Two-Dimensional Hyperfine Sublevel Correlation Spectroscopy Applied in the Study of aCu2+-[2-(r-Hydroxyethyl)thiamin Pyrophosphate]-[Pentapeptide] System as a Model ofThiamin-Dependent Enzymes

Gerasimos Malandrinos,† Maria Louloudi, † Yiannis Deligiannakis,*,‡ and Nick Hadjiliadis* ,†

Laboratory of Inorganic and General Chemistry, Department of Chemistry, UniVersity of Ioannina,45110 Ioannina, Greece, and Laboratory of Physical Chemistry, Department of EnVironmental andNatural Resources Management, UniVersity of Ioannina, Pyllinis 9, 30100 Agrinio, Greece

ReceiVed: December 4, 2000; In Final Form: March 19, 2001

To obtain structural information on the active-site of thiamin-dependent enzymes in solution, the ternaryCu2+-[Asp-Asp-Asn-Lys-Ile]-[2-(R-hydroxyethyl)thiamin pyrophosphate (HETPP)] system has been syn-thesized and studied by pulsed EPR (ESEEM and HYSCORE) spectroscopy in aqueous solution at physiologicalpH. HYSCORE proved to be especially useful in elucidating the coordination environment of the Cu2+ ion.The present data show that, in the ternary Cu2+-[pentapeptide]-[HETPP] system at physiological pH, thepeptide backbone offers three coordination sites to the metal ion and the coordination sphere is completed bytwo additional phosphate oxygens and the nitrogen N(1′) of the thiamin coenzyme. Thus the synthetic ternarysystem offers the first example of a reliable structural model of the active site of thiamin-dependent enzymesin solution. The importance of our findings concerning the N(1′) coordination in the Cu2+-[HETPP]-[pentapeptide] system is discussed in conjunction with the role of HETPP as an intermediate of thiamincatalysis.

Introduction

Thiamin enzymes catalyze the decarboxylation ofR-ketoacids and the transfer of aldehyde or acyl groups in vivo.1 Theholoenzymes depend on the cofactors thiamin pyrophosphate(TPP) and Mg2+ or Ca2+. See Scheme 1.

The mechanisms of TPP-dependent enzymes are dominatedby the chemistry of TPP cofactor and its intermediates.1 Mostspecifically, the importance of the pyrimidine ring has alwaysbeen recognized, although its participation in the catalyticprocess has been never understood in detail. In addition, theinteraction of active aldehyde derivatives of thiamin, 2-(R-hydroxybenzyl)- and 2-(R-hydroxy-R-cyclohexylmethyl)thiamin,with group IIB metals,2a as well as with first-row transitionmetals,2b are of particular interest. In all these cases, the metalions were found to be coordinated to the N(1′) atom of thepyrimidine moiety both in the solid state2a and in solution.2b

When the active aldehyde pyrophosphate derivatives of thiamin,2-(R-hydroxybenzyl)- and 2-(R-hydroxyethyl)thiamin pyrophos-phate, were used as ligands, metal coordination to both thepyrimidine N(1′) and the pyrophosphate group were detected.2c,d

Exceptionally, Hg2+ forms complexes with the active aldehydepyrophosphate derivatives of thiamin coordinated only to N(1′).2e

In all analyzed crystal structures of thiamin-dependentenzymes,3 the metal ions (Ca2+, Mg2+) were bound in anoctahedral coordination by two phosphate oxygens of the TPP,the side chains of Asp157 and Asn187, the main chain oxygenof residue 189, and a water molecule. This type of coordination

does not involve metal-N(1′) interaction and is conserved inpyruvate oxidase, pyruvate decarboxylase (PDC), transketolase,and benzyl formate decarboxylase (BFD).3 Moreover, a con-served interaction is the hydrogen bond between the side chainof Glu418 and the N(1′) atom of the pyrimidine ring.3 It waspreviously suggested4 that this hydrogen bond activates the 4′-NH2 group to act as an efficient proton acceptor for the C(2)proton, initiating in this way the catalytic cycle.

Despite the above-mentioned crystallographic data2a,c,e,3andsome molecular modeling studies,5 direct structural informationon the active site of thiamin-dependent enzymesin solution isscarce. In this context, based on enzymic studies in solutionwe recently have demonstrated that the HETPP-metal com-plexes, which present direct metal-N1′ and metal-pyrophos-phate oxygen bonds, exhibit coenzyme activity.6

In the present paper we report the synthesis and characteriza-tion of a model for the active site of thiamin-dependent enzymesin solution. Such a model, to be credible, had to incorporatethe structural key components, i.e., the protein, the cofactors(the thiamin and the metal ion), and the substrate. In this contextwe have synthesized the pentapeptide Asp-Asp-Asn-Lys-Ilewhich mimics the metal binding site Asp185-Asp186-Asn187-

* Addresscorrespondencetotheseauthors.E-mail(Y.D.): ideligia@cc.uoi.gr.E-mail (N.H.): nhadjil@cc.uoi.gr.

† Department of Chemistry.‡ Department of Environmental and Natural Resources Management.

SCHEME 1a

a R ) H; thiamin pyrophosphate (TPP); CH3C(OH)H-2-(R-hydroxy-ethyl)thiamin pyrophosphate (HETPP).

7323J. Phys. Chem. B2001,105,7323-7333

10.1021/jp004364p CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 07/07/2001

Lys188-Ile189 of transketolase and surrounds the pyrophosphatemoiety.3c,d Using HETPP, we have studied the tertiary system[Cu2+]-[pentapeptide]-[HETPP] in solution, at physiologicalpH.

The structure of the tertiary system was studied by usingelectron paramagnetic resonance (EPR) spectroscopy.7 Moreparticularly we have used electron spin-echo envelope modula-tion (ESEEM) spectroscopy8a,b to study the coordination envi-ronment of the [Cu2+]-[pentapeptide]-[HETPP] system infrozen solution. ESEEM is a pulsed EPR technique and is verywell suited for studying the coordination environment ofparamagnetic metal complexes in proteins, as well as in modelsystems.8b,9In previous work, ESEEM has been used extensivelyto study Cu(II)-(histidine)n complexes, although works on Cu-(II)-(non-histidine) complexes are rather scarce (for a reviewsee ref 9 and references therein). In the present work the studyof the [Cu2+]-[pentapeptide]-[HETPP] complex was mainlybased on hyperfine sublevel correlation (HYSCORE) spectros-copy. HYSCORE is a two-dimensional four-pulse ESEEMtechnique10a and is very helpful for disentangling complicatedoverlapping ESEEM spectra.10b-g In the case of the [Cu2+]-[pentapeptide]-[HETPP] complex, HYSCORE allowed theresolution, assignment, and quantitative analysis of nuclearcouplings. From these spectroscopic data structural models ofthis complicated system in solution were obtained. On the basisof this information, it was shown that the [Cu2+]-[pentapep-tide]-[HETPP] system provides a reliable model for the activesite of thiamin-dependent enzymes and the results can referdirectly to thiamin catalysis.

Experimental Section

Materials. Thiamin pyrophosphate was purchased fromSigma Chemical Co. and used without further purification.

Synthesis of the Compounds. Preparation of the Ligand2-(r-Hydroxyethyl)thiamin Pyrophosphate (HETPP) Chlo-ride and the Pentapeptide Asp-Asp-Asn-Lys-Ile.2-(R-Hy-droxyethyl)thiamin pyrophosphate chloride was prepared ac-cording to the literature.11 The ligand forms, (HETPPH2)0 and(HETPPH)-K+, were obtained by addition of 1 or 2 equiv of0.1 N KOH in alcoholic solutions. Any insoluble material wasremoved prior to use. The peptide Asp-Asp-Asn-Lys-Ile wassynthesized by solid-phase peptide synthesis using the 2-chlo-rotrityl chloride resin (substitution 1.1-1.6 mequiv g-1) as thesolid support.12a The standard Fmoc procedure was used.12b

Details on the synthesis of the pentapeptide will be publishedelsewhere.13 Peptide purity was controlled by TLC in thesystems CH3CN-H2O (5:1 v/v) and BuOH-CH3COOH-H2O(4:1:1 v/v), 400 MHz proton NMR spectroscopy, and potenti-ometry.

