PAPER www.rsc.org/dalton | Dalton Transactions

A new dinuclear heme-copper complex derived from functionalizedprotoporphyrin IX†

Corrado Dallacosta,a Wendel A. Alves,b Ana M. da Costa Ferreira,c Enrico Monzania and Luigi Casella*a

Received 2nd March 2007, Accepted 4th April 2007First published as an Advance Article on the web 19th April 2007DOI: 10.1039/b703240d

A new biomimetic model for the heterodinuclear heme/copper center of respiratory oxidases isdescribed. It is derived from iron(III) protoporphyrin IX by covalent attachment of a Gly-L-His-OMeresidue to one propionic acid substituent and an amino-bis(benzimidazole) residue to the otherpropionic acid substituent of the porphyrin ring, yielding the FeIII complex 1, and subsequent additionof a copper(II) or copper(I) ion, according to needs. The fully oxidized FeIII/CuII complex, 2, bindsazide more strongly than 1, and likely contains azide bound as a bridging ligand between FeIII and CuII.The two metal centers also cooperate in the reaction with hydrogen peroxide, as the peroxide adductsobtained at low temperature for 1 and 2 display different optical features. Support to this interpretationcomes from the investigation of the peroxidase activity of the complexes, where the activation ofhydrogen peroxide has been studied through the phenol coupling reaction of p-cresol. Here the presenceof CuII improves the catalytic performance of complex 2 with respect to 1 at acidic pH, where thepositive charge of the CuII ion is useful to promote O–O bond cleavage of the iron-boundhydroperoxide, but it depresses the activity at basic pH because it can stabilize an intramolecularhydroxo bridge between FeIII and CuII. The reactivity to dioxygen of the reduced complexes has beenstudied at low temperature starting from the carbonyl adducts of the FeII complex, 3, and FeII/CuI

complex, 4. Also in this case the adducts derived from the FeII and FeII/CuI complexes, that weformulate as FeIII–superoxo and FeIII/CuII–peroxo exhibit slightly different spectral properties, showingthat the copper center participates in a weak interaction with the dioxygen moiety.

Introduction

Cytochrome c oxidase (COX) is the terminal enzyme of themitochondrial respiratory chain catalyzing the four-electron/four-proton reduction of dioxygen to water.1–7 The functional unitcontains four redox-active metal centers (heme a, CuA, hemea3, CuB), two of which (heme a3, CuB) are in close proximityand constitute a unique dinuclear complex involved in dioxygenbinding and reduction, and proton pumping.8–11 Besides theporphyrin, the iron in heme a3 is axially bound to a histidineand three other histidines constitute the protein donor ligandsof CuB. The coupling between heme a3 and CuB in the oxidized,resting state of the enzyme is responsible for the absence of anEPR signal for this dinuclear unit. Spectroscopic and mechanisticinvestigations on the reduced enzyme suggest that, after a possibleinitial O2 interaction with CuB, a myoglobin-like Fe–O2 adduct(best described as a FeIII–O2

− superoxo species) forms andevolution of this species leads to a FeIV=O (ferryl) species.12–14

Although a transient peroxo-bridged FeIII–O22−–CuII intermediate

aDipartimento di Chimica Generale, Universita di Pavia, Via Taramelli 12,27100 Pavia, Italy. E-mail: bioinorg@unipv.it; Fax: +39 0382 528544bCentro de Ciencias Naturais e Humanas, Universidade Federal do ABC,09210-170 Santo Andre, SP, BrazilcInstituto de Quımica, Universidade de Sao Paulo, 05508-900 Sao Paulo,SP, Brazil† Electronic supplementary information (ESI) available: In Table 1S thecomplete electronic spectral data for complexes 1–8 is given. Fig. 1Sreports the paramagnetic proton NMR spectrum of compound 1. SeeDOI: 10.1039/b703240d

on the way to the ferryl species cannot be excluded a priori, suchan intermediate has not been observed so far. One of the CuB

imidazole ligands, which is cross-linked to a tyrosine, is thought toplay a key-role in electron shuttling and reduction of iron-bounddioxygen.12,15–19

The nature of integral membrane protein and the structuralcomplexity, due to the presence of several subunits, make thebiochemical and mechanistic studies on the enzyme particularlycomplex. Therefore, model studies of the dinuclear heme a–CuB

active site can be of great importance to improve our understand-ing of the properties of this biological heterodinuclear complexand the relevant dioxygen chemistry occurring at this site. Indeed,considerable progress has been made in the characterization ofO2 adducts of heme–Cu complexes, particularly by the groupsof Collman,20 Karlin,21–26 and Naruta,27,28 in the latter case alsowith the crystal structure determination of an unprecedented g2:g1-FeIII–O2

2−–CuII species.27 These model systems are generally basedon synthetic, non-natural porphyrins (e.g. substituted tetra-arylporphyrins) and it is interesting to note that in the two cases wherethe iron is bound to an axial ligand, a heme–superoxide adduct(FeIII–O2

−) is obtained,20,28 whereas in the other cases in whichiron lacks a medium- or strong-field axial ligand, some type ofheme/copper–peroxide adduct is formed.21–27

Some years ago we reported some heme–copper complexesderived from deuteroporphyrin modified at one or both propionateside arms with a poly(benzimidazole) residue (to act as a Cubinding ligand) and a histidine residue (to act as an iron axialligand).29–31 In this paper we expand upon these previous model

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systems by reporting complexes derived from a similarly modified,protoporphyrin IX ligand, 2L, and its mononuclear [(2L)FeIII]+

