Two-Dimensional Hyperfine Sublevel Correlation Spectroscopy

Thiamine models and perspectives on the mechanism of action of thiamine-dependent enzymes. Gerasimos Malandrinos , Maria Louloudi , Nick Hadjiliadis...
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J. Phys. Chem. B 2001, 105, 7323-7333

7323

Two-Dimensional Hyperfine Sublevel Correlation Spectroscopy Applied in the Study of a Cu2+-[2-(r-Hydroxyethyl)thiamin Pyrophosphate]-[Pentapeptide] System as a Model of Thiamin-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 and Natural 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 ternary Cu2+-[Asp-Asp-Asn-Lys-Ile]-[2-(R-hydroxyethyl)thiamin pyrophosphate (HETPP)] system has been synthesized and studied by pulsed EPR (ESEEM and HYSCORE) spectroscopy in aqueous solution at physiological pH. 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, the peptide backbone offers three coordination sites to the metal ion and the coordination sphere is completed by two additional phosphate oxygens and the nitrogen N(1′) of the thiamin coenzyme. Thus the synthetic ternary system offers the first example of a reliable structural model of the active site of thiamin-dependent enzymes in 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 thiamin catalysis.

SCHEME 1a

Introduction Thiamin enzymes catalyze the decarboxylation of R-keto acids and the transfer of aldehyde or acyl groups in vivo.1 The holoenzymes depend on the cofactors thiamin pyrophosphate (TPP) and Mg2+ or Ca2+. See Scheme 1. The mechanisms of TPP-dependent enzymes are dominated by the chemistry of TPP cofactor and its intermediates.1 Most specifically, the importance of the pyrimidine ring has always been recognized, although its participation in the catalytic process has been never understood in detail. In addition, the interaction of active aldehyde derivatives of thiamin, 2-(Rhydroxybenzyl)- and 2-(R-hydroxy-R-cyclohexylmethyl)thiamin, with group IIB metals,2a as well as with first-row transition metals,2b are of particular interest. In all these cases, the metal ions were found to be coordinated to the N(1′) atom of the pyrimidine 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 pyrophosphate, were used as ligands, metal coordination to both the pyrimidine N(1′) and the pyrophosphate group were detected.2c,d Exceptionally, Hg2+ forms complexes with the active aldehyde pyrophosphate derivatives of thiamin coordinated only to N(1′).2e In all analyzed crystal structures of thiamin-dependent enzymes,3 the metal ions (Ca2+, Mg2+) were bound in an octahedral coordination by two phosphate oxygens of the TPP, the side chains of Asp157 and Asn187, the main chain oxygen of residue 189, and a water molecule. This type of coordination * Addresscorrespondencetotheseauthors.E-mail(Y.D.): [email protected]. E-mail (N.H.): [email protected]. † Department of Chemistry. ‡ Department of Environmental and Natural Resources Management.

a R ) H; thiamin pyrophosphate (TPP); CH C(OH)H-2-(R-hydroxy3 ethyl)thiamin pyrophosphate (HETPP).

does not involve metal-N(1′) interaction and is conserved in pyruvate oxidase, pyruvate decarboxylase (PDC), transketolase, and benzyl formate decarboxylase (BFD).3 Moreover, a conserved interaction is the hydrogen bond between the side chain of Glu418 and the N(1′) atom of the pyrimidine ring.3 It was previously 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,3 and some molecular modeling studies,5 direct structural information on the active site of thiamin-dependent enzymes in solution is scarce. In this context, based on enzymic studies in solution we recently have demonstrated that the HETPP-metal complexes, which present direct metal-N1′ and metal-pyrophosphate oxygen bonds, exhibit coenzyme activity.6 In the present paper we report the synthesis and characterization of a model for the active site of thiamin-dependent enzymes in solution. Such a model, to be credible, had to incorporate the structural key components, i.e., the protein, the cofactors (the thiamin and the metal ion), and the substrate. In this context we have synthesized the pentapeptide Asp-Asp-Asn-Lys-Ile which mimics the metal binding site Asp185-Asp186-Asn187-

