Snapshots of a metamorphosing Cu(II) ground state in a galactose oxidase-inspired complex

The novel ligand 2,6-bis[S-(3,5-di-tert-butyl-2-hydroxyphenyl)sulfanylmethyl]pyridine (H(2)L1) and its copper(II) complex Cu(L1), 1, were synthesized with the aim of constructing a model of the active site of the enzyme galactose oxidase (GOase). Cyclic voltammetry studies show that 1 undergoes ligand-based quasi-reversible oxidations (phenolate/phenoxyl) and reversible metal-based reduction [copper(II)/copper(I)] similar to those of GOase, but at potentials much higher and lower, respectively, than those found for the enzyme. At room temperature, spectrophotometric titrations show that 1 binds strongly to 1 equiv of pyridine. In frozen solutions (77 K), 1 quantitatively binds both pyridine and ethers (e.g., 1,4-dioxane) as assessed by X- and Q-band EPR spectroscopy. Profound shifts in the pattern of g values result, from rhombic (g(1) > g(2) > g(3)) in toluene to either inverted axial patterns (g(1) = g(2) > g(3)) in the presence of ethers or a near-axial pattern (g(1) > g(2) > g(3)) in the presence of pyridine. Crystallographic analyses of the parent complex 1.MeCN, the dioxane-bridged dimer [(Cu(L1))(2)((mu-1,4)-1,4-dioxane)].(Me(2)CO)(2) (2), and the pyridine complex [Cu(L1)(pyridine)] (3) show that the pyridine and ether ligands bond to copper at a sixth octahedral position left vacant by the pentadentate NO(2)S(2) coordination mode of L1(2-) and induce perturbations of its geometry. Hybrid DFT calculations based on the crystallographic coordinates combined with perturbation theory expressions for the g values of a d(9) system correlate the results from EPR spectroscopy to the proportions of d(x)(2)(-)(y)(2) and d(z)(2) character in the relevant copper-centered unoccupied molecular orbital. The combination of spectroscopic, structural, and computational results for this set of copper(II) complexes provides a demonstrative example of the physical phenomena underlying rhombic EPR spectra of d(9) systems.     

Synthesis, Structure, and Properties of a Model for Galactose Oxidase

An active-site analog of the radical copper enzyme galactose oxidase has been prepared from a synthetic tripod chelate ((2-pyridylmethyl)[(2-hydroxy-3,5-dimethylphenyl)methyl][(2-hydroxy-5-methyl-3-(methylthio)phenyl)methyl]amine, duncamine (dnc)) that binds a single Cu(II) ion through phenolate, thioether-substituted phenolate, and pyridylamine arms. The Cu complex crystallizes as a dinucleated dimer bridged by phenolate oxygens, and the structure has been determined by X-ray crystallography. Addition of pyridine (or other coordinating bases) dissociates the complex into a monomeric derivative that has been characterized spectroscopically (optical absorption and EPR) and electrochemically. The model provides insight into the properties of a mutant form of galactose oxidase which retains the same copper ligand complement as the wild type protein but lacks catalytic activity.     

Synthesis and Crystal Structure of Copper(II) Complex with Mixed Bipyridine and 2-Hydroxy-1-naphthaldehyde Ligands

1 INTRODUCTION Galactose oxidase is a monomeric enzyme that catalyzes the stereospecific oxidation of a broad range of primary alcohol substrates and possesses a unique mononuclear copper site essential for catalyzing a two-electron transfer reaction during the oxidation of primary alcohol to corresponding aldehydes[1]. The catalytic reaction is shown in Eq. 1. RCH2OH + O2 RCHO + H2O2 (1) A recent report on the crystal structure of galactose oxidase reveals a unique mo…     

Dissecting an enzyme—model compounds for the galactose oxidase radical site

The active site of the enzyme galactose oxidase (GOase) contains square‐pyramidal monocopper site, one of whose ligands is a tyrosinate side‐chain that is oxidized to an unusually stable radical in the active enzyme. The structure of this non‐innocent tyrosinate is unique in two ways. First, the tyrosine ring is crosslinked to a neighboring cysteine residue, affording an orthoalkylsulfanyl‐substituted phenoxide ligand. Second, this assembly is protected by a π–π interaction to a tryptophan indole group. We describe here a series of compounds designed to model various aspects of the structure of this unusual cofactor. Our studies have shown that the thermodynamic stability of the GOase radical can be attributed almost exclusively to its thioether substituent, that the π–π interaction contributes little to this stability, and that the assignment of the optical spectrum of the GOase radical is more complex than had been previously suggested. © 2002 Wiley Periodicals, Inc. Heteroatom Chem 13:494–500, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10091     