(1) and dinuclear [(2L)FeIIICuII]3+ (2) complexes (Scheme 1). TheFeIII complex [(1L)FeIII]+, derived from protoporphyrin IX butcarrying only the histidine-containing residue attached at onepropionate side chain, is also reported for comparison purposes.These complexes represent a family of potentially useful modelsfor the heme–Cu site of cytochrome c oxidase because theyare derived from a natural porphyrin and although it is knownthat natural porphyrins are much more oxidation labile thansynthetic tetra-aryl porphyrins, we expect that the properties ofthese complexes (e.g. the spectral properties and the intrinsicFe reactivity) may be more directly associated with those ofthe enzyme heme a3–CuB complex. With respect to the previousdeuterohemin/Cu complexes, the present systems contain furtherstructural refinement in the histidine-containing arm attachedto the porphyrin propionate, which is now a protected Gly-His dipeptide, instead of a simple His residue, because we havepreviously shown that such a lengthening of the side arm enablesa stronger interaction between the axial imidazole and the hemeiron.32 This last feature is crucial because it allows the iron centerto have a coordinative environment similar to that present incytochrome c oxidase and in most heme-containing proteins. In

Scheme 1 (i): HOBt, HBTU, triethylamine, room temperature; (ii):Cu(ClO4)2.

the present model system, the copper and iron centers cooperatein binding exogenous ligands (e.g. azide ion) and small moleculesubstrates such as molecular oxygen and hydrogen peroxide. Atlow temperature, the reaction of the reduced forms of 1 and 2 withO2 gives rise to dioxygen adducts with different spectral featuresand, in addition, in the dimetallic system 2 the thermal degradationof the dioxygen adduct is significantly delayed compared to thecorresponding form derived from 1.

Results

Spectral properties and azide binding to [(2L)FeIII]+ and[(2L)FeIIICuII]3+

The electronic spectra of [(2L)FeIII]+ (1) and [(2L)FeIIICuII]+ (2)show the porphyrin Soret band (∼400 nm) and visible bands(∼485, 575, and 610 nm) at positions typical for FeIII in the high-spin state (see ESI†). The characteristic benzimidazole bands arealso observable below 300 nm. The binding of azide to 1 and 2was investigated by UV-Vis and EPR spectroscopies to assess thepossibility of simultaneous binding of the ligand to FeIII and CuII

in a bridging mode. The addition of sodium azide to a methanolsolution of 1 or 2 causes marked changes in the Soret region ofthe electronic spectra, due to the change in the spin state of FeIII,from high spin to low spin. Fig. 1 shows, as an example, the UV-Vis spectral changes occurring upon titration of [(2L)FeIIICuII]3+

with a solution of N3−. The difference spectra show a minimum

at 400 and a maximum at 420 nm, as a result of the shift of theSoret band towards lower energy. The plot of D absorbance (420–400 nm) vs. [N3

−] for complexes 1 and 2 is reported in the insetof Fig. 1. The data could not be fitted considering the binding ofa single azide anion, while the binding isotherm considering twoconsecutive equilibria of azide binding to the iron center fitted thedata well, with the first step accounting for a large part of thespectral changes. The two binding constants (K1 and K2) obtainedfor 1 and 2 are reported in Table 1. It should be noted that it cannotbe excluded that further azide ions can bind to the copper centerof 2; in fact, the changes of the Soret band can only reflect changesat the iron site. An azide ion binding to copper(II) would produce

Fig. 1 Difference spectra observed for complex [(2L)FeIIICuII]3+ (about6.7 lM) upon titration with sodium azide in methanol. Inset: plot ofD absorbance (420–400 nm) vs. azide concentration for complexes 1(diamonds) and 2 (circles), showing the fitting of the data with twoconsecutive binding equilibria.

2198 | Dalton Trans., 2007, 2197–2206 This journal is © The Royal Society of Chemistry 2007

Table 1 Association constants for the binding of azide to complexes 1and 2 in methanol solution

Complex K1/M−1 K2/M−1

[(2L)FeIII]+ (1) 32 000 ± 2000 1700 ± 100[(2L)FeIIICuII]3+ (2) 169 000 ± 6000 4400 ± 200

LMCT spectral changes too weak to be appreciated under theintense porphyrin absorption.

EPR spectra

The copper signal in the frozen solution EPR spectrum of 2 is simi-lar to that of the CuII complex derived from N-acetyl-N,N-bis[2-(1-methylbenzimidazol-2-yl)ethyl]amine, [Cu(AcBB)]2+,33 containingthe same flexible bidentate residue, although the analysis of thesignal is complicated by the presence of an iron signal near 2900 G(Fig. 2A). A better comparison between the copper signals of thetwo complexes can be made in the EPR spectra recorded at highertemperatures, where the iron signal disappears. As the CuII spin–lattice relaxation time is long, its signals can be detected even at170 K, as shown for complex 2 in Fig. 2B. For both [Cu(AcBB)]2+33

Fig. 2 (A) EPR spectra of [Cu(AcBB)]2+ and [(2L)FeIIICuII]3+ (2) (highfield portion) recorded in frozen methanol solution at 77 K; (B) Compari-son of the EPR spectra of [Cu(AcBB)]2+ (at 77 K) and [(2L)FeIIICuII]3+ (at170 K) recorded in frozen methanol-solution, after baseline correction.

and 2 the |A‖| values below 150 × 10−4 cm−1 suggest the presenceof a five-coordinated CuII species, with three additional solventmolecules bound to the metal. The EPR spectra of the iron centersin 1 and 2 in frozen methanol solution (77 K) are shown in Fig. 3.Both spectra show rhombic distortion and a mixture of a majorhigh-spin species, with a broad signal near g = 5.8, and a minorlow-spin species, with g values of 2.70, 2.29 and 1.74, respectively.The low-spin species is not observed in the electronic spectrumand is therefore associated with the much higher concentrationof the EPR sample. In fact, upon dilution of the solution, theratio between the high-spin and low-spin components in the EPRspectra increases. Although for complex 1 we can hypothesize thatone of the benzimidazole groups acts as the iron sixth ligand, thispossibility seems excluded by the identity of the low-spin signalsfor complexes 1 and 2, where the benzimidazole groups are boundto CuII. We thus assume that this low-spin species results fromaggregation of the iron–porphyrin complexes, as is often observedfor sterically non-protected porphyrins.30

Fig. 3 EPR spectra of [(2L)FeIII]+ (1) and [(2L)FeIIICuII]3+ (2) recorded infrozen methanol solution at 77 K. The components of the iron and copperEPR signals are indicated by vertical dashed lines.