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

7324 J. Phys. Chem. B, Vol. 105, No. 30, 2001 Lys188-Ile189 of transketolase and surrounds the pyrophosphate moiety.3c,d Using HETPP, we have studied the tertiary system [Cu2+]-[pentapeptide]-[HETPP] in solution, at physiological pH. The structure of the tertiary system was studied by using electron paramagnetic resonance (EPR) spectroscopy.7 More particularly we have used electron spin-echo envelope modulation (ESEEM) spectroscopy8a,b to study the coordination environment of the [Cu2+]-[pentapeptide]-[HETPP] system in frozen solution. ESEEM is a pulsed EPR technique and is very well suited for studying the coordination environment of paramagnetic metal complexes in proteins, as well as in model systems.8b,9 In previous work, ESEEM has been used extensively to study Cu(II)-(histidine)n complexes, although works on Cu(II)-(non-histidine) complexes are rather scarce (for a review see ref 9 and references therein). In the present work the study of the [Cu2+]-[pentapeptide]-[HETPP] complex was mainly based on hyperfine sublevel correlation (HYSCORE) spectroscopy. HYSCORE is a two-dimensional four-pulse ESEEM technique10a and is very helpful for disentangling complicated overlapping ESEEM spectra.10b-g In the case of the [Cu2+][pentapeptide]-[HETPP] complex, HYSCORE allowed the resolution, assignment, and quantitative analysis of nuclear couplings. From these spectroscopic data structural models of this complicated system in solution were obtained. On the basis of this information, it was shown that the [Cu2+]-[pentapeptide]-[HETPP] system provides a reliable model for the active site of thiamin-dependent enzymes and the results can refer directly to thiamin catalysis. Experimental Section Materials. Thiamin pyrophosphate was purchased from Sigma Chemical Co. and used without further purification. Synthesis of the Compounds. Preparation of the Ligand 2-(r-Hydroxyethyl)thiamin Pyrophosphate (HETPP) Chloride and the Pentapeptide Asp-Asp-Asn-Lys-Ile. 2-(R-Hydroxyethyl)thiamin pyrophosphate chloride was prepared according to the literature.11 The ligand forms, (HETPPH2)0 and (HETPPH)-K+, were obtained by addition of 1 or 2 equiv of 0.1 N KOH in alcoholic solutions. Any insoluble material was removed prior to use. The peptide Asp-Asp-Asn-Lys-Ile was synthesized by solid-phase peptide synthesis using the 2-chlorotrityl chloride resin (substitution 1.1-1.6 mequiv g-1) as the solid support.12a The standard Fmoc procedure was used.12b Details on the synthesis of the pentapeptide will be published elsewhere.13 Peptide purity was controlled by TLC in the systems CH3CN-H2O (5:1 v/v) and BuOH-CH3COOH-H2O (4:1:1 v/v), 400 MHz proton NMR spectroscopy, and potentiometry. EPR and ESEEM Spectra. Continuous-wave (CW) EPR spectra were recorded at liquid helium temperatures with a Bruker ER 200D X-band spectrometer equipped with an Oxford Instruments cryostat. The microwave frequency and the magnetic field were measured with a microwave frequency counter HP 5350B and a Bruker ER035M NMR gaussmeter, respectively. Orientation-selective pulsed EPR experiments were performed with a Bruker ESP380 spectrometer with a dielectric resonator. In the three-pulse (π/2-τ-π/2-T-π/2) ESEEM data the amplitude of the stimulated echo as a function of τ + T was measured at a frequency near 9.6 GHz at various magnetic field settings across the field-swept echo-detected EPR spectrum. Data manipulations were performed as described earlier.14b The HYSCORE spectra recorded for the Cu2+ complexes studied