Autoredox interconversion of two galactose oxidase forms GOase(ox) and GOase(semi) with and without dioxygen

Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOase(ox)(Cu(II)-Tyr(*)) and GOase(semi)(Cu(II)-Tyr), as a result of processes involving the formation and decay of the Cu(II)-coordinated tyrosyl radical (Tyr(*)). In this work the two reactions have been studied by UV-vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOase(ox) to GOase(semi), which in air-free conditions is 100% complete. Rate constants (k(red)) are dependent on pH, and previously defined acid dissociation constants pK(1a) = 5.7 (exogenous H(2)O ligand), and pK(2a) = 8.0 (axial H(+)Tyr-495) apply. Values of k(red)(25 degrees C) range from 1.55 x 10(-4) s(-1) (pH 5.5) to 2.69 x 10(-4) s(-1) (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N(3)(-) or NCS(-) present in amounts sufficient to give >98% binding at the substrate binding (exogenous) site, while CH(3)CO(2)(-) and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 degrees C) is 161 M(-1) at pH 6.4 for acetate (previous value 140 M(-)(1)) and 46 M(-1) at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 A from the Cu) is chemically modified with HSPO(3)(2-), and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOase(semi) to GOase(ox) only occurs with O(2) present, when a first-order dependence on [O(2)] is observed, giving k(ox)(25 degrees C) = 0.021 M(-1) s(-1) at pH 7.5. This process is unaffected by NCS(-) or N(3)(-) bound at the exogenous site, and a mechanism involving outer-sphere reaction of O(2) to O(2)(-) followed by a fast step O(2) to H(2)O(2) is proposed. As GOase(ox) is formed, autoreduction back to GOase(semi) occurs, and at pH 7.5 with O(2) in large excess (1.13 mM) the maximum conversion to GOase(ox) is 69%. The k(ox) reaction proceeds to completion with >98% N(3)(-) bound at the exogenous site.     

Pyridazine-bridged copper(I) complexes of bis-bidentate ligands: tetranuclear [2 x 2] grid versus dinuclear side-by-side architectures as a function of ligand substituents

Eight bis-bidentate Schiff-base ligands, derived from 3,6-diformylpyridazine and substituted amino-benzenes, have been prepared. A variety of electron donating/withdrawing and/or sterically demanding/undemanding substituents were employed. Two ligands and five of the six pure copper(I) complexes have been structurally characterised. The sterically unhindered ligand derived from 3,5-difluoroaniline, (m,m-F), was almost completely flat whereas the very sterically hindered ligand derived from trimethylaniline, (o,o,p-Me), was severely twisted. The only dinuclear side-by-side complex obtained, [Cu(I)(2)((o-Ph))(2)](PF(6))(2), was of the ligand derived from 2-aminobiphenyl. All five of the other complexes are believed to be [2 x 2] tetranuclear grid complexes, and this was unequivocally shown to be the case for four of these complexes, [Cu(I)(4)((p-Me))(4)](PF(6))(4), [Cu(I)(4)((o,p-Me))(4)](PF(6))(4), [Cu(I)(4)((m,m-F))(4)](PF(6))(4) and [Cu(I)(4)((m,m-Cl))(4)](PF(6))(4). In all cases the copper(I) centres are substantially distorted from tetrahedral, with the most severe distortion present in the side-by-side complex. In the absence of any special effects, tetracopper(I) [2 x 2] grid architectures are observed to be the favored outcome for 1 : 1 reactions of these bis-bidentate ligands with copper(I) ions. Only when the aromaticity of the ligand was extended by employing a phenyl substituent on the phenyl rings, (o-Ph), did a dicopper(I) side-by-side architecture result. Cyclic voltammetry in acetone revealed that the free ligands did not undergo reduction until potentials below -0.8 V, whereas between three and four reversible one electron reductions were observed, between +0.16 and -0.71 vs. AgCl/Ag, for the tetranuclear copper(I) [2 x 2] grid complexes. The redox potentials observed for these complexes are highly dependent on the nature of the ligand phenyl ring substituent(s). The side-by-side complex had one irreversible reduction process, E(pc)ca.-0.5 V.     