Addition of N3− to a methanolic solution of 2 results in the

disappearance of both the high-spin and low-spin iron signals, andin the appearance of a new signal of low spin type. The processcan be monitored by the decrease of the signal at g = 5.80 andthe growth of signals at g = 2.78, 2.20 and 1.74 (2395, 3025 and3830 G), which correspond to the formation of a low-spin FeIII–N3

− species (Fig. 4). Also, the EPR signal of CuII is perturbedby azide binding and N3

− causes an apparent decrease in theintensities of both the CuII and FeIII EPR signals (approximately20%), likely due to an interaction between the paramagneticcenters. In the case of complex 1, titration with azide produces theprogressive decrease of the high-spin component and increase inthe low-spin signal, but with g values similar to the initial low-spincomponent.

NMR spectra—Measurement of magnetic moments

The NMR spectra of complexes 1 and 2 in deuterated DMSO orCD3OD are consistent with high spin iron(III)–porphyrin systems,where the paramagnetic signals are distributed in the −10 to+80 ppm interval. The spectra are complicated by the presence oftwo isomers of the compounds with substitution patterns 2(18) and

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Fig. 4 EPR spectra of [(2L)FeIIICuII]3+ (2) recorded in frozen methanolsolution at 77 K upon titration with sodium azide. Complex concentrationwas 2 mM, azide concentration was (from bottom to top) 0, 0.95 mM,1.88 mM, 2.78 mM, 4.53 mM, 7.00 mM, and 9.31 mM, respectively.Vertical dashed lines highlight the high field shift of the major componentof the low-spin iron signal.

18(2) of the histidine and benzimidazole side arms at the porphyrinperiphery, as it is always found when the porphyrin derivative isobtained by functionalization of the propionate side chains,29–32

making the spectra little informative for structural purpose (seefor instance the spectrum of 1 in the ESI†). In addition, due to therelatively high concentration of the samples, the spectra exhibitpeaks of low intensity in the region between 27 and 19 ppm,attributable to porphyrin methyl groups, which are indicative ofthe presence of a minor fraction of low spin species. The attemptto follow the binding of azide to complexes 1 and 2 in CD3OD byNMR was unsuccessful because at the concentrations requiredto record reliable NMR spectra, the addition of azide causesprecipitation of the complexes.

The NMR technique has been used also to evaluate the magneticmoment exhibited by the complexes in solution through the Evans’method.34 The magnetic moment determined for 1 in CD3OD (5.33lB) agrees with a high-spin iron(III) species. The limited solubilityof 2 in CD3OD does not allow a precise determination of itsmagnetic moment; the estimated value (∼6.2 lB) is compatiblewith the presence of non-interacting, or very weakly interacting,high spin iron(III) and copper(II) centers. The precipitation causedby addition of azide prevented the analysis of the effect of thisligand on the magnetic moment of the complexes.

Peroxidase activity

Hemin complexes are able to catalyze the oxidation of typical per-oxidase substrates, such as phenolic compounds, in the presence of

hydrogen peroxide, through a catalytic cycle which is similar to thatof peroxidases and microperoxidases.35–45 The oxidation of p-cresolto phenol coupling dimers with H2O2 catalyzed by [(2L)FeIII]+ (1),[(2L)FeIIICuII]3+ (2), or [(1L)FeIII]+ was studied through the initialrates method to neglect the significant inactivation undergone bythe complexes during the reactions. The kinetics were investigatedat pH 5.0, 7.0 and 9.0 under conditions in which the rates do notdepend on the substrate concentration but are linearly dependenton [H2O2].35–37 This means that the rate-determining step of thereaction, characterized by the rate constant k1, is the formationof a high valent state of the complex, while the reaction with thesubstrate occurs in a fast step. This allowed the determination ofk1 for 1, 2, and [(1L)FeIII]+ from the slope of the rate vs. [H2O2]plot (Table 2). At pH 5.0 the rate constants increase in the order[(1L)FeIII]+ < 1 < 2. At pH 7.0 and 9.0 the reactions are muchfaster than at pH 5.0 for all the complexes but, interestingly, thereactivity order of the complexes is reversed with respect to pH 5.0,with [(1L)FeIII]+ reacting faster than 1 and 2. Though, the rateconstants undergo a marked reduction from pH 7.0 to pH 9.0.