EPR and ESEEM Spectra.Continuous-wave (CW) EPRspectra were recorded at liquid helium temperatures with aBruker ER 200D X-band spectrometer equipped with an OxfordInstruments cryostat. The microwave frequency and the mag-netic field were measured with a microwave frequency counterHP 5350B and a Bruker ER035M NMR gaussmeter, respec-tively. Orientation-selective pulsed EPR experiments wereperformed with a Bruker ESP380 spectrometer with a dielectricresonator. In the three-pulse (π/2-τ-π/2-T-π/2) ESEEM datathe amplitude of the stimulated echo as a function ofτ + Twas measured at a frequency near 9.6 GHz at various magneticfield settings across the field-swept echo-detected EPR spectrum.Data manipulations were performed as described earlier.14b TheHYSCORE spectra recorded for the Cu2+ complexes studied

here contained cross-peaks from nuclear transitions correspond-ing to either14N, 31P, or1H, some of them having considerablehyperfine anisotropy. For each complex several HYSCOREspectra were recorded at variousτ-values, to compensate forthe blind spots due toτ-suppression effect.15a Thus, thefrequency-domain HYSCORE spectra recorded atτ ) 88, 120,and 136 ns (all the other experimental parameters being keptsimilar) were added together in order to minimize blind spots.

Analysis of the ESEEM Spectra.The ESEEM spectra forthe complexes studied here show nuclear transitions fromvarious nuclei (1H(I ) 1/2), 31P(I ) 1/2), 14N(I ) 1)) havingeither weak (that is,Aiso< 2νI) or strong (that is,Aiso > 2νI)hyperfine interactions. In general, the measured three-pulseESEEM spectra were congested consisting of overlapping weakfeatures. In this context, HYSCORE spectroscopy proved to bevery useful since it allowed both the assignment and quantitativeanalysis of the nuclear couplings. In the case ofI ) 1/2 nucleithe spectra were analyzed based on analytical expressions whichare derived in the Appendix. The values estimated from theexperimental HYSCORE spectra were further refined by nu-merical simulations of the three-pulse ESEEM which wereperformed as described earlier.10d,e

Theory and Method of Analysis of the HYSCORE LineShapes forI ) 1/2, S) 1/2 for an Axial g-Tensor. In general,the frequency-domain HYSCORE spectra consist of correlationcross-peaks whose coordinates are nuclear frequencies ((νR,(νâ) from opposite electron spin manifolds.15,16aIn the case ofI ) 1/2 analytical expressions are available for the HYSCOREspectrum in the time domain.15a In the frequency domain,analytical expressions for the line shapes of the HYSCOREspectra have been derived by Dikanov and Bowman16a,b,c forthe case of an isotropic electrong-tensor. This method is ofgreat practical use because it is computationally much easierthan the time-domain calculations and it provides a compre-hensive overview of the cross-peak line shape in HYSCOREspectra. For example, these analytical expressions have beenproven to be of practical value even in cases of very complicatedHYSCORE spectra containing multiple contributions fromnuclei havingAiso< 2νI, Aiso ∼ 2νI, andAiso> 2νI.16d

The EPR spectra for the Cu2+ complexes studied here arecharacterized by axialg-tensors. In such a case, analyticalexpressions for the line shapes of the HYSCORE spectra arenot available. Thus, in the present paper the pertinent expressionsare derived (see Appendix). In the following we use theseexpressions to discuss HYSCORE line shapes which will beused for the analysis of the experimental spectra.

Consider the principal axes system (PAS) of theg-tensor,whereg| lies alongZ andθ0 is the angle between the externalmagnetic fieldH0 and theg| direction; see Figure 1. At a givenθ0 the effectiveg-value is given by

We assume that the anisotropic electron-nuclear interaction canbe described by the point-dipole approximation. The electronspin is at the origin of the axes, and the position of the nucleusis described by a vectorR which lies in theXZ plane at angleθI from Z; see Figure 1.

Then the correlation between the nuclear frequencies in twoopposite electron spin manifoldsR andâ is given by eq A12:

geff ) [(g⊥ sin θ0)2 + (g| cosθ0)

2]1/2

ν2R(â) ) ν2

â(R)QR(â) + GR(â) +

QR(â)[12C1 - 14C2] + [12C1 + 1

4C2]

7324 J. Phys. Chem. B, Vol. 105, No. 30, 2001 Malandrinos et al.

The derivation of this equation together with the definitionsof the various terms and further details are given in theAppendix.

Orientation Selective Experiments.In eq A12, the termsC1 and C2 are nonzero at noncanonical orientations, see eqsA10 and A11. Thus in cases when the HYSCORE experimentis performed at field positions which correspond to noncanonicalorientations, the correlation cross-peaks are expected to be ofcomplicated form, as discussed in the Appendix. On the otherhand, in orientation selective experiments one can select arestricted range ofθ0 values by appropriate setting of themeasuring magnetic field within the EPR resonances. In thecase of Cu2+ complexes where theg-tensor is usually axial, ornearly axial, it is convenient to record the ESEEM spectrum atthe g⊥ value of the EPR spectrum, i.e., forθ0 ) 90°. Theadvantage of selecting theg⊥ value is 2-fold: one is theimproved signal/noise ratio of the echo, and the second is thesimplification of the analysis of the cross-peaks as we discussin the following.

In the case ofθ0 ) 90° the parametersC1 andC2 are 0; theneq A12 becomes

The form of eq 1 is similar to the expression of Dikanov andBowman for the isotropicg-tensor, and shows that in theorientation selective experiments atθ0 ) 90° the cross-peaksare expected to be arc-shaped. However, apart from thissimilarity, certain differences should be pointed out betweenthe isotropic and the axialg-tensor. One is the different way inwhich the hyperfine coupling parametersAiso and AT and theLarmor frequencyνI determine the parametersQR(â) andGR(â).This can be seen by referring to eqs A1, A7-A8, and A13-A14. The second important factor is that in the case of the axialg-tensor, the geometrical disposition of the nuclear spin, i.e.,the angleθI in the PAS of theg-tensor, plays a key role. Therelative importance of these parameters can be evaluated byexamining some representative theoretical spectra in Figure 2,which we have calculated17 based on the analytical expressions.The spectra have been calculated for two types of hyperfinecouplings, i.e., weak for the protons (Figure 2A) and strongerfor the phosphorus (Figure 2B,C). These are pertinent for theanalysis of the experimental HYSCORE spectra encounteredin the present case for the Cu2+ complexes under study.

Figure 2A displays theoretical1H HYSCORE spectra calcu-lated for three sets of1H hyperfine couplings corresponding toprogressively increasing hyperfine anisotropyAani ) g⊥T. InFigure 2A, we observe two characteristic trends of the correla-tion features as a function of theAani values: (1) For increasinghyperfine anisotropy there is a progressive spreading of thefeatures. This reflects the dispersion of the nuclear frequenciesfor increasingAani values. (2) For increasing hyperfine aniso-tropy values there is a progressive increase of the maximumshift from the (-νI, νI) antidiagonal which is marked by thesolid line in Figure 2A. To simplify the discussion, in Figure2A the maximum shift∆νmax is indicated at the left of eachspectrum.

This shift may be of practical use since it allows a quantitativeestimation of the nuclear couplings directly from experimentalHYSCORE spectra.15b,16eMore specifically, for nuclei withθI

> 45° the anisotropic hyperfine interaction is calculated directlyfrom the maximum vertical shift∆νmax of the cross-peak ridgesestimated from the HYSCORE spectrum according to16e

From eq 2a it is seen that the estimated dipolar couplingAani isproportional to the Larmor frequencyνI. Thus, in real experi-ments, a given experimental∆νmax value will correspond tolarger estimated dipolar couplings for nuclei with larger LarmorfrequencyνI. The inset in Figure 2A is a plot of the estimatedAani as a function of∆νmax according to eq 2a, for two typicalI ) 1/2 cases i.e.,1H and31P nuclei. According to these plotsthe method works better for protons than for phosphorus. Fromthe present experimental data an upper error limit for theestimation of∆νmax is about 0.025 MHz, and this can result inan error of up to 0.6-1.0 MHz in the estimatedAani valuedepending on the type of nucleus; see inset in Figure 2A.