Oxygen-Depleted Calixarenes as Ligands for Molecular Models of Galactose Oxidase

A calix[4]arene ligand, in which two of the phenol functions are replaced by pyrazole units has been employed to mimic the His₂–Tyr₂ (His: histidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase (GO). The calixarene backbone forces the corresponding copper(II) complex into a see-saw-type structure, which is hitherto unprecedented in GO modelling chemistry. It undergoes a one-electron oxidation that is centered at the phenolate donor leading to a copper-coordinated phenoxyl radical like in the GO. Accordingly, the complex was tested as a functional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl-radical equivalents as oxidants. Finally, the results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pockets that allow for the creation of novel types of model compounds.     

含5-氯水杨醛Schiff碱配体的镍和铜配合物的合成及晶体结构

合成了2个含三齿Schiff碱配体和单齿N-杂环分子的多核过渡金属配合物:1个含5-氯水杨醛缩对硝基苯甲酰腙(H2L1)和吗啡啉(Mf)的镍髤配合物[Ni(L1)(Mf)](1),1个含5-氯水杨醛缩水杨酰腙(H2L2)和吡啶(Py)的铜髤配合物[Cu2(L2)2(Py)2](2),并通过元素分析、红外光谱、紫外光谱以及单晶衍射等手段进行表征。在配合物1中,中心Ni髤与酰腙配体(L12-)的酚氧、亚胺氮、去质子酰胺氧原子以及中性吗啡啉氮原子配位形成平面四方形的N2O2配位构型,相邻配合物通过分子间氢键作用构筑成一维超分子链状结构。配合物2中含有2个晶体学上独立的双核铜髤配合物,相邻配合物分子的酚氧原子分别桥联2个[Cu(L2)(Py)]基本单元,形成2个含有Cu2(μ-O)2核心的配合物。每个Cu髤原子具有五配位的NONO(O)四角锥配位构型。     

Heavily fluorinated carbohydrates as enzyme substrates: oxidation of tetrafluorinated galactose by galactose oxidase

Galactose oxidase (GOase) was shown to oxidise several C2/C3 fluorinated galactose analogues. Interestingly, the enzyme was able to distinguish between the 2,3-tetrafluorinated galactose and its epimeric glucose analogue, and this represents the first reported biotransformation of a heavily fluorinated sugar.     

Models for the active site in galactose oxidase: Structure, spectra and redox of copper(II) complexes of certain phenolate ligands

Galactose oxidase (GOase) is a fungal enzyme which is unusual among metalloenzymes in appearing to catalyse the two electron oxidation of primary alcohols to aldehydes and H₂O₂. The crystal structure of the enzyme reveals that the coordination geometry of mononuclear copper(II) ion is square pyramidal, with two histidine imidazoles, a tyrosinate, and either H₂O (pH 7.0) or acetate (from buffer,pH 4-5) in the equatorial sites and a tyrosinate ligand weakly bound in the axial position. This paper summarizes the results of our studies on the structure, spectral and redox properties of certain novel models for the active site of the inactive form of GOase. The monophenolato Cu(II) complexes of the type [Cu(L1)X][H(L1) = 2-(bis(pyrid-2-ylmethyl)aminomethyl)-4-nitrophenol and X⁻ = Cl⁻ 1, NCS⁻ 2, CH₃COO⁻ 3, ClO₄ ⁻ 4] reveal a distorted square pyramidal geometry around Cu(II) with an unusual axial coordination of phenolate moiety. The coordination geometry of 3 is reminiscent of the active site of GOase with an axial phenolate and equatorial CH₃COO⁻ ligands. All the present complexes exhibit several electronic and EPR spectral features which are also similar to the enzyme. Further, to establish the structural and spectroscopic consequences of the coordination of two tyrosinates in GOase enzyme, we studied the monomeric copper(II) complexes containing two phenolates and imidazole/pyridine donors as closer structural models for GOase. N,N-dimethylethylenediamine and N,N’-dimethylethylenediamine have been used as starting materials to obtain a variety of 2,4-disubstituted phenolate ligands. The X-ray crystal structures of the complexes [Cu(L5)(py)], (8) [H₂(L5) = N,N-dimethyl-N’,N’-bis(2-hydroxy-4-nitrobenzyl) ethylenediamine, py = pyridine] and [Cu(L8)(H₂O)] (11), [H₂(L8) = N,N’-dimethyl-N,N’-bis(2-hydroxy-4-nitrobenzyl)ethylenediamine] reveal distorted square pyramidal geometries around Cu(II) with the axial tertiary amine nitrogen and water coordination respectively. Interestingly, for the latter complex there are two different molecules present in the same unit cell containing the methyl groups of the ethylenediamine fragmentcis to each other in one molecule andtrans to each other in the other. The ligand field and EPR spectra of the model complexes reveal square-based geometries even in solution. The electrochemical and chemical means of generating novel radical species of the model complexes, analogous to the active form of the enzyme is presently under investigation.     