Low temperature spectroscopic studies. Oxygenation of reducedcomplexes

A very mild reduction of [(2L)FeIII]+ was obtained by bubblingcarbon monoxide into the complex solution, at room temperatureand under anaerobic conditions, in the presence of an excess ofpiperidine (Scheme 2). The high affinity of CO for five-coordinatedheme species increases the E◦(FeIII/FeII), thus allowing the metalto be reduced by piperidine. The [(2L)FeIICO] (3) species whichforms in these conditions exhibits a sharp Soret band at 418 nm,typical for imidazole-bound protoheme carbonyl species,46 and aand b bands at 568 and 535 nm, respectively (see ESI†). Addition ofdioxygen to 3 at −60 ◦C causes a shift of the Soret band to 412 nm,with smaller shifts of the a and b bands (to 575 and 539 nm,respectively), producing the dioxygen adduct [(2L)FeII(O2)] (5)(Fig. 5A). Overall, the spectral features of [(2L)FeIICO]+ and[(2L)FeII(O2)] are similar to those of the corresponding adducts

Scheme 2 (i): CO, piperidine; (ii): [CuI(CH3CN)4]PF6; (iii): −60 ◦C, O2;(iv): −60 ◦C, H2O2–NEt3.

Table 2 Active species formation constants for the heme complexes at 25 ± 0.1 ◦C in 200 mM acetate buffer at pH 5.0, a mixture of methanol/aqueousphosphate at pH 7.0, and borate buffer 10 mM (7 : 3, v/v) at pH 9.0, respectively

Complex k1/M−1 s−1, pH 5 k1/M−1 s−1, pH 7 k1/M−1 s−1, pH 9

[(1L)FeIII]+ 20 ± 2 3160 ± 80 590 ± 10[(2L)FeIII]+ (1) 74 ± 1 1230 ± 30 228 ± 6[(2L)FeIIICuII]3+ (2) 102 ± 3 1100 ± 10 46 ± 1

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Fig. 5 Low-temperature oxygenation experiments of the reduced com-plexes in DMF solution. (A) Electronic spectra of [(2L)FeIII]+ (1) at25◦C (dashed line), [(2L)FeIICO]+ (3) at 25 ◦C (dashed and dotted line),and [(2L)FeII(O2)] (5) at −60 ◦C (solid line). (B) Electronic spectra of[(2L)FeIIICuII]3+ (2) at 25 ◦C (dashed line), [(2L)FeIICOCuI]+ (4) at 25 ◦C(dashed and dotted line), and [(2L)FeIICuI(O2)]+ (6) at −60 ◦C (solid line).

of hemoglobin and myoglobin, but with Soret bands slightly blueshifted with respect to the protein bands.47

The above reported mild procedure for the reduction of theiron(III) center of 1 does not produce a reduction of the copper(II)ion in the dinuclear complex [(2L)FeIIICuII]3+. Therefore, a fully re-duced form of 2 was obtained from [(2L)FeIICO] by the addition ofone equiv. [Cu(CH3CN)4]+, thus producing the [(2L)FeIICOCuI]+

complex 4 (Scheme 2). The UV-Vis spectrum of [(2L)FeIICOCuI]+

is very similar to that of [(2L)FeIICO], displaying bands at 418,542 and 574 nm. Addition of dioxygen to 4, at −60 ◦C, causesthe Soret band to shift to 414 nm, with slight shifts in the a andb bands, to 577 and 542 nm, respectively, producing the dioxygenadduct [(2L)FeIICuI(O2)] (6) (Fig. 5B). It can be noted that theoxygenated compounds 5 and 6 bear slightly different electronicspectra, indicating that copper exerts at least a weak interactionwith the dioxygen moiety.

Reaction of hydrogen peroxide with oxidized complexes

The reactivity of compounds 1 and 2 towards H2O2 was alsoinvestigated at low temperature, both for obtaining a comparison

with the behavior of the complexes in the oxygenation of theirreduced forms and because the interaction with hydrogen peroxideis an important step in the peroxidase-like activity exhibited by thecomplexes. Addition of 3 equiv. H2O2 and 6 equiv. triethylamine,as a base, to solutions of 1 and 2 in DMF at −60 ◦C gave riseto spectral changes that can be interpreted with the formationof peroxide adducts of the complexes upon binding to the Feand Fe/Cu centers, respectively. The Soret band of the newspecies occurs at 412 nm and is much sharper for that derivedfrom complex 1 than from complex 2 (Fig. 6). Larger differencesbetween the spectra of the peroxide adducts of 1 and 2, that welabel as complex 7 and complex 8, respectively, are observed inthe range between 500 and 650 nm (see ESI†). In particular, theposition of the a and b bands occurs at lower energy for 7 (538 and576 nm) than for 8 (548 and 578 nm) and the relative intensity ofthe two bands is opposite in the spectra of the two adducts.

Fig. 6 Comparative low-temperature spectra (−60 ◦C) of the peroxideadducts derived from 1 (i.e. 7) (solid line) and 2 (i.e. 8) (dashed line) inDMF solution.

Degradation studies

To assess the capability of the copper center to interact withiron-bound dioxygen in the dinuclear complex [(2L)FeIICuI(O2)]+

6, comparative degradation experiments were performed on theoxygenated complexes 5 and 6. The low-temperature solutions ofthe dioxygen adducts were brought to 0 ◦C, and the evolution ofthe optical spectrum vs. time was monitored (Fig. 7). Evolutionof complex 5 occurred with a small decrease and a blue-shift to405 nm of the Soret band, whereas complex 6 underwent a slowerdegradative process, also signaled by a decrease in the intensity butno appreciable shift of the Soret band. The occurring processesare connected to both iron and copper oxidation22 and porphyrindisruption. During the first 10 min, the system underwent thermalsettlement, therefore, the data collected within this time intervalwere neglected. The degradative processes for complexes 5 and6 followed an exponential behavior, with rate constants (kobs) of>0.069(7) min−1 and 0.014(1) min−1, respectively (Fig. 7). As arelevant part of degradation of the oxygenated complex 5 occurredduring the thermal settlement, the kobs can only be considered alower limit of the real value. The presence of copper clearly slowsdown the degradation, which needed a time of about 300 min to

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Fig. 7 First order degradation process undergone by the oxygenatedcomplexes 5 (triangles) and 6 (circles) at 0 ◦C in DMF solution.

reach completion. The difference in the degradation rate of 5 and6 can be attributed to some cooperation between iron and copperin binding dioxygen in 6, which likely stabilizes the oxygenatedadduct.

