively low level of overall sequence similarity between the model and the vicilin structures, the conservation of amino acids important in maintaining the scaffold of the β-barrel lends confidence to the juxtaposition of the histidine residues. The cluster is similar structurally to those found in copper amine oxidase and other proteins, leading to the suggestion that it defines a metal-binding location within the oxalate oxidase active site. It is also proposed that the structural elements involved in intermolecular interactions in vicilins may play a role in oligomer formation in germin/oxalate oxidase. Received: 25 April 1997 / Accepted: 29 July 1997     

Study of copper(II) ternary complexes with phosphocreatine and some polyamines in aqueous solution

Ternary systems of Cu(II) with phosphocreatine (PCr) and the polyamines (PAs), ethylenediamine (en), 1,3-diaminopropane (tn), putrescine (Put), spermidine (Spd), and spermine (Spm), were investigated in aqueous solution through potentiometry, ultraviolet-visible, EPR and Raman spectroscopy. The binary complex CuPCr was also studied by Raman spectroscopy, and the calculation of the minimum stabilization energy was done assuming this molecule in aqueous solution. The stability constants of the CuPCrPA ternary complexes were determined by potentiometry (T = 25 °C, I = 0.1 mol L^− 1, KNO₃). The stability order determined was CuPCrSpm > CuPCrSpd > CuPCren > CuPCrtn > CuPCrPut, the same order of the corresponding binary complexes of Cu(II) with these polyamines. The evaluation of intramolecular PA-PCr interactions in protonated and deprotonated species of ternary complexes was carried out using the equation Δlog K = log β_{CuPCrPAHq + p} − (log β_CuPAHq + log β_CuPCrHp). All of the CuPCrPA ternary complexes have a square planar structure and are bonded to PCr through the nitrogen atom of the guanidine group and the oxygen atom of the phosphate group, and to the PAs through two nitrogen atoms of the amine groups. The structure of the complex CuPCrSpm is planar with distortion towards tetrahedral. Calculation of the minimum stabilization energy for the CuPCr and CuPCrenH complexes confirmed the proposed coordination mode.     

Transition-metal complexes as alternatives to proteolytic enzymes. Regioselective cleavage of myoglobin by palladium(II) aqua complexes

 The palladium(II) aqua complexes [Pd(H₂O)₄]²⁺, cis-[Pd(en)(H₂O)₂]²⁺, and cis-[Pd(dtco-OH)(H₂O)₂]²⁺ effect hydrolytic cleavage of horse myoglobin in aqueous solution. The conditions were optimized with the third complex. Its structure was determined by X-ray crystallographic analysis of its precursor, the square-planar complex cis-[Pd(dtco-OH)Cl₂], in which the chelating ligand adopts a boat-chair conformation. A weak interaction between the hydroxyl group and the palladium(II) atom seems to improve the stability of the reagent. The yield of cleavage after a 24-h incubation at 60  °C increases from 39% to 85% as the pH decreases from 6.2 to 3.2. The protein fragments are separated by SDS-PAGE electrophoresis and HPLC separation methods, and identified by ESIMS and MALDI-TOF mass spectrometric methods and by determination of terminal amino-acid sequences. Most of the 13 cleavage sites are clustered around the methionine, arginine, and some of the histidine residues, whose side chains can bind to palladium(II). Cleavage tends to occur at the peptide bonds one to three positions removed from the binding residues; the scissile bonds usually lie on the amino-terminal side, seldom on the carboxy-terminal side, of the binding residues. Removal of the heme and unfolding of the protein do not drastically alter the pattern of cleavage. The ability of palladium(II) aqua complexes to cleave proteins at relatively few sites, with explicable selectivity, with good to very good yield, and in weakly acidic and nearly neutral solutions, bodes well for their future use in biochemical and bioanalytical practice. Received: 23 December 1997 / Accepted: 14 April 1998     

Hydrogen atom abstraction by Cu(II)- and Zn(II)-phenoxyl radical complexes, models for the active form of galactose oxidase

The Cu(II) and Zn(II) complexes of phenoxyl radical species [M(II)(L1*)(NO3)]+ (M=Cu or Zn, L1H: 2-methylthio-4-tert-butyl-6-[[bis[2-(2-pyridyl)ethyl]amino]methyl]phenol ) and [M(II)(L2*)(NO3)]+ (M=Cu or Zn, L2H: 2,4-di-tert-butyl-6-[[bis[2-(2-pyridyl)ethyl]amino]methyl]phenol) are prepared as model complexes of the active form of galactose oxidase (GAO). Hydrogen atom abstraction of 1,4-cyclohexadiene and tert-butyl substituted phenols by the GAO model complexes proceeds very efficiently to give benzene and the corresponding phenoxyl radical or its C-C coupling dimer as the oxidation products, respectively. Kinetic analyses on the oxidation reactions have shown that the hydrogen atom abstraction of the phenol substrates is significantly enhanced by the coordinative interaction of the OH group to the metal ion center of the complex, providing valuable insight into the enzymatic mechanism of the alcohol oxidation. Details of the substrate-activation process have been discussed based on the activation parameters (deltaH* and deltaS*) of the reactions.     

On the relative stability of tetragonal and trigonal Cu(II) complexes with relevance to the blue copper proteins

 The role of the cysteine thiolate ligand for the unusual copper coordination geometry in the blue copper proteins has been studied by comparing the electronic structure, geometry, and energetics of a number of small Cu(II) complexes. The geometries have been optimised with the density functional B3LYP method, and energies have been calculated by multiconfigurational second-order perturbation theory (the CASPT2 method). Most small inorganic Cu(II) complexes assume a tetragonal geometry, where four ligands make σ bonds to a Cu 3d orbital. If a ligand lone-pair orbital instead forms a π bond to the copper ion, it formally occupies two ligand positions in a square coordination, and the structure becomes trigonal. Large, soft, and polarisable ligands, such as SH^– and SeH^–, give rise to covalent copper-ligand bonds and structures close to a tetrahedron, which might be trigonal or tetragonal with approximately the same stability. On the other hand, small and hard ligands, such as NH₃, OH₂, and OH^–, give ionic bonds and flattened tetragonal structures. It is shown that axial type 1 (blue) copper proteins have a trigonal structure with a π bond to the cysteine sulphur atom, whereas rhombic type 1 and type 2 proteins have a tetragonal structure with σ bonds to all strong ligands. The soft cysteine ligand is essential for the stabilisation of a structure that is close to a tetrahedron (either trigonal or tetragonal), which ensures a low reorganisation energy during electron transfer. Received: 9 July 1997 / 26 November 1997