In suitable cases, the isotropic hyperfine coupling, Aiso, canbe estimated from the position of the inner end of the ridges(νR, νâ) and (νâ, νR) which correspond to the canonicalorientations of the hyperfine tensor15b,16a

In our experimental spectra the inner-end positions are onlyvaguely resolved; therefore, application of eq 2b allowed onlyan approximate estimation of the isotropic coupling. In thepresent case we have adopted the following strategy for theanalysis of the1H HYSCORE spectra: the dipolar coupling wasestimated to first order from the maximum shift of thecorrelation ridges according to eq 2a. Based on this information,the anisotropic and isotropic coupling were determined bysimulations of HYSCORE spectra based on the expressionsderived in the Appendix.

In the case of31P, the observed hyperfine couplings havesmall anisotropy and correspond toAiso values comparable to2νI(31P); therefore, the evaluation of the hyperfine couplingsfrom ∆νmax was not applicable. Instead we have simulated theexperimental31P spectra based on the analytical expressionsderived in the Appendix. Pertinent theoretical HYSCOREspectra for31P are displayed in Figure 2B and Figure 2C forAiso< 2νI(31P) andAiso > 2νI(31P), respectively. Despite the smallanisotropy, we found that the shape of the theoretical cross-peaks for31P was sensitive to the angleθI. For example, in thespectra in Figure 2A,B the horn-shaped features were generated

Figure 1. Reference axis system.H is the external magnetic field;Rdescribes the position of the nuclear spin interacting with an electronspin located at the origin of the axes.

θ0 ) 90°: ν2R(â) ) ν2

â(R)QR(â) + GR(â) (1)

Aani ≡ g⊥T ) 23x8∆νmaxνI

x2(2a)

Aiso )|νR - νâ|

2+ g⊥T (2b)

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System J. Phys. Chem. B, Vol. 105, No. 30, 20017325

by settingθI of 60° in Figure 2A and 55° in Figure 2B. Theseparticular spectra remained invariant only forθI values within(10°. In structural terms, theseθI values correspond to nucleilocated near, although not exactly on, theXY plane of theg-tensor of the Cu2+.

ResultsThe synthesized pentapeptide has the structure shown in

Scheme 2.

Full NMR assignments of the peptide were done based onone- and two-dimensional1H-1H TOCSY, 1H-13C HMQC,and1H-13C HMBC spectral analysis and details will be reportedelsewhere.13

Potentiometric and CW EPR Studies.Potentiometric ti-trations for the peptide and the Cu2+-HETPP, Cu2+-peptide,and Cu2+-[peptide]-[HETPP], as well as UV-vis and CWEPR data show that the Cu2+-peptide-HETPP system is the

Figure 2. (A) Theoretical frequency-domain 2D-HYSCORE line shapes for1H(I ) 1/2) nucleus interacting with anS ) 1/2 electron spin. Thehorizontal and vertical dashed lines mark the nuclear Larmor frequency. The antidiagonal dotted lines mark the maximum shift (∆νmax) of thecross-peaks. The spin Hamiltonian parameters used areT ) 4.3 MHz (equivalent toR ) 2.1 Å according to eq A2) in (I),T ) 3.2 MHz (R ) 2.3Å) in (II), and T ) 1.8 MHz (R ) 2.8 Å) in (III), with T defined as in eq A2. Common parameters in all cases:νI ) 14.26 MHz,g⊥) 2.02,g| )2.25, θI ) 90°, Aiso ) 6.7 MHz. Inset: Plot of the dipolar couplingAani ) g⊥T, as a function of the maximum shift (∆νmax) of the cross-peaksaccording to eq 2a. The two plots correspond to Larmor frequencies at 3350 G,νI(1H) ) 14.26 MHz andνI(31P) ) 5.77 MHz. (B) Theoreticalfrequency-domain HYSCORE line shapes for a31P(I ) 1/2) nucleus interacting with anS ) 1/2 electron spin. The horizontal and vertical dashedlines mark the nuclear Larmor frequencyνI ) 5.77 MHz. The spin Hamiltonian parameters used areAiso ) -9.2 MHz,R) 3.4 Å, which correspondsto Aani ) 1.12 MHz,θI ) 55°, g⊥ ) 2, g| ) 2.25, andνI ) 5.77 MHz.(C) Same as in (B). The spin Hamiltonian parameters used areAiso ) -14.0MHz, R ) 3.2 Å, which corresponds toAani ) 1.36 MHz,θI ) 50°, g⊥ ) 2, g| ) 2.25, andνI ) 5.77 MHz.

7326 J. Phys. Chem. B, Vol. 105, No. 30, 2001 Malandrinos et al.

most stable species at pH 5-8.13 The CW EPR spectra aretypical of monomeric Cu2+ complexes with axial symmetry andno evidence for magnetic interactions, i.e., line broadening orsplittings, between copper complexes in solution. At pH 7-7.2,the CW EPR parameters are (g| ) 2.22,g⊥ ) 2.025,A| ) 203G, A⊥ ) 12 G) for Cu2+-[peptide]-[HETPP], (g| ) 2.25,g⊥) 2.02,A| ) 145 G,A⊥ ) 14 G) for Cu2+-[HETPP], and (g|

) 2.22, g⊥ ) 2.08, A| ) 198 G,A⊥ ) 30 G) for the Cu2+-[peptide].13

HYSCORE Spectroscopy.(i) Cu2+-HETPP. A HYSCOREspectrum recorded for the Cu2+-HETPP complex at pH 7.5 isdisplayed in Figure 3. The spectrum is characterized bypronounced ridges with considerable intensity in the (+, +) aswell as in the (+, -) quadrant, and this indicates that the Cu2+-HETPP complex contains nuclei with both weak and stronghyperfine couplings. The size of these couplings and theirassignment are discussed in the following.

1H(I ) 1/2) Couplings. The ridges disposed symmetricallyaround the point at [+14.3 MHz, +14.3 MHz] in the (+, +)quadrant are assigned to protons coupled to the Cu2+ electronspin, withAiso(1H) , 2νI(1H) ) 28.6 MHz. The form of the1Hcross-peaks in Figure 3 is directly comparable to those in thetheoretical HYSCORE spectra in Figure 2A. More particularly,the arc-shaped cross-peak shifted by∆νmax )1.8 ( 0.1 MHzfrom the ν1 ) -ν2 off-diagonal axis (dashed line in the1H

region in Figure 3) resembles those in the theoretical spectrum,marked as∆νmax(I) in Figure 2A. According to eq 2a, the shiftof ∆νmax ) 1.8 MHz corresponds to a proton withAani ) 7.9MHz. Starting from thisAani value, the best simulation for thisproton is obtained by usingAani ) 7.6 MHz andAiso ) 6.5MHz, which is listed in Table 1.

31P(I ) 1/2) Couplings. The cross-peaks assigned to31Pa and31Pb in Figure 3 are similar to those reported earlier forHYSCORE spectra for31P coupled to VO2+ in the case of TF1-ATPase18a and recently in vanadyl coordinated to phosphate inbone.18b In Figure 3, the ridges disposed symmetrically aroundthe point with frequency coordinates [+5.7 kHz,+5.7 MHz],i.e., the Larmor frequency of31P(I ) 1/2), can be simulated(see Figure 2B) by usingAiso(31P) ) -9.2 ( 0.2 MHz18d andAani ) 1.2 ( 0.1 MHz. In the (+, -) quadrant of theexperimental spectrum in Figure 3, a set of cross-peaks centeredat [2.5 MHz,-13.4 MHz] and [13.4 MHz,-2.6 MHz], termed31Pb, have an average frequency distance of 13.4- 2.5 MHz)10.9 MHz which is close to 2νI(31P) ) 11.4 MHz. A goodsimulation (see Figure 2C) is obtained for aAiso ) -14.0 (0.218d MHz andAani ) 1.4 ( 0.2 MHz.