Discussion

The oxygen reduction mechanism, and particularly the role of CuB

in the redox process catalyzed by COX is still a matter of debate.Biomimetic models of the heme a3-CuB site have been widely usedin an attempt to unravel this problem, due to the fact that theyare easier to handle and usually much less reactive than proteinsthemselves. Many efforts have been made to develop suitablemodels and most of them interact with dioxygen forming transientspecies that in some cases have been isolated and characterized.20–28

The dinuclear complex 2 contains a bis(benzimidazole)–copperdomain linked to a heme b domain with a Gly-L-His-OMe armconnected to one of the carboxylate groups and providing theiron axial ligand. The first problem to clarify is whether theresidue carrying the bis(benzimidazole) arm has enough flexibilityto approach the heme, allowing the copper center to cooperatewith iron in ligand binding, as well as in dioxygen ligation andreduction. Previous studies on deuterohemin–copper complexesshowed that the presence of an imidazole group axially ligated tothe FeIII center promotes the folding of the bis(benzimidazole) armtowards the opposite part of the porphyrin plane.29–31 Titration of1 and 2 with azide reveals in both cases the successive binding oftwo azide ions to the FeIII center. The first azide binding processcharacterized by K1 is most relevant to this work, and it is ofnote that complex 2 exhibits a K1 value five times larger thanthat exhibited by 1 (Table 1). Such a difference in affinity can beaccounted for considering that the CuII center assists the azidebinding process to FeIII. The large increase in the binding constantsuggests that the CuII ion gives more than a simple electrostaticcontribution, rather it likely participates in the coordination toform a bridged azide complex.

The constant K2 is connected to the binding of a second azidemolecule to either 1 or 2, likely by displacement of the ironcoordinated imidazole of the Gly-His-OMe arm. Only minorspectral changes are observed in this case, because the complexes

remain in the low-spin state. A larger K2 value is again observedfor 2 (Table 1). This can be explained considering that the bindingof the first azide anion as a bridge between the iron and coppercenters of 2 keeps the Cu-ligated bis(benzimidazole) group farfrom the heme in the distal side; therefore, the steric hindrancearound the proximal face of the heme decreases with respect to 1,thus facilitating the binding of the second N3

− anion.The EPR experiments support the formation of a bridged,

weakly-coupled FeIII–N3−–CuII complex by 2. In the oxidized

state of COX, the magnetic properties1,48,49 and the absence ofan EPR signal for odd-spin species,1,50 are consistent with a strongantiferromagnetic interaction (−J > 200 cm−1) between FeIII andCuII sites, yielding an S = 2 ground-spin state. Although, accordingto other data,51 Fe–Cu coupling may be very weak (|J| <1 cm−1),in agreement with the relatively large separation between theiron and copper centers found in the structure of the enzyme. Incomplex 2, the two metal centers are not magnetically coupled, oronly weakly magnetically coupled, but the addition of azide causesan appreciable decrease in intensity of the CuII and FeIII EPRsignals, which testifies an antiferromagnetic interaction betweenthe two paramagnetic centers (Fig. 4).

The cooperation between the FeIII and CuII of 2 also emergesfrom the analysis of the peroxidase reactivity of complexes[(1L)FeIII]+, 1, and 2. At pH 5.0 the rate constant k1 is larger forboth 1 and 2 than for [(1L)FeIII]+. This can be explained by the needof acid–base catalysis for an efficient heterolytic cleavage of thehydrogen peroxide O–O bond.37,41,45 At pH 5.0, the benzimidazoleresidues of 1 are protonated and can exert a positive effect onthe O–O cleavage. The formation of the active intermediate isfurther increased in complex 2, where the presence of CuII canassist both peroxide ligation to the heme and heterolytic O–Obond cleavage. At pH 7.0 all the rates are markedly larger, butthe reactivity order of the complexes is reversed with respectto pH 5.0. Here deprotonation of hydrogen peroxide becomesnon-negligible, facilitating its coordination to the heme iron.Also the phenol is partly deprotonated, making the oxidationof phenolate to phenoxy radical faster; but since it reacts ina fast step of the catalytic cycle (the rate determining step isthe activation of hydrogen peroxide), the effect on the reactionrate is negligible. The higher reactivity of [(1L)FeIII]+ with respectto complex 1 is likely due to the presence, in the latter, of thebenzimidazole groups, which participate in stacking interactionswith the porphyrin ring, thereby hindering the approach of H2O2

to the iron (and reducing k1). The further reduction of the rateconstant for 2 can be accounted for considering that at pH 7.0 afraction of the heterodinuclear complex may be present in solutionwith a hydroxide ion bridged between the two metal centers.Since the coordination of the peroxide to the iron must occurwith displacement of the bridged hydroxide, the rate constant isreduced. The rate constants are all markedly reduced at pH 9.0,in spite of the larger fraction of deprotonated hydrogen peroxidewith respect to pH 5.0 and 7.0. Here the competition by both thephenolate and hydroxide with hydrogen peroxide in the bindingto the iron center is stronger and has the effect of decreasingthe rate. In complex 1, the steric hindrance and the p–p stackinginteractions exerted by the benzimidazoles have further negativeeffects, while the lower reactivity of complex 2, compared to[(1L)FeIII]+ and 1, can be explained by the establishment of ahydroxo bridge between FeIII and CuII.