Redox-adaptable copper hosts. Pyridazine-linked cryptands accommodate copper in a range of redox States

A series of structurally characterized copper complexes of two pyridazine-spaced cryptands in redox states + (I,I), (II,I), (II), (II,II) are reported. The hexaimine cryptand L(I) [formed by the 2 + 3 condensation of 3,6-diformylpyridazine with tris(2-aminoethyl)amine (tren)] is able to accommodate two non-stereochemically demanding copper(I) ions, resulting in [Cu(I)(2)L(I)](BF(4))(2) 1, or one stereochemically demanding copper(II) ion, resulting in [Cu(II)L(I)()](BF(4))(2) 3. Complex 3 crystallizes in two forms, 3a and 3b, with differing copper(II) ion coordination geometries. Addition of copper(I) to the monometallic complex 3 results in the mixed-valence complex [Cu(I)Cu(II)L(I)](X)(3) (X = PF(6)(-), 2a; X = BF(4)(-), 2b) which is well stabilized within this cryptand as indicated by electrochemical studies (K(com) = 2.1 x 10(11)). The structurally characterized, octaamine cryptand L(A), prepared by sodium borohydride reduction of L(I), is more flexible than L(I) and can accommodate two stereochemically demanding copper(II) ions, generating the dicopper(II) cryptate [Cu(II)(2)L(A)](BF(4))(4) 4. Electrochemical studies indicate that L(A) stabilizes the copper(II) oxidation state more effectively than L(I); no copper redox state lower than II,II has been isolated in the solid state using this ligand.     

A tris(mu-hydroxy)tricopper(II) complex as a model of the native intermediate in laccase and its relationship to a binuclear analogue

The reaction of a copper(I) complex with a sterically demanding secondary diamine ligand and O2 yields a tris(mu-hydroxy)tricopper(II) complex. This complex is a structural model of the proposed native intermediate in multicopper oxidases, with interesting structural, magnetic, and solution properties.     

含取代芳酰腙和单齿N-杂环分子铜(II)配合物的合成、结构及荧光性质

合成了1个含有取代芳酰腙和单齿N-杂环分子的三元铜配合物[Cu(L)(ampy)](H₂L=5-溴水杨醛苯甲酰腙，ampy=2-氨基吡啶)，并通过IR、UV、荧光光谱和循环伏安进行了性质研究。[Cu(L)(ampy)]的晶体结构分析表明，中心金属通过酰腙配体的酚基氧原子、亚胺基氮原子、去质子酰胺氧原子以及中性杂环分子的氮原子形成平面四方形的N₂O₂配位环境。配合物通过N-H…O和N-H…N氢键作用形成一维链状结构。     