In the present case the orientation-selective simulations showthat for hyperfine couplings such as those for31Pa and31Pb anacceptable simulation of the experimental spectrum consistentlykeeps the angleθI in the range 50°-70°. This indicates thatthe two interacting phosphorus nuclei are located close to theequatorial plane of theg-tensor of Cu2+-HETPP. The estimated31P anisotropic hyperfine couplings, when interpreted as point-dipole interactions, imply Cu-31P distances of, e.g., 3.2 and3.4 Å for 31Pa and 31Pb, respectively. Such distances areconsistent with coordination of the two phosphate groups viathe oxygens.

14N Couplings.In addition to the1H and the31P couplingsdiscussed above, the spectrum in Figure 3 contains additionallow-frequency cross-peaks with maximum intensity at [+2.1-2.4 MHz,+4.1-4.5 MHz]. These features are characteristic of14N(I ) 1) nuclei coupled to the electron spin with hyperfinecoupling comparable to 2νI(14N) ) 2.06 MHz.10b,c,f,g In thiscontext, the resolved features in Figure 3 are assigned tocorrelations between the so-called “double quantum transiitons”10c

νdq(+1/2) andνdq(-1/2), respectively: for one14N nucleus theνdq(+1/2) frequency can be approximated by8b,14a

whereP ≡ K2(3 + η2) is determined by the quadrupole tensorof the nitrogen, andA is the hyperfine interaction at the directiondetermined by the selectedg-value.14a This expression isapplicable only for hyperfine couplings such that the inequality(3b) holds.

In the case of the Cu2+-HETPP complex, the observed weaknitrogen coupling originates from the nitrogens of the HETPP;see Scheme 1. In copper(II) complexes it is well documentedthat the directly coordinated14N nuclei are not resolved atX-band ESEEM (for a review see ref 9). In contrast, it isexpected that the noncoordinated14N nuclei would contributeto ESEEM at the X-band.8b,9Thus we consider that the observed14N coupling in Cu2+-HETPP originates from a nitrogen ofHETPP which is close but not directly coordinated to copper.Since the coordination of the HETPP to copper is expected tobe through the N(1′), then the plausible candidates for the14Ncoupling seen in Figure 3 are the N(3′) nitrogen or the NH2 of

Figure 3. Contour plots of HYSCORE of experimental frequency-domain HYSCORE spectrum for the Cu-HETPP complex. Experi-mental conditions:H ) 3351 G, sample temperature 4.1 K. Timeinterval between successive pulse sets, 3 ms; microwave frequency,9.64 GHz. Frequency-domain spectra were obtained after Fouriertransform in the magnitude mode, of 2D time-domain patternscontaining 200× 200 points with a step of 16 ns. The displayedspectrum was obtained by numerical addition of three frequency-domainspectra recorded atτ ) 88, 120, and 136 ns, respectively.

SCHEME 2

νdq(+1/2)∼ 2[(A/2 + νI)2 + P]1/2 (3a)

|A - 2νI| < 4K/3νI (3b)

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System J. Phys. Chem. B, Vol. 105, No. 30, 20017327

the pyrimidine ring. TheP value for the nitrogen N(3′),estimated from earlier ESEEM studies on Cu-thiochrome,14b

is (0.79)2(3 + (0.25)2) ) 1.9 MHz2 while for the NH2 inaminopyrimidine14c P ) (0.82)2(3 + (0.40)2) ) 2.1 MHz2. Forthese twoP values usingνdq(+1/2) ) 4.3 MHz from eq 3a wegetA ∼ 1.2 MHz (for N3′) or A ∼ 1.0 MHz (for NH2). Amongthese two options, the assignment of the observed14N couplingvalues to N(3′) is favored because the estimatedA value of 1.2MHz fulfills the condition 3b; i.e., the deviation|A - 2νI| is0.92 MHz, which is smaller than 4K/3νI ) 1.03 MHz. Incontrast, for NH2 the |A - 2νI| is 1.12 MHz, which is largerthan 4K/3νI ) 0.99 MHz. This analysis favors the assignmentof the 14N coupling seen in the HYSCORE spectrum of theCu2+-HETPP to a weak coupling of the Cu2+ electron withthe N(3′) nitrogen.

In summary, the HYSCORE data show that, in the Cu2+-HETPP system, specific interacting1H, 31P, and14N nuclei arein the near vicinity of copper. From the estimated couplingslisted in Table 1, further assignment of these nuclei can be donebased on reference values. This will be presented in thediscussion.

(ii) Cu2+-Pentapeptide.The HYSCORE spectrum of theCu2+-pentapeptide complex at pH 7.2 (Figure 4) is character-ized by two sets of pronounced ridges, marked I and II in Figure4, located in the (+, +) quadrant. They are directed perpen-dicular to theν1 ) ν2 frequency diagonal and are disposedsymmetrically around the point with frequency coordinates[+14.3 MHz,+14.3 MHz] which correspond to the1H Larmorfrequency at the field setting (3351 G) of the experiment.Accordingly the ridges I and II in Figure 4 are assigned toprotons coupled to the Cu2+ electron spin. For their analysiswe first use the maximum shift∆νmax and then the simulatedspectra.

In Figure 4 the maximum shift for the ridges I does not exceed0.1 MHz. By setting∆ν(I)

max∼ 0.1 MHz, withνI ) 14.27 MHz,eq 2a gives an upperAani value of∼1.9 MHz. However, thisvalue is based on a very small shift and might be prone to errors.An independent estimate of theAani value can be obtained fromthe total width of the projection of the ridges I on eitherfrequency axis. From the expressions for the nuclear frequencies,

TABLE 1: Nuclear Coupling Parameters of the Three Cu2+ Complexes Determined from the HYSCORE Spectra, andLiterature Data

literature datac

complex nucleus Aiso, Aania,b (MHz) Aiso (MHz) Aani (MHz) ref

Cu2+-pentapeptide 1H(I) 4.3, 1.6 Cu2+-H2O in Mg-Tuton salt 0.20-0.9 3.4-3.9 20a

1H(II) 6.3, 5.9 Cu2+-H2O in MCM-41 1.2-2.2 4.1-4.8 20c

Cu2+-glycine inR-glycine host 3.2 1.5 (CH2) 20b

Cu2+-glycine inR-glycine host 6.3 5.8 (NH2)d 20b

Cu2+-glycine inR-glycine host 3.5 4.8 (NH2) 20b

Cu2+ (CH2) in triglycine sulfate 3.6-3.7 1.2-1.3 20b

Cu2+ (CH2) in triglycine sulfate 5.7 5.6 (NH2) 20b

Cu2+ (CH2) in triglycine sulfate 3.5-4.0 5.0-5.2 (NH2) 20b

Cu2+-HETPP 1H 6.5, 7.431Pa -9.2, 1.1 31P-VO2+ -14.3 1.9 18b31Pb -14.2, 1.4 31P-VO2+ -9.2 1.5 18b

31P-VO2+ 8.7 18a

31P-VO2+ 15.5 18a31P-Cu2+- 8.2 18c

14N A ) 1.2 MHz;K2(3 + η2) ) 1.9 MHz2

Cu-Thiochrome(N3′);Aiso ) 1.33 MHz;K2(3 + η2) ) 1.9 MHz2

14b

Cu2+-HETPP-pentapeptide 1H(I) 6.4, 8.01H(II) 4.4, 1.931Pa -9.1, 1.331Pb -14.0, 1.214N A ) 2.1 MHz;

K2(3 + η2) ) 1.9 MHz2

a In our caseAani is calculated asg⊥T, whereT is defined according to eq A2.b Average errors(0.6 MHz forAiso andAani.c In cases where three

principal valuesA1, A2, A3 of the hyperfine tensor have been reported,Aiso andT have been calculated asAiso ) (A1 + A2 + A3)/3, Aani ) (A2 + A3)/2- Aiso.

d The reported1H ENDOR data for the two1H’s of NH2 give A(2) ) (A1,A2,A3) ) (-11.1 MHz,-5.6 MHz, 6.1 MHz) andA(3) ) (A1,A2,A3)) (-9.7 MHz, -13.9 MHz, 4.7 MHz). These tensors are strongly rhombic due to delocalization of the unpaired electron (see discussion in p 74of ref 23b). In our case the HYSCORE line shape in Cu-[pentapeptide] is indeed compatible with such rhombicity. However, to keep thingstractable here, we approximate the proton couplings in terms of a simple dipolar tensor. Thus, in the case of the NH2 protons we consider asreference values the absolute values ofAiso,derived from (A1 + A2 + A3)/3, and ofAani ) (A1 + A2)/2 - Aiso.