2202 | Dalton Trans., 2007, 2197–2206 This journal is © The Royal Society of Chemistry 2007

At low temperature, the intermediate formed upon reaction ofcomplexes 1 and 2 with hydrogen peroxide can be isolated. Thereactions generate low-spin adducts, as shown by the position ofthe a, b and Soret bands in the electronic spectra (Fig. 6), butinterestingly, the spectra of the peroxide adducts obtained in thepresence or absence of CuII are different. A possible explanationis that 1 gives a hydroperoxo complex 7, [(2L)FeIII(OOH)], whilewith 2 also CuII is involved in peroxide binding giving complex8, [(2L)FeIIICuII(O2

2−)]+, where various coordination modes arepossible (Scheme 3).21 Unfortunately, unlike the systems basedon the more lipophilic tetra-aryl porphyrins, these complexesare poorly soluble in non-polar or weakly polar solvents, likedichloromethane, especially at low temperature, and this preventsinvestigation of the medium polarity effect on the stabilization ofone particular form of the adduct.23

Scheme 3

To investigate the reactivity of the model systems with dioxygen,a mild reduction method for the metal centers of complexes 1 and2 was found to be convenient. While strong reducing agents suchas sodium borohydride and dithionite have several drawbacks,reaction of the precursor complexes with a small amount ofpiperidine in the presence of CO led smoothly to the heme–carbonyl complexes (Scheme 2). Once FeIII is reduced to FeII, thehigh affinity of CO for FeII precludes to piperidine the possibilityof binding to FeII. Another advantage of piperidine is that it doesnot interfere with the oxygenation process of the heme–carbonylcomplexes. On the other hand, piperidine is not able to reduceCuII to CuI; for this reason the fully-reduced, binuclear complex[(2L)FeIICOCuI]+ (4) was obtained from 3 by addition of a CuI salt.Complexes 3 and 4 exhibit similar electronic spectra, consistentwith the formulation of low-spin carbonyl hemes46 (Fig. 5). Likein most heme-containing proteins, CO reversibly binds to theheme and can thus be removed in vacuum or by another ligand.Hence, upon exposing solutions of 3 and 4 to molecular oxygenat low temperature, the oxygenated adducts 5 and 6 are formed(Scheme 2). These species are relatively stable at −60 ◦C butundergo degradation when the temperature is raised. This showsthat the dioxygen adducts are reactive or, more likely, evolve toproduce other reactive intermediates.

The optical features of the oxygenated adducts 5 and 6, at firstsight, are attributable to low-spin, myoglobin-like, “superoxo”FeIII–O2

− complexes. This is also one of the (fast) intermediatesobserved during the normal COX catalytic cycle,1 whereas noevidence for a l-peroxo FeIII–CuII bridged intermediate is currentlyavailable.7,13 Though, the slight differences observed in the elec-tronic spectra of 5 and 6 may also imply a structural difference, oreven an equilibrium between the superoxo species (FeIII–O2

− CuI)and a peroxo species (FeIII–O2

2−–CuII) (Scheme 3). Comparing thespectra of the adducts 5 and 6 (Fig. 5) with those of 7 and 8(Fig. 6) it is apparent that the spectral differences between thevarious types of bound dioxygen species are not large. Perhapsworthy of note is that the intensity ratio between the b and abands is >1 for the mononuclear complexes 5 (FeIII–O2

−) and7 (FeIII–OOH), and <1 for the dinuclear complexes 6 (FeIII–O2

−

CuI/FeIII–O22−–CuII) and 8 (FeIII–O2

2−–CuII). Unfortunately, allthe attempts to characterize the bound dioxygen species in thesecompounds through Raman experiments were unsuccessful so far,possibly because of fluorescence problems.

The degradation experiments on the oxygenated complexes 5and 6 confirm that the two dioxygen adducts are different. Thethermal degradation process is much faster for 5 than 6 (Fig. 7).This is an important observation as it implies that Cu influencesand prolongs the life-time of the active species. The cooperationbetween FeII and CuI metal ions in binding O2 confers stabilityto the oxygenated species at the higher temperatures. In addition,the release of radical species, deriving from intermediate steps inthe reduction of dioxygen and responsible for several degradativereactions, is significantly reduced, a feature that could also be veryimportant for COX. However, it is clear that the stability of thedioxygen adducts described here is much lower than that of heme–copper complexes derived from substituted tetra-aryl porphyrins,which can be generally handled at higher temperatures, and insome cases are even stable at room temperature.20,23,27 Protectionof the porphyrin meso positions by the aryl substituents is criticalfor the thermal stability of the dioxygen adducts, while the useof pyridine, benzimidazole or imidazole residues for binding thecopper center is not expected to affect the stability of the complexesto a large extent, as it is known from copper–dioxygen chemistrythat pyridine and benzimidazole ligands are good substitutes ofthe natural imidazole donors.57–59

In conclusion, the synthesis and characterization of a heme–copper model system based on b-type heme, in which the iron isfive-coordinated with a histidine ligand have been described. Thepresence of a tethered histidine arm is an important structuralfeature of the model, since the axial ligand is responsible forproviding electron density to the heme–iron and affects itsreactivity. The additional flexible arm carrying the copper centerallows the two metal ions to cooperate in the binding of exogenousligands. The preliminary characterization carried out here, inparticular, suggests that Fe and Cu cooperate both in the bindingof O2 to the reduced FeII/CuI form, and in peroxide binding tothe oxidized FeIII/CuII form of the heme–copper model. Althoughthe present FeO2 and FeO2Cu adducts are intrinsically less stablethan those derived from the substituted tetra-aryl porphyrins, theirspectral properties are more directly comparable to those of theproteins. For instance, the spectra of 5, 6, 7 and 8 exhibit Soretbands in the same range as oxymyoglobin or oxyhemoglobin,47

and are significantly blue shifted with respect to those of all

This journal is © The Royal Society of Chemistry 2007 Dalton Trans., 2007, 2197–2206 | 2203