Malandrinos et al. here contained cross-peaks from nuclear transitions corresponding to either 14N, 31P, or 1H, some of them having considerable hyperfine anisotropy. For each complex several HYSCORE spectra were recorded at various τ-values, to compensate for the blind spots due to τ-suppression effect.15a Thus, the frequency-domain HYSCORE spectra recorded at τ ) 88, 120, and 136 ns (all the other experimental parameters being kept similar) were added together in order to minimize blind spots. Analysis of the ESEEM Spectra. The ESEEM spectra for the complexes studied here show nuclear transitions from various nuclei (1H(I ) 1/2), 31P(I ) 1/2), 14N(I ) 1)) having either weak (that is, Aiso< 2νI) or strong (that is, Aiso > 2νI) hyperfine interactions. In general, the measured three-pulse ESEEM spectra were congested consisting of overlapping weak features. In this context, HYSCORE spectroscopy proved to be very useful since it allowed both the assignment and quantitative analysis of the nuclear couplings. In the case of I ) 1/2 nuclei the spectra were analyzed based on analytical expressions which are derived in the Appendix. The values estimated from the experimental HYSCORE spectra were further refined by numerical simulations of the three-pulse ESEEM which were performed as described earlier.10d,e Theory and Method of Analysis of the HYSCORE Line Shapes for I ) 1/2, S ) 1/2 for an Axial g-Tensor. In general, the frequency-domain HYSCORE spectra consist of correlation cross-peaks whose coordinates are nuclear frequencies ((νR, (νβ) from opposite electron spin manifolds.15,16a In the case of I ) 1/2 analytical expressions are available for the HYSCORE spectrum in the time domain.15a In the frequency domain, analytical expressions for the line shapes of the HYSCORE spectra have been derived by Dikanov and Bowman16a,b,c for the case of an isotropic electron g-tensor. This method is of great practical use because it is computationally much easier than the time-domain calculations and it provides a comprehensive overview of the cross-peak line shape in HYSCORE spectra. For example, these analytical expressions have been proven to be of practical value even in cases of very complicated HYSCORE spectra containing multiple contributions from nuclei having Aiso< 2νI, Aiso ∼ 2νI, and Aiso> 2νI.16d The EPR spectra for the Cu2+ complexes studied here are characterized by axial g-tensors. In such a case, analytical expressions for the line shapes of the HYSCORE spectra are not available. Thus, in the present paper the pertinent expressions are derived (see Appendix). In the following we use these expressions to discuss HYSCORE line shapes which will be used for the analysis of the experimental spectra. Consider the principal axes system (PAS) of the g-tensor, where g| lies along Z and θ0 is the angle between the external magnetic field H0 and the g| direction; see Figure 1. At a given θ0 the effective g-value is given by

geff ) [(g⊥ sin θ0)2 + (g| cos θ0)2]1/2 We assume that the anisotropic electron-nuclear interaction can be described by the point-dipole approximation. The electron spin is at the origin of the axes, and the position of the nucleus is described by a vector R which lies in the XZ plane at angle θI from Z; see Figure 1. Then the correlation between the nuclear frequencies in two opposite electron spin manifolds R and β is given by eq A12:

ν2R(β) ) ν2β(R)QR(β) + GR(β) + 1 1 1 1 QR(β) C1 - C2 + C1 + C2 2 4 2 4

[

] [

]

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System

J. Phys. Chem. B, Vol. 105, No. 30, 2001 7325

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

The derivation of this equation together with the definitions of the various terms and further details are given in the Appendix. Orientation Selective Experiments. In eq A12, the terms C1 and C2 are nonzero at noncanonical orientations, see eqs A10 and A11. Thus in cases when the HYSCORE experiment is performed at field positions which correspond to noncanonical orientations, the correlation cross-peaks are expected to be of complicated form, as discussed in the Appendix. On the other hand, in orientation selective experiments one can select a restricted range of θ0 values by appropriate setting of the measuring magnetic field within the EPR resonances. In the case of Cu2+ complexes where the g-tensor is usually axial, or nearly axial, it is convenient to record the ESEEM spectrum at the g⊥ value of the EPR spectrum, i.e., for θ0 ) 90°. The advantage of selecting the g⊥ value is 2-fold: one is the improved signal/noise ratio of the echo, and the second is the simplification of the analysis of the cross-peaks as we discuss in the following. In the case of θ0 ) 90° the parameters C1 and C2 are 0; then eq A12 becomes

θ0 ) 90°:

ν2R(β) ) ν2β(R)QR(β) + GR(β)

(1)

The form of eq 1 is similar to the expression of Dikanov and Bowman for the isotropic g-tensor, and shows that in the orientation selective experiments at θ0 ) 90° the cross-peaks are expected to be arc-shaped. However, apart from this similarity, certain differences should be pointed out between the isotropic and the axial g-tensor. One is the different way in which the hyperfine coupling parameters Aiso and AT and the Larmor frequency νI determine the parameters QR(β) and GR(β). This can be seen by referring to eqs A1, A7-A8, and A13A14. The second important factor is that in the case of the axial g-tensor, the geometrical disposition of the nuclear spin, i.e., the angle θI in the PAS of the g-tensor, plays a key role. The relative importance of these parameters can be evaluated by examining 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 hyperfine couplings, i.e., weak for the protons (Figure 2A) and stronger for the phosphorus (Figure 2B,C). These are pertinent for the analysis of the experimental HYSCORE spectra encountered in the present case for the Cu 2+ complexes under study.