Figure 4. Contour plots of HYSCORE of experimental frequency-domain HYSCORE spectrum for the Cu-pentapeptide complex.Experimental conditionsH ) 3351 G, sample temperature 4.2 K. Otherconditions as in Figure 3.

7328 J. Phys. Chem. B, Vol. 105, No. 30, 2001 Malandrinos et al.

eq A3, it can be shown that forθ0 ) 90°, æ0 ) 90° the widthof the projection is16e

From the total width of the projection of the ridges I in Figure4, we estimate∆ν ∼ 1.0 MHz; thus by using eq 4, we getAani

∼ 0.7 MHz. Therefore, we consider that theAani value for thisproton should lie between 0.7 and 1.9 MHz orAani ) 1.3( 0.6MHz. Then, by using an average value ofAani ) 1.3 MHz fromthe inner-end position of ridges I at∼17.3 MHz and∼11.7MHz from eq 2b, we getAiso ) 4.1 ( 0.6 MHz. Taking intoaccount the simulation of the HYSCORE spectrum, we concludethat the valuesAani ) 1.6 MHz, Aiso ) 4.3 MHz are in goodagreement with the ridges I in Figure 4. These values are listedin Table 1.

In a similar manner we proceed with the analysis of thecoupling parameters for the proton coupling giving rise to theridges II in Figure 4. In this case the maximum shift is∆ν(II)

max

) 1.0 ( 0.1 MHz (see Figure 4), which according to eq 2acorresponds to aAani value of 5.9( 1.9 MHz. This largehyperfine anisotropy results in extended ridges whose inner-end positions can be discerned near the diagonal i.e., at [∼13.7MHz, ∼14.6 MHz] in Figure 4. From eq 2b we getAiso ) 6.3MHz. The ridges II are horn-shaped, which might indicate eitherthe pertinent1H hyperfine tensor is rhombic or a distributionin the hyperfine coupling parameters. To keep things as simpleas possible, we keep the main elements of the hyperfine tensor;i.e., we consider that for proton II we have (Aiso, Aani) ) (6.3MHz, 5.9 MHz); see Table 1.

In summary, the Cu2+-pentapeptide complex is characterizedby two types of1H couplings listed in Table 1. No other nuclearcouplings, i.e.,14N, are evidenced in the ESEEM and HY-SCORE spectra of this complex. The assignment of the observed1H couplings will be discussed together with the data for theother complexes studied in the present work.

(iii) Cu2+-HETPP-Pentapeptide.The HYSCORE spectrumof the Cu2+-HETPP-pentapeptide complex at pH 7.5 ispresented in Figure 5. To facilitate the discussion, the observed

cross-peaks have been labeled according to their origin. Beforewe proceed to the analytical estimation of the couplingparameters, we observe that the low-frequency part of theHYSCORE spectrum in Figure 5 (Cu2+-HETPP-pentapeptidecomplex) bears similarities to the spectrum in Figure 3 (Cu2+-HETPP complex). More specifically, the31P features at [+2.6MHz, -13.4 MHz] and [+13.4 MHz,-2.6 MHz] of Figure 5look homologous, i.e., in terms of shape and frequency position,to the features of31Pb in Figure 3, while the31P features [+2.2MHz, +11.2 MHz] and [+11.2 MHz,+2.2 MHz] of Figure 5look homologous to the features of31Pa in Figure 3. Followingthe same reasoning as that for the assignment of the31Pfrequencies in Figure 3, we conclude that in the Cu2+-[HETPP]-[pentapeptide] complex there are two types of31Pcouplings.

Low-frequency cross-peaks, labeled as14N, are also observedin Figure 5. The main intensity of these cross-peaks is at the(+, +) quadrant, at [+2.2-2.5 MHz, +5.0-5.2 MHz] and[+5.1-5.2 MHz, +2.2-2.5 MHz]. A difference between the14N features in Figure 3 compared to Figure 5 is that the14Ncross-peaks in Figure 5 have nonzero intensity at the (+, -)quadrant. The frequency positions of the14N features in the(+, +) and (+, -) in Figure 5 are similar, and this favors theirassignment to one14N coupling with hyperfine coupling closeto the cancellation condition, i.e., whenA(14N) ∼ 2νI(14N).8b,10b,c

For example, this case has been previously verified boththeoretically and experimentally by us10d,f and others.10b,c Onthe basis of eq 3a, we proceed with the calculation of thehyperfine couplings. To do this, we consider the possible14Ntypes, i.e., the peptide backbone nitrogens, NH, of the pen-tapeptide (K ) 0.80,η ) 0.45), the NH2 (K ) 0.82,η ) 0.40)of the pentapeptide or the HETPP, and the N(3′) (K ) 0.79,η) 0.25) of HETPP. For these cases, settingνdq(+) ) 5.1 MHzfrom eq 3a, we estimateA ) 2.07 MHz (for NH), A ) 2.14MHz (for NH2), or A ) 2.10 MHz (for N3′). This calculationshows that, irrespective of the type of the14N atom, theestimatedA values are comparable and all three are close tothe cancellation condition. This has the immediate implicationthat the+2.2-2.5 MHz frequency should be a good approxima-tion of ν+ ) K(3 + η), i.e., the double quantum frequency inthe spin manifold where the nuclear Zeeman and the hyperfineterms have opposite signs. In this context, from the three setsof K, η values for NH, NH2, and N(3′), respectively, we estimateK(3 + η) values of 2.76 MHz for NH, 2.79 MHz for NH2, and2.57 MHz for N3′. Among them the value for NH and NH2 areclearly higher than the uncertainty in the frequency position ofthe 2.2-2.5 MHz peak. On the other hand, the value for N(3′)fits reasonably with the experimentally observed value. Ac-cordingly, we assign the14N feature in Figure 5 to the N(3′) ofthe pyrimidine ring of HETPP.

The1H region in Figure 5 contains two types of cross-peaks,both located at the (+, +) quadrant. The first set,1H(I), ischaracterized by strong anisotropy which leads to a considerableshift of ∼1.7 MHz, and this according to eq 2a corresponds toAani ) 8.2 MHz. Further refinement by the simulation givesAiso ) 6.4 MHz, Aani ) 8.0 MHz listed in Table 1 for1H(I).The cross-peaks centered at [11.2 MHz, 17.8 MHz] and [17.7MHz, 11.1 MHz] are characterized by small anisotropy. Theirmaximum shift is 0.1-0.2 MHz, which according to eq 2a givesAani ) 1.8-2.6 MHz. Starting from this value, the simulationfor this proton leads toAiso ) 4.4 MHz,Aani ) 1.9 MHz, listedin Table 1 for1H(II).

In summary, in the three Cu2+ complexes studied here theHYSCORE data allowed the resolution, assignment, and

Figure 5. Contour plots of HYSCORE of experimental frequency-domain HYSCORE spectrum for the tertiary Cu-HETPP-pentapeptidecomplex. Experimental conditionsH ) 3352 G, sample temperature4.2 K. Other conditions as in Figure 3.

∆ν ∼ (3/2)Aani (4)

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System J. Phys. Chem. B, Vol. 105, No. 30, 20017329

quantitative analysis of specific1H, 31P, and14N nuclei in thenear vicinity of the copper atom. From the spectroscopic pointof view, taking into account the remarkably complicated natureof the coordination environment of the present complexes,especially the [Cu2+-HETPP-pentapeptide] complex, thepresent work demonstrates the power of HYSCORE in address-ing such structural problems.

Discussion

Assignment of Nuclear Couplings.In the preceding para-graphs, the resolved nitrogen couplings have been assignedbased on the14N quadrupole coupling parameters. It is foundthat in both Cu2+-[HETPP] and the Cu2+-[HETPP]-[pen-tapeptide] the observed14N coupling is due to N(3′) of thepyrimidine ring. In the Cu2+-pentapeptide complex, no14Nmodulation is resolved by ESEEM. Comparison of the experi-mentalg andA(63Cu(I ) 3/2) values of the Cu2+-pentapeptide,estimated from CW EPR, with literature data19 suggests thatthe coordination environment of Cu2+ should contain at leasttwo coordinated nitrogens and two oxygens. For the coordinatednitrogens their hyperfine coupling is expected to be strong, i.e.,∼40 MHz (for a detailed discussion, see ref 9). Thus the absenceof 14N echo modulations from the coordinated nitrogens in theCu-pentapeptide complex is due to the mismatch of theirhyperfine coupling and the14N Larmor frequency at X-band.On the same grounds, in the Cu2+-[HETPP]-[pentapeptide]the coordinated nitrogens would not contribute to the ESEEM.