FeO2 and FeO2Cu heme–Cu model compounds derived fromsubstituted tetra-aryl porphyrins that have been reported so far,which generally occur above 420 nm.20–28 The Soret spectrum of theoxy derivative of the COX heme a3–CuB complex is not known, dueto the very short lifetime,12–14,21 but based on the analogy betweenthe spectra of the FeO2 and FeO2Cu species found here we expectthat it will also occur in the same range. Our current efforts aredirected to further improve the structure of the heme–Cu modelcompound by introducing a tridentate binding residue as a Cuchelating arm, instead of the bidentate residue present in 1.

Experimental

General

Dimethylformamide was stored over BaO and distilled underreduced pressure over CaH2 before use. 1-Hydroxybenzotriazole(HOBt) and H-Gly-His-OH·2 HCl were obtained from Aldrich.O-(Benzotriazol-1-yl)-N,N,N ′,N ′-tetramethyluronium hexafluo-rophosphate (HBTU) was obtained from Fluka. Hydrogen per-oxide solutions were prepared by diluting a 30% w/w solutionin water and were standardized with a literature procedure.35 Thebuffer solutions were prepared using Milli-Q R© water. All the otherreagents and solvents were of the highest grade available. HPLCruns were performed on a Jasco HPLC system equipped with twoPU-1580 pumps and a MD-1510 diode array detector. UV/Visspectra were recorded on a HP8452A or a HP8453 diode arrayspectrophotometer. Low temperature optical spectra were takenusing a custom-designed optical fiber probe from Hellma of 0.5cm-path length. All the solutions were thermostated by a JulaboFT 901 temperature control system at ±0.5 ◦C. Mass spectrawere recorded with a Finnigan LCQ ion trap mass spectrometer.Atomic emission analysis was performed using an ICP-AES, JobinYvon-JY138 Ultrace instrument.

Preparation of ligands and complexes

H-Gly-His-OMe·2HCl was synthesized according to a slightmodification of a literature procedure.52 N,N-Bis[2-(1-methylben-zimidazol-2-yl)ethyl]amine (2-BB) and N-acetyl-N,N-bis[2-(1-methylbenzimidazol-2-yl)ethyl]amine (AcBB) were obtained fol-lowing a published method.53

The synthesis of hemin-2(18)-glycyl-L-histidine methyl ester([(1L)FeIII]+) was carried out essentially following a recently re-ported procedure.46 A mixture of 800 mg hemin, 640 mg HOBt and465 mg HBTU was dissolved in 10 cm3 of freshly distilled DMFcontaining 0.80 cm3 of triethylamine. After 10 min incubation,304 mg of H-Gly-His-OMe·2HCl were added and the solutionkept under stirring for 10 h. The solution was dried under vacuumand the solid residue washed several times with water and diethylether. The crude material was purified first on a 4 × 40 cm silica gelcolumn using butanol–acetic acid–water 20/1/1 (v/v/v) as eluent.The unreacted hemin eluted as the first fraction; [(1L)FeIII]+ (as anequimolar mixture of isomeric hemins derivatized at the 6 or 7position of the porphyrin ring) eluted as the second fraction whilethe bis-condensation product remained in the column. [(1L)FeIII]+

was further purified by preparative HPLC chromatography usinga SUPELCO C18 reverse phase column in 3 : 1 (v/v) 0.1%TFA–water/0.1% TFA–CH3CN (TFA = trifluoroacetic acid). The

compound was characterized by MS (m/z = 824) and 1H NMRin d4-methanol.

The mononuclear complex [(2L)FeIII]+ (1) was prepared asfollows (Scheme 1). HBTU (26 mg, 0.071 mmol) was added to5 cm3 of a dry DMF solution containing [(1L)FeIII]+ (60 mg,0.071 mmol), 2-BB (25 mg, 0.074 mmol), dried HOBt (38 mg,0.37 mmol), and triethylamine (0.37 mmol). The solution wasstirred for 2 h at room temperature and then added dropwiseto ice-cooled diethyl ether (50 cm3) to precipitate the product.The complex was chromatographed on a 4 × 40 cm silica gelcolumn using a mixture of CH2Cl2–MeOH (9 : 1 v/v) as eluent.The unreacted [(1L)FeIII]+ was retained in the column, whilethe eluted product 1 was further purified by preparative HPLCchromatography using a reverse phase C18 column and elutingwith 1 : 1 (v/v) 0.1% TFA–water/0.1% TFA–CH3CN (yield 30%).Complex 1 was finally characterized by MS (m/z = 1139).

The dinuclear complex [(2L)FeIIICuII]3+, 2, was obtained byadding 1.1 equiv. of Cu(ClO4)2 to 10 cm3 of a methanolic solutionof 1 (10 mM) and stirring for 30 min. The solvent was then rotaryevaporated and 2 was washed with water and diethyl ether toeliminate the small excess of copper(II) (Scheme 1). The complexwas then characterized by mass spectroscopy (m/z = 1203) andatomic emission analysis, which confirmed the total incorporationof copper(II) ion in the dinuclear complex (Fe/Cu ratio = 1.0).