Figure 2A displays theoretical 1H HYSCORE spectra calculated for three sets of 1H hyperfine couplings corresponding to progressively increasing hyperfine anisotropy Aani ) g⊥T. In Figure 2A, we observe two characteristic trends of the correlation features as a function of the Aani values: (1) For increasing hyperfine anisotropy there is a progressive spreading of the features. This reflects the dispersion of the nuclear frequencies for increasing Aani values. (2) For increasing hyperfine anisotropy values there is a progressive increase of the maximum shift from the (-νI, νI) antidiagonal which is marked by the solid line in Figure 2A. To simplify the discussion, in Figure 2A the maximum shift ∆νmax is indicated at the left of each spectrum. This shift may be of practical use since it allows a quantitative estimation of the nuclear couplings directly from experimental HYSCORE spectra.15b,16e More specifically, for nuclei with θI > 45° the anisotropic hyperfine interaction is calculated directly from the maximum vertical shift ∆νmax of the cross-peak ridges estimated from the HYSCORE spectrum according to16e

Aani ≡ g⊥T )

x

2 3

8∆νmaxνI

x2

(2a)

From eq 2a it is seen that the estimated dipolar coupling Aani is proportional to the Larmor frequency νI. Thus, in real experiments, a given experimental ∆νmax value will correspond to larger estimated dipolar couplings for nuclei with larger Larmor frequency νI. The inset in Figure 2A is a plot of the estimated Aani as a function of ∆νmax according to eq 2a, for two typical I ) 1/2 cases i.e., 1H and 31P nuclei. According to these plots the method works better for protons than for phosphorus. From the present experimental data an upper error limit for the estimation of ∆νmax is about 0.025 MHz, and this can result in an error of up to 0.6-1.0 MHz in the estimated Aani value depending on the type of nucleus; see inset in Figure 2A. In suitable cases, the isotropic hyperfine coupling, Aiso, can be estimated from the position of the inner end of the ridges (νR, νβ) and (νβ, νR) which correspond to the canonical orientations of the hyperfine tensor15b,16a

Aiso )

|νR - νβ| + g⊥T 2

(2b)

In our experimental spectra the inner-end positions are only vaguely resolved; therefore, application of eq 2b allowed only an approximate estimation of the isotropic coupling. In the present case we have adopted the following strategy for the analysis of the 1H HYSCORE spectra: the dipolar coupling was estimated to first order from the maximum shift of the correlation ridges according to eq 2a. Based on this information, the anisotropic and isotropic coupling were determined by simulations of HYSCORE spectra based on the expressions derived in the Appendix. In the case of 31P, the observed hyperfine couplings have small anisotropy and correspond to Aiso values comparable to 2νI(31P); therefore, the evaluation of the hyperfine couplings from ∆νmax was not applicable. Instead we have simulated the experimental 31P spectra based on the analytical expressions derived in the Appendix. Pertinent theoretical HYSCORE spectra for 31P are displayed in Figure 2B and Figure 2C for Aiso< 2νI(31P) and Aiso > 2νI(31P), respectively. Despite the small anisotropy, we found that the shape of the theoretical crosspeaks for 31P was sensitive to the angle θI. For example, in the spectra in Figure 2A,B the horn-shaped features were generated