The 1H couplings resolved in the present work are ofparticular interest since their proper understanding contributesimportant structural information. Therefore, it is worth discuss-ing these1H couplings in some detail. In this context it ispertinent to compare the measured hyperfine couplings, listedin Table 1, with reference values from the literature. These datacome from pertinent1H ENDOR measurements and are alsoincluded in Table 1.

Cu2+-HETPP. The strong1H coupling resolved for theCu2+-HETPP complex at pH 7.5 (see Table 1) is unique amongthe cases studied here. Comparison of this coupling with the1H coupling in Cu2+-H2O20a,b leads to the conclusion that inthe case of Cu2+-HETPP this hyperfine coupling is not due toa H2O bound to Cu2+. Among the other candidates, from thestructure of the HETPP molecule we consider the methyl protonsat the 2′-position, the H(6′) proton, and the NH2 protons of thepyrimidine ring of HETPP. The CH3 hyperfine tensors areusually isotropic,20b,g and this disfavors the assignment of theobserved anisotropic1H hyperfine tensor to CH3. On the otherhand, this anisotropic coupling is qualitatively compatible with1H tensors reported for either NH2 protons20bor for theR-protonsof pyridine coordinated to copper.20c At this point, the observedparamagnetic broadening of the1H NMR signals in the Cu2+-[HETPP] system is helpful. These NMR data13 show that theC(6′)-H adjacent to N(1′) is the one most affected by the Cu2+

spin. This favors the assignment of the strong1H couplingdetected in the Cu2+-HETPP complex to the H(6′) proton ofthe pyrimidine ring. The assignment to this proton is also inagreement with the Cu-H(6′) distance limits imposed by thestrong anisotropic couplingAani(1H) ) 7.6 MHz: if thisanisotropic hyperfine coupling is due to dipolar interaction withthe copper, then eq 2b gives an effective Cu-H(6′) distance ofR ∼ 2.1 Å. This distance estimate is a lower limit only, sincelocal π-spin densities on the neighboring C(6′) atom might alsocontribute to the1H dipolar interaction.20e Taking into accountthis effect, we estimate20ea Cu-H(′6) distance ofR∼ 2.5-2.8Å. In conclusion this analysis poses the distance limits between

the copper and the interacting H(6′) to be 2.1-2.8 Å. This canbe envisaged only if the pyrimidine is coordinated to copperthrough the N(1′) atom.

The measuredAiso(1H) of 6.5 MHz for the H(6′) correspondsto a s-spin density of∼6.5/1420) 4.5‰20d delocalized on thisproton. Thus the strong1H coupling requires that a considerableamount of the unpaired electron spin is delocalized over thepyrimidine ring. In line with this is the observation of the N(3′)coupling (A ) 1.2 MHz) which requires that a fraction, equalto ∼1.2/1809 ) 0.6‰,20d of the electron spin density isdelocalized through the pyrimidine ring to the N3′ atom. Thisamount of delocalized spin density is in agreement with thevalues reported in the literature for noncoordinated nitrogensof rings liganded to copper.10c,d,f,g Overall, the present dataprovide strong evidence that in Cu2+-HETPP the pyrimidinering of HETPP is directly coordinated to the copper most likelythrough the N(1′) atom.

The two 31P couplings observed in Cu2+-HETPP areassigned to the two phosphorus atoms of HETPP. By comparingthe 31P couplings estimated for Cu2+-HETPP with pertinentdata in the literature,18 we conclude that the two phosphorusatoms are coordinated to Cu2+ via the oxygens. These31Phyperfine couplings in Cu2+-HETPP are inequivalent, and thisindicates that the coordination modes of the two phosphateoxygen atoms to Cu2+ are different.

In conclusion, the structural picture emerging from theHYSCORE data is that in the Cu2+-HETPP complex at pH7.5 the coordination sphere of the Cu2+ atom contains twophosphate oxygens and the N(1′) atom of HETPP. This iscorroborated by the CW EPR data,g| vs A| parameters, whichis compatible with a 1N and 3O coordination.19b In this context,it is possible that an oxygen solvent atom completes thecoordination sphere.

Cu2+-Pentapeptide.The 1H couplings resolved for theCu2+-pentapeptide complex at pH 7.2 can originate either fromthe protons of the solvent (H2O) or from the pentapeptide.Comparison of the hyperfine couplings in Table 1 with thosereported for H2O bound to Cu2+ 20a,brenders the assignment ofeither coupling to solvent protons unlikely. On the other hand,we see that the hyperfine tensor I (see Table 1), which ischaracterized by relatively small anisotropy and small isotropiccoupling, is similar to that reported for CH2 protons in Cu-glycine complex.20b Accordingly, we suggest the assignmentof this proton to a CH2 group of the pentapeptide. The stronglycoupled proton II (see Table 1) has a hyperfine coupling tensorsimilar to that reported for the NH2 protons in the Cu-glycinecomplex.20b Interestingly the NH2 proton couplings in the Cu-glycine complex are characterized by nonnegligible rhombicity,which is in agreement with the observation of horn-shapedfeatures in the HYSCORE spectrum in Figure 4. In conclusion,we assign the two proton tensors in Table 1 to the CH2 proton-(s) and NH2 proton(s) of the pentapeptide coordinated to Cu2+.Accordingly, based on the structure of the pentapeptide, wesuggest that at pH 7.2 the pentapeptide is coordinated to Cu2+

via the terminal amino group and theâ-carboxylate of aspartateresidues.

Cu2+-HETPP-Pentapeptide.The presence of the weak1Hcoupling in the Cu2+-[HETPP]-[pentapeptide] system, as-signed to the CH2 group of the aspartate, indicates that in thissystem the pentapeptide is also coordinated to the copper. Itshould be noticed at this point that the strong NH2 couplingobserved in the Cu2+-[pentapeptide] (see Table 1) is absent inthe Cu2+-[HETPP]-[pentapeptide] system. This indicates thatin the tertiary system the NH2 coordination is modified, i.e., is

7330 J. Phys. Chem. B, Vol. 105, No. 30, 2001 Malandrinos et al.

severely weakened. Most likely the NH2 group occupies an axialposition. This is based on the observation of a ca. 9 nm blueshift of the d-d transition of the Cu2+-[HETPP]-[pentapep-tide] compared to those of Cu2+-[pentapeptide], which can beattributed to the coordination of a N atom in an axial positionresulting in an elongated Cu-N bond.21

Proceeding in a similar way as in the case of the Cu2+-HETPP, we assign the strong1H coupling observed in theCu2+-[HETPP]-[pentapeptide] system to the H(6′) of HETPP.This H(6′) interaction together with the observed N(3′) couplingimplies a coordination of the HETPP via the N(1′). The twoinequivalent 31P couplings resolved in Cu2+-HETPP areconsistently resolved in the Cu2+-[HETPP]-[pentapeptide]system as well. Accordingly, they are assigned to the twophosphorus atoms of HETPP. In Cu2+-[HETPP]-[pentapep-tide], the 31P couplings are only slightly modified whencompared to the Cu2+-HETPP complex. This small differenceindicates that the main characteristics of the coordination modeof the phosphate’s oxygen atoms to Cu2+ are not perturbed bythe presence of the pentapeptide.

Overall these data show that in the Cu2+-[HETPP]-[pentapeptide] complex the pentapeptide is coordinated via theaspartateâ-carboxylate(s) and possibly via the terminal NH2 atan axial position, while the HETPP molecule is coordinated tothe copper via two phosphate oxygen atoms and the N(1′) ofthe pyrimidine.

Based on this structural information, a more completestructural model of the Cu2+-[HETPP]-[pentapeptide] insolution can be proposed; see Scheme 3.