Pulse radiolysis studies on galactose oxidase

Single-Cu-containing galactose oxidase in the GOase(semi) state (Cu(II), no Tyr(*) radical) reacts with pulse radiolysis generated formate radicals CO(2)(*-) to give an intermediate UV-vis spectrum assigned as RSSR(*-), peak at 450 nm (epsilon = 8100 M(-1) cm(-1)). From a detailed kinetic analysis at 450 nm, pH 7.0, the following steps have been identified. First the strongly reducing CO(2)(*-) (-1.9V) reduces GOase(semi) (k(0) > or = 6.5 x 10(8) M(-1) s(-1)) to a species GOase(semi)(*-). This is followed by biphasic reactions (i) GOase(semi)(*-) + GOase(semi) (k(1) = 1.6 x 10(7) M(-1) s(-1)) to give GOase(semi) + P(*-) and (ii) P(*-) + GOase(semi) (k(2) = 6.7 x 10(6) M(-1) s(-1)) to give GOase(semi)RSSR(*-). There are no significant absorbance changes for the formation of GOase(semi)(*-) and P(*-), which are Cu(I) (or related) species. However, GOase(semi)RSSR(*-) has an absorption spectrum which differs significantly from that of GOase(semi). The 450 nm peak is characteristic of an RSSR(*-) radical with two cysteines in close sequence proximity and is here assigned to Cys515-Cys518, which is at the GOase surface and 10.2 A from the Cu. On chemical modification of the RSSR group with HSPO(3)(2-) to give RSSPO(3)H(-) and RS(-), absorbance changes are approximately 50% of those previously observed. The decay of RSSR(*-) (0.17 s(-1)) results in the formation of GOase(red). No RSSR(*-) formation is observed in the reaction of GOase(semi) Tyr495Phe with CO(2)(*-), and a single process giving GOase(red)Tyr495Phe occurs. Similarly in the reaction of GOase(ox) with CO(2)(*-), a single-stage reaction gives GOase(semi).     

Reaction of beta-diketiminate copper(II) complexes and Na2S2

Reaction of beta-diketiminate copper(II) complexes and Na2S2 resulted in formation of (mu-eta2:eta2-disulfido)dicopper(II) complexes (adduct formation) or beta-diketiminate copper(I) complexes (reduction of copper(II)) depending on the substituents of the supporting ligands. In the case of sterically less demanding ligands, adduct formation occurred to provide the (mu-eta2:eta2-disulfido)dicopper(II) complexes, whereas reduction of copper(II) took place to give the corresponding copper(I) complexes with sterically more demanding beta-diketiminate ligands. Spectroscopic examinations of the reactions at low temperature using UV-vis and ESR as well as kinetic analysis have suggested that a 1 : 1 adduct LCuII-S-SNa with an end-on binding mode is initially formed as a common intermediate, from which different reaction pathways exist depending on the steric environment of the metal-coordination sphere provided by the ligands. Thus, with the sterically less demanding ligands, rearrangement of the disulfide adduct from end-on to side-on followed by self-dimerisation occurs to give the (mu-eta2:eta2-disulfido)dicopper(II) complexes, whereas such an intramolecular rearrangement of the disulfide co-ligand does not take place with the sterically more demanding ligands. In this case, homolytic cleavage of the CuII-S bond occurs to give the reduced copper(I) product. The steric effects of the supporting ligands have been discussed on the basis of detailed analysis of the crystal structures of the copper(II) starting materials.     

Synthesis, crystal structure, magnetic properties and theoretical studies on a one-dimensional polynuclear copper(II) complex [Cu2(mu1,3-SCN)2(mu'1,3-SCN)2(MPyO)2]n

A new one-dimensional polynuclear copper(II) complex [Cu(2)(mu(1,3)-SCN)(2)(mu'(1,3)-SCN)(2)(MPyO)(2)](n)(where MPyO = 4-methylpyridine N-oxide) has been synthesized and its crystal structure determined by X-ray crystallography. In the complex there exist two kinds of bridging coordination modes, namely, mu(1,3)-SCN(-) equatorial-equatorial (EE) bridging ligand and micro'(1,3)-SCN(-) equatorial-axial (EA) bridging ligand. Two micro(1,3)-SCN(-) EE bridging ligands coordinate two copper(II) ions in a binuclear unit, and the S atoms from the micro'(1,3)-SCN(-) EA bridging ligands as axial coordinated atoms link the binuclear units into one-dimensional chains. The ESR spectra have been investigated, and variable temperature (4-300 K) magnetic measurements were analyzed using a binuclear Cu(ii) magnetic interaction formula and indicate the existence of strong antiferromagnetic coupling with 2J=- 216.00 cm(-1) between bridged copper(II) ions. Density functional calculations have been carried out on this binuclear unit, yielding a similar singlet-triplet splitting. The mechanism of strong antiferromangetic interaction is revealed according to the calculations.     