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Figure 2. (A) Theoretical frequency-domain 2D-HYSCORE line shapes for 1H(I ) 1/2) nucleus interacting with an S ) 1/2 electron spin. The horizontal and vertical dashed lines mark the nuclear Larmor frequency. The antidiagonal dotted lines mark the maximum shift (∆νmax) of the cross-peaks. The spin Hamiltonian parameters used are T ) 4.3 MHz (equivalent to R ) 2.1 Å according to eq A2) in (I), T ) 3.2 MHz (R ) 2.3 Å) 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 coupling Aani ) g⊥T, as a function of the maximum shift (∆νmax) of the cross-peaks according to eq 2a. The two plots correspond to Larmor frequencies at 3350 G, νI(1H) ) 14.26 MHz and νI(31P) ) 5.77 MHz. (B) Theoretical frequency-domain HYSCORE line shapes for a 31P(I ) 1/2) nucleus interacting with an S ) 1/2 electron spin. The horizontal and vertical dashed lines mark the nuclear Larmor frequency νI ) 5.77 MHz. The spin Hamiltonian parameters used are Aiso ) -9.2 MHz, R ) 3.4 Å, which corresponds to Aani ) 1.12 MHz, θI ) 55°, g⊥ ) 2, g| ) 2.25, and νI ) 5.77 MHz. (C) Same as in (B). The spin Hamiltonian parameters used are Aiso ) -14.0 MHz, R ) 3.2 Å, which corresponds to Aani ) 1.36 MHz, θI ) 50°, g⊥ ) 2, g| ) 2.25, and νI ) 5.77 MHz.

by setting θI of 60° in Figure 2A and 55° in Figure 2B. These particular spectra remained invariant only for θI values within (10°. In structural terms, these θI values correspond to nuclei located near, although not exactly on, the XY plane of the g-tensor of the Cu2+. Results The synthesized pentapeptide has the structure shown in Scheme 2.

Full NMR assignments of the peptide were done based on one- and two-dimensional 1H-1H TOCSY, 1H-13C HMQC, and 1H-13C HMBC spectral analysis and details will be reported elsewhere.13 Potentiometric and CW EPR Studies. Potentiometric titrations for the peptide and the Cu2+-HETPP, Cu2+-peptide, and Cu2+-[peptide]-[HETPP], as well as UV-vis and CW EPR data show that the Cu2+-peptide-HETPP system is the

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System

Figure 3. Contour plots of HYSCORE of experimental frequencydomain HYSCORE spectrum for the Cu-HETPP complex. Experimental conditions: H ) 3351 G, sample temperature 4.1 K. Time interval between successive pulse sets, 3 ms; microwave frequency, 9.64 GHz. Frequency-domain spectra were obtained after Fourier transform in the magnitude mode, of 2D time-domain patterns containing 200 × 200 points with a step of 16 ns. The displayed spectrum was obtained by numerical addition of three frequency-domain spectra recorded at τ ) 88, 120, and 136 ns, respectively.

SCHEME 2

most stable species at pH 5-8.13 The CW EPR spectra are typical of monomeric Cu2+ complexes with axial symmetry and no evidence for magnetic interactions, i.e., line broadening or splittings, between copper complexes in solution. At pH 7-7.2, the CW EPR parameters are (g| ) 2.22, g⊥ ) 2.025, A| ) 203 G, 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 HYSCORE spectrum recorded for the Cu2+-HETPP complex at pH 7.5 is displayed in Figure 3. The spectrum is characterized by pronounced ridges with considerable intensity in the (+, +) as well as in the (+, -) quadrant, and this indicates that the Cu2+HETPP complex contains nuclei with both weak and strong hyperfine couplings. The size of these couplings and their assignment are discussed in the following. 1H(I ) 1/2) Couplings. The ridges disposed symmetrically around the point at [+14.3 MHz, +14.3 MHz] in the (+, +) quadrant are assigned to protons coupled to the Cu2+ electron spin, with Aiso(1H) , 2νI(1H) ) 28.6 MHz. The form of the 1H cross-peaks in Figure 3 is directly comparable to those in the theoretical HYSCORE spectra in Figure 2A. More particularly, the arc-shaped cross-peak shifted by ∆νmax )1.8 ( 0.1 MHz from the ν1 ) -ν2 off-diagonal axis (dashed line in the 1H