In this structure, both the peptide and the thiamin bindingmust impose a distorted geometry. The coordination of thiaminpyrophosphate group and N(1′) is supported due to formationof a macrochelate ring. On the other hand, bidentate coordinationof peptide in Cu2+-[HETPP]-[pentapeptide] would involvebinding of terminal NH2-Asp1 group, of -OOCâ-Asp1 or-OOCâ-Asp2 carboxylates, and finally of a deprotonatedpeptide nitrogen N- (Scheme 3). The d-d transition of thiscomplex (centered at 621 nm) and the CW EPR parameters (g|

) 2.22,A| ) 203 G) are compatible with 2N coordination intothe equatorial plane as in Cu2+-[pentapeptide] (g| ) 2.22,A|

) 198 G).13

Conclusion

The present data show that in the Cu2+-[HETPP]-[pen-tapeptide] system at physiological pH the peptide [Asp-Asp-Asn-Lys-Ile], which mimics the protein environment of the

metal binding site [Asp185-Asp186-Asn187-Lys188-Ile189] oftransketolase, offers three coordination sites to the metal ion,i.e., the terminal amino group, the side chain of one Asp, andone main chain nitrogen. The coordination sphere is completedby two phosphate oxygens of the coenzyme as in the crystalstructures of the enzymes,3 and instead of a water molecule,the N(1′) of the pyrimidine ring is bound. This system includesa polypeptide plus the HETPP moiety, which is a covalentadduct of cofactor TPP with the substrate, common to all TPP-catalyzed reactions, and metal ions. Thus, the present studyconstitutes the first example of the synthesis and characterizationof a reliable model of the active site of thiamin-dependentenzymes in solution.

The main mechanistic implications of these findings are asfollows: (i) In solution, the high basicity of N(1′) results incoordination of this site. (ii) This N(1′) coordination does notcause a major change at the metal coordination environmentwhen compared to published crystal structures of TPP-dependentenzymes. The main difference is that the N(1′) replaces onewater molecule. (iii) As already mentioned, recent studiesshowed that HETPP-metal complexes, which present a directN(1′)-metal bond, are able to release acetaldehyde.6 All theseobservations indicate that N(1′) is a potential metal-cofactorbinding site in the thiamin catalytic cycle. Moreover, theysubstantiate our previous proposal2a that if N(1′) is to be usedas a metal coordination siteduring the catalytic cycle, then thecoordination to N(1′) should happenafter the formation of the“active aldehyde” intermediates.

The main structural information of the system Cu2+-[HETPP]-[pentapeptide] studied here was obtained based onHYSCORE, and this demonstrates that this technique can beused very efficiently for the structural study of particularlycomplicated coordination spheres. In this context the analyticalexpressions derived for the HYSCORE line shapes forI ) 1/2in the case of axialg-tensor offer a facile tool for analyzing thespectra.

Appendix

In the principal axis system of the axially symmetricg-tensor,the vector of the external magnetic fieldH is defined by itsdirection cosinesh ≡ sin θ0 cosæ0, h2 ≡ sin θ0 sin æ0, h3 ≡cosθ0 and vectorR by n1 ≡ cosθI, n2 ≡ 0, h3 ≡ sin θI; seeFigure 1.

Having defined the vectors in this way, and assuming thepoint-dipole approximation,22 then the matrix of the hyperfinecoupling tensorA can be written in the form

where we define

Then, the nuclear transition frequenciesνR, νâ for oneI ) 1/2

SCHEME 3

|T| ≡ (µ0

4π)gnânâe

R3(A2)

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System J. Phys. Chem. B, Vol. 105, No. 30, 20017331

nucleus can be written22

where R and â refer to the twoMs ) (1/2 electron spinmanifolds, respectively, and

On the basis of eqs A1-A6, we can derive the nuclearfrequencies at the canonical orientations:

Then by combining eqs A3, A7, and A9 the nuclear frequenciescan be written as

where

Equation A9 can then be rearranged in the following form:

whereQ andG are defined as

and ν2| and ν2

⊥ are defined by expressions A7 and A8,respectively.

Equation A12 describes the correlation between the nuclearfrequencies in opposite electron spin manifoldsR andâ. In aνR vs νâ plot, keeping angleφ0 as a constant and varyingθ0, eq

A12 leads to arc-shaped cross-peaks. Differentφ0 values leadto different arcs; all these arcs have a common point atθ0 )90°. Thus in a powder spectrum the line shapes of a HYSCOREspectrum with an axial effectiveg-tensor are horn-shaped ridges.The details of the line shapes depend on the orientation selection,the hyperfine coupling, and the nuclear disposition. The formsof eqs A12, A13, and A14 bear similarities to expressions 4and 5 of Dikanov and Bowmann which were derived for theisotropicg-tensor.16aIt can be easily proven that relations A12-A14 are simplified to those of Dikanov and Bowmann for anisotropic g-tensor.17a The extra termsC1 and C2 appearing inA12, are due to the nonisotropicg-tensor. From eqs A9 andA10 it is seen thatC1 and C2 are zeroed at the canonicalorientations, i.e., when the magnetic field becomes parallel toany of theX, Y, or Z axes. However, at intermediate angles theextra termsC1 andC2 are in general nonzero, and this influencesthe accumulation of intensity of the cross-peaks.16b

The analytical expressions have been used to generaterepresentative HYSCORE spectra, displayed in Figure 2. Togenerate these spectra, we have calculated the various parametersin eqs A9-A14 by using the spreadsheet software QuartoPro6.0. At each position the intensity of the cross-peak wasweighted by the transition probabilities of the allowed and thesemiforbidden transitions, respectively:16a

The analytical methodology described here is simpler thanthe calculation of the time-domain signal and its Fouriertransformation. The latter allows additional factors, i.e., apartfrom spin-Hamiltonian parameters, to be taken into account,which influence the appearance of real spectra. These are thetime-domain effects, i.e.,τ-suppression, windowing, and deadtime. For comparison we have also calculated the time-domainHYSCORE spectra for the cases described in Figure 2 (thecomputational details of our program have been described inref 10d,e). This comparison (not shown) shows that, in practicalterms, the analytical expressions allow to a good approximationa credible determination of the coupling parameters. Thus,among the aims of the present work is to point out that theanalytical approach, although less sound than the time-domaincalculations, gives an easy way for generation of the HYSCOREspectra and that this is of practical use even by non EPRspecialists.

References and Notes

(1) (a) Schellenberg, A.Angew. Chem., Int. Ed. Engl.1967, 6, 1024.(b) Kluger, R.Chem. ReV. 1987, 87,863. (c) Kluger, R.Pure Appl. Chem.1997, 69,1957. (d) Schellenberger, A.Biochim. Biophys. Acta1998, 1385,177.

(2) (a) Louloudi, M.; Hadjiliadis, N.; Feng, J. A.; Sukumar, S.; Bau,R. J.J. Am. Chem. Soc.1990, 112,7233. (b) Louloudi, M.; Hadjiliadis, N.J. Chem. Soc. Dalton Trans.1992, 1635. (c) Malandrinos, G.; Louloudi,M.; Mitsopoulou, C. A.; Butler, I. S.; Bau, R.; Hadjiliadis, N.J. Biol. Inorg.Chem. 1998, 3, 437. (d) Dodi, K.; Louloudi, M.; Malandrinos, G.;Hadjiliadis, N.J. Inorg. Biochem.1999, 73, 41. (e) Louloudi, M.; Hadjiliadis,N. Coord. Chem. ReV. 1994, 135,429 and references therein.

(3) (a) Muller, Y. A.; Schulz, G. E.Science1993, 259,965. (b) Dyda,F.; Furey, W.; Swaminathan, S.; Sax, M.; Farrenkopf, B.; Jordan, F.Biochemistry1993, 32, 6165. (c) Lindqvist, Y.; Schneider, G.; Ermler, U.;Sundstrom, M.EMBO J.1992, 11, 2373. (d) Nikkola, M.; Lindqvist, Y.;Schneider, G.J. Mol. Biol. 1994, 238, 387. (e) Hasson, M. S.; Muscate,A.; McLeish, M. J.; Polovnikova, L. S.; Gerlt, J. A.; Kenyon, G. L.; Petsko,G. A.; Ringe, D.Biochemistry1998, 37, 9918.