The carbonyl complex [(2L)FeIICO] (3) was obtained by bub-bling CO under anaerobic conditions into a degassed solutionof [(2L)FeIII]+ in DMF for 30 min, followed by the addition ofan excess of piperidine (58 mm3). The fully reduced, dinuclearcarbonyl complex [(2L)FeIICOCuI]+ (4) was prepared from 3by adding, under CO-saturated anaerobic conditions, 1 equiv.of [CuI(CH3CN)4]PF6 dissolved in a small amount of degassedacetonitrile (Scheme 2).

The concentrations of [(2L)FeIII]+, [(1L)FeIII]+ and[(2L)FeIIICuII]3+ solutions were determined spectrophotometricallyusing an extinction coefficient e = 120 000 dm3 mol−1 cm−1 for theSoret band of the compounds in DMF solution. Such a value wascalculated from the combination of quantitative atomic emissionanalysis of both iron and iron/copper metals in the complexesand the pyridine hemochromogen method.54

Ligand binding

Ligand binding experiments of azide to [(2L)FeIII]+ and[(2L)FeIIICuII]3+ were performed spectrophotometrically by mon-itoring the spectral changes upon addition of small amounts ofconcentrated solutions of azide to dilute solutions of 1 and 2in methanol. The binding constants were calculated as describedpreviously.55

Kinetic experiments

The catalytic activity exhibited by [(1L)FeIII]+, [(2L)FeIII]+ and[(2L)FeIIICuII]3+ in the H2O2-catalyzed oxidation of p-cresol wasstudied by monitoring the development of the band of thephenol coupling dimers in the optical spectrum near 300 nm.The reactions were performed in either a 1 : 1 (v/v) mixtureof methanol–200 mM aqueous acetate buffer pH 5, or in 1 : 1(v/v) methanol–10 mM phosphate buffer pH 7, or 1 : 1 (v/v)methanol–10 mM borate buffer pH 9, using a magnetically stirred,

2204 | Dalton Trans., 2007, 2197–2206 This journal is © The Royal Society of Chemistry 2007

thermostated optical cell. The temperature was kept at 25.0 ±0.1 ◦C in all the experiments. The reaction was generally initiatedby addition of H2O2 as the last reactant. The concentration ofthe complexes in the cuvette ranged from 1 lM to 2 lM, thatof p-cresol was 0.8 mM, while the concentration of H2O2 wasvaried between 0.2 and 3 mM. The initial rates were evaluatedfrom the slope of the absorbance vs. time plots in the first fewseconds of the reaction, in order to apply the pseudo first-orderapproximation and neglect the catalyst inactivation. The kineticdata were transformed from absorbance s−1 to dm3 mol−1 s−1

by using the difference between the extinction coefficient of theproducts and the reactant at the reading wavelength. In theexperiments at pH 5 and pH 7 the reactions were followed at300 nm using a De value of 2350 dm3 mol−1 cm−1;56 in theexperiments at pH 9 the monitored wavelength was 316 nm anda De value of 7300 dm3 mol−1 cm−1 was used.55 The rates werenormalized dividing each value by the catalyst concentration.

Low temperature experiments

The reactions of the reduced complexes [(2L)FeIICO] (3) and[(2L)FeIICOCuI]+ (4) with O2 were studied in DMF solution at−60 ◦C. In these experiments, compounds 3 and 4 were preparedin situ, as described above, in freshly-distilled and degassed DMF(8 cm3) to a final 10 lM concentration. Dioxygen was bubbleddirectly into the solution at low temperature, causing a red shift inthe Soret bands of the complexes. Bubbling at low temperature wascontinued until the optical spectra of the oxygenated compounds5, [(2L)FeII(O2)], and 6, [(2L)FeIICuI(O2)]+, fully developed.

The reactions of [(2L)FeIII]+ (1) and [(2L)FeIIICuII]3+ (2) withH2O2 were also performed in DMF at −60 ◦C. After dissolvingcompounds 1 and 2 in DMF (8 cm3), 3 equiv. of a H2O2–triethylamine mixture in 1 : 2 molar ratio (100 mm3, 3 mM H2O2–6 mM triethylamine) were added to the complex solution at lowtemperature, and the UV-Vis spectral changes were followed withtime as above.

Thermal degradation experiments of the oxygenated species 5and 6 were performed with a temperature jump method. Afterobtaining 5 and 6 at −60 ◦C as described above, the degradationwas initiated by placing the solution on ice at 0 ◦C and observingthe changes in the Soret band of the complex. The plot of Dabsorbance (379–417 nm) vs. time was obtained from the differencespectra. Fitting the data by an exponential decay equation gave thekinetic constants for degradation of the three complexes. The first10 min of the degradative process were neglected due to thermalstabilization. In the case of complex 5, a relevant part of thedegradation occurred during the thermal stabilization; therefore,the rate constant obtained is affected by a large error.

NMR and EPR spectra

NMR experiments were carried out in DMSO-d6 and CD3ODsolutions at 298 K. Proton NMR spectra were recorded usinga Bruker AVANCE 400 spectrometer operating at 400.13 MHzproton Larmor frequency. Data acquisition and processing wereperformed using a standard Bruker software package (XWIN-NMR).

EPR spectra were recorded with a Bruker EMX instrument,operating at X-band frequency, using standard Wilmad quartz

tubes. The spectra were taken at 77 K, in frozen methanol,acetonitrile, DMSO, or in methanol–acetonitrile (4 : 1, v/v)solution. For variable temperature studies, an Oxford accessorywith continuous flow of liquid nitrogen and a temperature controlsystem was used.

Acknowledgements

The authors thank the Italian MIUR, through a PRIN project,CIRCMSB, and the University of Pavia for support. W. A. Alvesand A. M. D. C. Ferreira thank the Brazilian sponsor FAPESPfor grant (Proc. 01/09127-6) and fellowship (Proc. 00/11862-3).

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