Synthesis and Crystal Structure of a Dinuclear Cu(II) Complex with Tridentate Schiff Base and Azido Bridge

1 INTRODUCTION The azido ligand is an efficient superexchange path-way for propagating magnetic interaction between theparamagnetic centers, such as copper(II), giving di-nuclear, tetranuclear, 1D, 2D and 3D complexes[1～4].The versatility of this ligand due to its diverse bin-ding modes leads to the variation in magnetic pro-perties that depend on its orientation with respect tothe magnetic centers. In general, the bridging modesobserved for the azido group are endtoend and en-don. In…     

Binding of Ru(bpy)2(eilatin)2+ to matched and mismatched DNA

The DNA-binding properties of Ru(bpy)2(eilatin)(2+) have been investigated to determine if the sterically expansive eilatin ligand confers specificity for destabilized single-base mismatches in DNA. Competitive DNA photocleavage experiments employing a sequence-neutral metallointercalator, Rh(bpy)2(phi)(3+) (phi = 9,10-phenanthrenequinonediimine), and a mismatch-specific metalloinsertor, Rh(bpy)2(chrysi)(3+) (chrysi = chrysene-5,6-quinonediimine), reveal that the eilatin complex binds to a CC mismatched site with an apparent binding constant of 2.2(2) x 10(6) M(-1). Nonetheless, the selectivity in binding mismatched DNA is not high: competitive titrations with Rh(bpy)2(phi)(3+) show that the complex binds also to well-matched B-form sites. Thus, Ru(bpy)2(eilatin)(2+), despite containing the extremely expansive eilatin ligand, displays lower selectivity for the mismatch than does Rh(bpy)2(chrysi)(3+), a metalloinsertor containing the smaller, though still bulky, chrysene-5,6-quinonediimine ligand. In summary, the size and shape of the eilatin ligand allow stacking with both well-matched and mismatched DNA.     

配合物[Cu_2(TS)(H_2O)]·H_2O的自组装制备、晶体结构和电化学性质

利用单核配合物Na2 CuTS·H2 O在溶剂中的不稳定性质 ,自分解并重新组装得到了双核配合物 [Cu2 (TS)(H2 O) ]·H2 O .配合物为单斜晶系 ,空间群P2 1/C ,晶胞参数为a =0 .80 830 (16 )nm ,b =1.70 79(3)nm ,c =1.4 0 73(3)nm ,β =10 5 .0 8(3)° ,V =1.86 5 4 (6 )nm3 ,Z =4 ,最终一致性因子R =0 .0 6 4 4 .该分子结构为双核单元 ,金属原子通过两个酚氧原子桥联在一起 .结合晶体结构对配合物作了电化学研究     

Intramolecular charge transfer and biomimetic reaction kinetics in galactose oxidase model complexes

One-electron oxidation of two structurally similar CuII-diphenolate complexes, 1 and 2, creates EPR-silent CuII-phenoxyl complexes [1]+ and [2]+ that mimic the oxidized form of the enzyme galactose oxidase (GOase). Both model complexes display novel NIR absorptions assigned to phenolate-phenoxyl charge transfer that resemble a tyrosinate-tyrosyl charge-transfer band observed in the enzymatic system. [1]+ and [2]+ react with benzyl alcohol to form 0.5 equivs of benzaldehyde per complex; biomimetic reduction to CuI-phenol complexes is not observed, but such species may exist transiently. Initial kinetic studies show that [2]+ reacts faster with benzyl alcohol than does [1]+, despite being a significantly weaker oxidant (DeltaE degrees = 370 mV). This acceleration is ascribed to mechanistic differences: [2]+ appears to bind substrate prior to the rate-determining step. Large, nonclassical kinetic isotope effects confirm C-H bond cleavage as the rate-determining step in the reactions of both [1]+ and [2]+ with benzyl alcohol, as is the case for GOase.