(4) Kern, D.; Kern, G.; Neef, H.; Tittmann, K.; Killenberg-Jabs, M.;Wikner, C.; Schneider, G.; Hu¨bner, G.Science1997, 275,67.

ν2R,â ) (MsA1 - νIh1)

2 + (MsA2 - νIh2)2 + (MsA3 - νIh3)

2

(A3)

A1 ≡ Tgeff

(g⊥h1A11 + g|h3A13) (A4)

A2 ≡ Tgeff

(g⊥h2A22) (A5)

A3 ≡ Tgeff

(g⊥h1A31 + g|h3A33) (A6)

θ0 ) 0°: ν2|(R,â) ) (MsTA33 - νI)

2 + (MsTA13)2 (A7)

θ0 ) 90°: ν2⊥(R,â) ) (MsTA11 - νI)

2 cos2 φ0 +

(MsTA22 - νI)2 sin2

φ0 + (MsTA31)2 cos2 φ0 (A8)

ν2R(â) ) [( g|

geff)2

ν2| R(â) - ( g⊥

geff)2

ν2⊥ R(â)] cos2 θ0 +

( g⊥

geff)2

ν2⊥ R(â) + [MsC1 + Ms

2C2] (A9)

C1 ≡ 2T

geff[A11h1

2g⊥(1 -g⊥

geff) + A22h2

2g⊥(1 -g⊥

geff) +

A33h32g|(1 -

g|

geff) + h1h3νI(A13g| + A31g⊥)] (A10)

C2 ≡ 2( Tgeff

)2h1h3g|g⊥[A13A33 + A11A31] (A11)

ν2R(â) ) ν2

â(R)QR(â) + GR(â)[12C1 - 14C2] + [12C1 + 1

4C2](A12)

QR(â) ≡ν2

| R(â) - ν2⊥ R(â)

ν2| â(R) - ν2

⊥ â(R)

(A13)

GR(â) ≡ν2

⊥ â(R)ν2| R(â) - ν2

⊥R(â)ν2| â(R)

ν2| â(R) - ν2

⊥ â(R)

(A14)

I ∼ 4s2c2 ) |νI2 - (1/4)(νR + νâ)

2||νI2 -

(1/4)(νR - νâ)2|/( νRνâ)

2 (A15)

7332 J. Phys. Chem. B, Vol. 105, No. 30, 2001 Malandrinos et al.

(5) (a) Shin, W.; Oh, D. G.; Chae, C. H.; Yoon, T. S.J. Am. Chem.Soc.1993, 115, 12238. (b) Lobell, M.; Crout, D. H. G.J. Am. Chem. Soc.1996, 118, 1867.

(6) (a) Malandrinos, G.; Louloudi, M.; Koukkou, A. I.; Sovago, I.;Drainas, K.; Hadjiliadis, N.J. Biol. Inorg. Chem.2000, 5, 218. (b) Kluger,R.; Trachsel, M. R.Bioorg. Chem.1990, 18, 136.

(7) Pilbrow, J. R.Transiiton Ion Electron Paramagnetic Resonance;Clarendon Press: Oxford, 1990.

(8) (a) Rowan, G. L.; Hahn, E.; Mims, W. B.Phys. ReV. A 1965, 138,4. (b) Dikanov, S. A.; Tsvetkov, Y. D.ESEEM Spectroscopy; CRC Press:Boca Raton, 1992.

(9) Deligiannakis Y.; Louloudi, M.; Hadjiliadis, N.Coord. Chem. ReV.2000, 204,1.

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(11) (a) For preparation of the ligand see ref 4d and references therein.(b) Shin, W.; Pletcher, J.; Blank, G.; Sax, M.J. Am. Chem. Soc.1977, 99,3491.

(12) (a) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G.Int. J. Pept.Protein Res. 1991, 37, 513. (b) Stavropoulos, G.; Karagiannis, K.; Vynios,D.; Papaioannou, D.; Aksnes, D. W.; Froystein, N. A.; Francis, G. W.ActaChem. Scand.1991, 45, 1047.

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(17) In the ideal case of narrow excitation only the Cu2+ complexeswith their g⊥ axes parallel to external magnetic field contribute to theHYSCORE spectrum. In real experiments, however, a broad range of

orientations contribute to the spectrum. In the case of our experimens, basedon the CW EPR spectrum, the orientation selection estimated as describedpreviously14b is 65° < θ0 < 90° within (5°. For simplicity hereafter werefer to the experimental spectra recorded in the field of 3345-3355 G asthe g⊥ position.

(18) (a) Buy, C.; Matsui, T.; Adrianambinitsoa, S.; Sigalat, C.; Girault,G.; Zimmerman, J.-L.Biochemistry1996, 35, 14821. (b) Dikanov, S. A.;Liboiron, B. D.; Thompson, K. H.; Vera, E.; Yuen, V. G.; McNeil, J. H.;Orvig, C. J. Am. Chem. Soc.1999, 121, 11004. (c) Mo¨hl, W.; Schweiger,A.; Motschi, H.Inorg. Chem.1990, 29, 1536. (d) The calculations in Figure2A,B have been performed by using a negative isotropic coupling. Thisnegative value means that the unpaired spin density at the phosphorus isgenerated via spin polarization. It should be pointed out, however, that thechoice of the sign ofAiso cannot be dictated by the HYSCORE spectraonly. For example, acceptable simulations of the experimental31P featurescan be obtained by choosing positiveAiso of +6.3-6.6 MHz and largeranisotropy, e.g., 3.6-3.8 MHz. We favor the negative sign ofAiso/smalleranisotropy, i.e., the values listed in Table 1, for two reasons: One is thatthis is in good agreement with the earlier observation of negativeAiso inref 18d. Second, the dipolar anisotropy of 3.6-3.8 MHz would require aCu-31P distance of e.g. 2 Å, and this would imply a rather unusual geometryof the phosphate’s coordination to copper.

(19) (a) Hathaway, B. J.; Billing, D. E.Coord. Chem. ReV. 1970, 5,143. (b) Peisach, J.; Blumberg, W. E.Arch. Biochem. Biophys.1974, 165,691.

(20) (a) Atherton, N. M.; Horsewill, A. J.Mol. Phys.1979, 37, 1349.(b) Schweiger, A.Struct. Bonding1982, 51, 1. (c) Poppl, A.; Hartmann,M.; Bohlmann, W.; Bo¨ttcher, R.J. Phys. Chem. 1998, 102, 3599. (d)Morton, J. R.; Preston, K. F.J. Magn. Reson.1978, 30, 577. (e) Accordingto the McConnell spin-polarization mechanism,20f an isotropic coupling atthe R-proton H(6′) can be generated byπ-spin density at the C(6′) carbonof the pyrimidine. Application of the McConnell equation allows anestimation of theπ-spin density at the neighboring carbon, i.e.,F[C(6′)] )Aiso[1H(6′)]/Q, whereQ is the McConnell constant. If we assume aQ valueof 70 (see Chapter 3 in ref 20g), then theAiso[1H(6′)] of 6.5 MHz wouldimply F[C(6′)] ) 6.5/70) 9%. A unit π-spin density at the carbon wouldgenerate an anisotropic coupling ofAani ∼ 30-40 MHz (see Chapter 5 inref 20g) at the (C)-H proton. Thus theF[C(6′)] ) 9% would account forAani ) 2.7-3.6 MHz. In this limit, the part of the observedAani ) 7.6 MHzdue to dipolar coupling is 4.9-3.7 MHz which corresponds toR(Cu-1H) )2.5-2.8 Å. (f) McConnell, H.J. Chem. Phys.1985, 24, 764. (g) Atherton,N. M. Principles of Electron Spin Resonance; Ellis Horwood: Chichester,UK, 1993.

(21) (a) Billo, E. J.Inorg. Nucl. Chem. Lett.1974, 10,613. (b) Stephens,A. K. W.; Orvig, C. J. Chem. Soc., Dalton Trans.1998, 3049.

(22) Hutchison, C. A.; McKay, D. B.J. Chem. Phys.1977, 66, 3311.

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System J. Phys. Chem. B, Vol. 105, No. 30, 20017333