J. Phys. Chem. B, Vol. 105, No. 30, 2001 7327 region in Figure 3) resembles those in the theoretical spectrum, marked as ∆νmax(I) in Figure 2A. According to eq 2a, the shift of ∆νmax ) 1.8 MHz corresponds to a proton with Aani ) 7.9 MHz. Starting from this Aani value, the best simulation for this proton is obtained by using Aani ) 7.6 MHz and Aiso ) 6.5 MHz, which is listed in Table 1. 31P(I ) 1/2) Couplings. The cross-peaks assigned to 31P and a 31P in Figure 3 are similar to those reported earlier for b HYSCORE spectra for 31P coupled to VO2+ in the case of TF1ATPase18a and recently in vanadyl coordinated to phosphate in bone.18b In Figure 3, the ridges disposed symmetrically around the point with frequency coordinates [+5.7 kHz, +5.7 MHz], i.e., the Larmor frequency of 31P(I ) 1/2), can be simulated (see Figure 2B) by using Aiso(31P) ) -9.2 ( 0.2 MHz18d and Aani ) 1.2 ( 0.1 MHz. In the (+, -) quadrant of the experimental spectrum in Figure 3, a set of cross-peaks centered at [2.5 MHz, -13.4 MHz] and [13.4 MHz, -2.6 MHz], termed 31P , have an average frequency distance of 13.4 - 2.5 MHz ) b 10.9 MHz which is close to 2νI(31P) ) 11.4 MHz. A good simulation (see Figure 2C) is obtained for a Aiso ) -14.0 ( 0.2 18d MHz and Aani ) 1.4 ( 0.2 MHz. In the present case the orientation-selective simulations show that for hyperfine couplings such as those for 31Pa and 31Pb an acceptable simulation of the experimental spectrum consistently keeps the angle θI in the range 50°-70°. This indicates that the two interacting phosphorus nuclei are located close to the equatorial plane of the g-tensor of Cu2+-HETPP. The estimated 31P anisotropic hyperfine couplings, when interpreted as pointdipole interactions, imply Cu-31P distances of, e.g., 3.2 and 3.4 Å for 31Pa and 31Pb, respectively. Such distances are consistent with coordination of the two phosphate groups via the oxygens. 14N Couplings. In addition to the 1H and the 31P couplings discussed above, the spectrum in Figure 3 contains additional low-frequency cross-peaks with maximum intensity at [+2.12.4 MHz, +4.1-4.5 MHz]. These features are characteristic of 14N(I ) 1) nuclei coupled to the electron spin with hyperfine coupling comparable to 2νI(14N) ) 2.06 MHz.10b,c,f,g In this context, the resolved features in Figure 3 are assigned to correlations between the so-called “double quantum transiitons”10c νdq(+1/2) and νdq(-1/2), respectively: for one 14N nucleus the νdq(+1/2) frequency can be approximated by8b,14a

νdq(+1/2) ∼ 2[(A/2 + νI)2 + P]1/2

(3a)

where P ≡ K2(3 + η2) is determined by the quadrupole tensor of the nitrogen, and A is the hyperfine interaction at the direction determined by the selected g-value.14a This expression is applicable only for hyperfine couplings such that the inequality (3b) holds.

|A - 2νI| < 4K/3νI

(3b)

In the case of the Cu2+-HETPP complex, the observed weak nitrogen coupling originates from the nitrogens of the HETPP; see Scheme 1. In copper(II) complexes it is well documented that the directly coordinated 14N nuclei are not resolved at X-band ESEEM (for a review see ref 9). In contrast, it is expected that the noncoordinated 14N nuclei would contribute to ESEEM at the X-band.8b,9 Thus we consider that the observed 14N coupling in Cu2+-HETPP originates from a nitrogen of HETPP which is close but not directly coordinated to copper. Since the coordination of the HETPP to copper is expected to be through the N(1′), then the plausible candidates for the 14N coupling seen in Figure 3 are the N(3′) nitrogen or the NH2 of

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TABLE 1: Nuclear Coupling Parameters of the Three Cu2+ Complexes Determined from the HYSCORE Spectra, and Literature Data literature datac complex Cu2+-pentapeptide

nucleus 1H(I) 1H(II)

Cu2+-HETPP

1

4.3, 1.6 6.3, 5.9

H Pa 31 Pb

6.5, 7.4 -9.2, 1.1 -14.2, 1.4

14N

A ) 1.2 MHz; K2(3 + η2) ) 1.9 MHz2

1H(I)

6.4, 8.0 4.4, 1.9 -9.1, 1.3 -14.0, 1.2 A ) 2.1 MHz; K2(3 + η2) ) 1.9 MHz2

31

Cu2+-HETPP-pentapeptide

Aiso, Aania,b (MHz)

1H(II) 31P 31P

a

b 14N

Aiso (MHz) Cu2+-H

2O in Mg-Tuton salt Cu2+-H2O in MCM-41 Cu2+-glycine in R-glycine host Cu2+-glycine in R-glycine host Cu2+-glycine in R-glycine host Cu2+ (CH2) in triglycine sulfate Cu2+ (CH2) in triglycine sulfate Cu2+ (CH2) in triglycine sulfate 31

P-VO2+ P-VO2+ 31 P-VO2+ 31P-VO2+ 31 P-Cu2+Cu-Thiochrome(N3′); Aiso ) 1.33 MHz; K2(3 + η2) ) 1.9 MHz2 31

0.20-0.9 1.2-2.2 3.2 6.3 3.5 3.6-3.7 5.7 3.5-4.0 -14.3 -9.2 8.7 15.5 8.2

Aani (MHz)

ref

3.4-3.9 4.1-4.8 1.5 (CH2) 5.8 (NH2)d 4.8 (NH2) 1.2-1.3 5.6 (NH2) 5.0-5.2 (NH2)

20a 20c 20b 20b 20b 20b 20b 20b

1.9 1.5

18b 18b 18a 18a 18c 14b

b c In cases where three a In our case A ani is calculated as g⊥T, where T is defined according to eq A2. Average errors (0.6 MHz for Aiso and Aani. principal values A1, A2, A3 of the hyperfine tensor have been reported, Aiso and T have been calculated as Aiso ) (A1 + A2 + A3)/3, Aani ) (A2 + A3)/2 - Aiso. d The reported 1H ENDOR data for the two 1H’s of NH2 give A(2) ) (A1,A2,A3) ) (-11.1 MHz, -5.6 MHz, 6.1 MHz) and A(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 74 of ref 23b). In our case the HYSCORE line shape in Cu-[pentapeptide] is indeed compatible with such rhombicity. However, to keep things tractable here, we approximate the proton couplings in terms of a simple dipolar tensor. Thus, in the case of the NH2 protons we consider as reference values the absolute values of Aiso,derived from (A1 + A2 + A3)/3, and of Aani ) (A1 + A2)/2 - Aiso.

the pyrimidine ring. The P 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 in aminopyrimidine14c P ) (0.82)2(3 + (0.40)2) ) 2.1 MHz2. For these two P values using νdq(+1/2) ) 4.3 MHz from eq 3a we get A ∼ 1.2 MHz (for N3′) or A ∼ 1.0 MHz (for NH2). Among these two options, the assignment of the observed 14N coupling values to N(3′) is favored because the estimated A value of 1.2 MHz fulfills the condition 3b; i.e., the deviation |A - 2νI| is 0.92 MHz, which is smaller than 4K/3νI ) 1.03 MHz. In contrast, for NH2 the |A - 2νI| is 1.12 MHz, which is larger than 4K/3νI ) 0.99 MHz. This analysis favors the assignment of the 14N coupling seen in the HYSCORE spectrum of the Cu2+-HETPP to a weak coupling of the Cu2+ electron with the N(3′) nitrogen. In summary, the HYSCORE data show that, in the Cu2+HETPP system, specific interacting 1H, 31P, and 14N nuclei are in the near vicinity of copper. From the estimated couplings listed in Table 1, further assignment of these nuclei can be done based on reference values. This will be presented in the discussion. (ii) Cu2+-Pentapeptide. The HYSCORE spectrum of the Cu2+-pentapeptide complex at pH 7.2 (Figure 4) is characterized by two sets of pronounced ridges, marked I and II in Figure 4, located in the (+, +) quadrant. They are directed perpendicular to the ν1 ) ν2 frequency diagonal and are disposed symmetrically around the point with frequency coordinates [+14.3 MHz, +14.3 MHz] which correspond to the 1H Larmor frequency at the field setting (3351 G) of the experiment. Accordingly the ridges I and II in Figure 4 are assigned to protons coupled to the Cu2+ electron spin. For their analysis we first use the maximum shift ∆νmax and then the simulated spectra.

Figure 4. Contour plots of HYSCORE of experimental frequencydomain HYSCORE spectrum for the Cu-pentapeptide complex. Experimental conditions H ) 3351 G, sample temperature 4.2 K. Other conditions as in Figure 3.

In Figure 4 the maximum shift for the ridges I does not exceed 0.1 MHz. By setting ∆ν(I)max ∼ 0.1 MHz, with νI ) 14.27 MHz, eq 2a gives an upper Aani value of ∼1.9 MHz. However, this value is based on a very small shift and might be prone to errors. An independent estimate of the Aani value can be obtained from the total width of the projection of the ridges I on either frequency axis. From the expressions for the nuclear frequencies,

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System

J. Phys. Chem. B, Vol. 105, No. 30, 2001 7329

Figure 5. Contour plots of HYSCORE of experimental frequencydomain HYSCORE spectrum for the tertiary Cu-HETPP-pentapeptide complex. Experimental conditions H ) 3352 G, sample temperature 4.2 K. Other conditions as in Figure 3.

eq A3, it can be shown that for θ0 ) 90°, φ0 ) 90° the width of the projection is16e

∆ν ∼ (3/2)Aani

(4)

From the total width of the projection of the ridges I in Figure 4, we estimate ∆ν ∼ 1.0 MHz; thus by using eq 4, we get Aani ∼ 0.7 MHz. Therefore, we consider that the Aani value for this proton should lie between 0.7 and 1.9 MHz or Aani ) 1.3 ( 0.6 MHz. Then, by using an average value of Aani ) 1.3 MHz from the inner-end position of ridges I at ∼17.3 MHz and ∼11.7 MHz from eq 2b, we get Aiso ) 4.1 ( 0.6 MHz. Taking into account the simulation of the HYSCORE spectrum, we conclude that the values Aani ) 1.6 MHz, Aiso ) 4.3 MHz are in good agreement with the ridges I in Figure 4. These values are listed in Table 1. In a similar manner we proceed with the analysis of the coupling parameters for the proton coupling giving rise to the ridges 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 2a corresponds to a Aani value of 5.9 ( 1.9 MHz. This large hyperfine anisotropy results in extended ridges whose innerend positions can be discerned near the diagonal i.e., at [∼13.7 MHz, ∼14.6 MHz] in Figure 4. From eq 2b we get Aiso ) 6.3 MHz. The ridges II are horn-shaped, which might indicate either the pertinent 1H hyperfine tensor is rhombic or a distribution in the hyperfine coupling parameters. To keep things as simple as possible, we keep the main elements of the hyperfine tensor; i.e., we consider that for proton II we have (Aiso, Aani) ) (6.3 MHz, 5.9 MHz); see Table 1. In summary, the Cu2+-pentapeptide complex is characterized by two types of 1H couplings listed in Table 1. No other nuclear couplings, i.e., 14N, are evidenced in the ESEEM and HYSCORE spectra of this complex. The assignment of the observed 1H couplings will be discussed together with the data for the other complexes studied in the present work. (iii) Cu2+-HETPP-Pentapeptide. The HYSCORE spectrum of the Cu2+-HETPP-pentapeptide complex at pH 7.5 is presented in Figure 5. To facilitate the discussion, the observed

cross-peaks have been labeled according to their origin. Before we proceed to the analytical estimation of the coupling parameters, we observe that the low-frequency part of the HYSCORE spectrum in Figure 5 (Cu2+-HETPP-pentapeptide complex) bears similarities to the spectrum in Figure 3 (Cu2+HETPP complex). More specifically, the 31P features at [+2.6 MHz, -13.4 MHz] and [+13.4 MHz, -2.6 MHz] of Figure 5 look homologous, i.e., in terms of shape and frequency position, to the features of 31Pb in Figure 3, while the 31P features [+2.2 MHz, +11.2 MHz] and [+11.2 MHz, +2.2 MHz] of Figure 5 look homologous to the features of 31Pa in Figure 3. Following the same reasoning as that for the assignment of the 31P frequencies in Figure 3, we conclude that in the Cu2+[HETPP]-[pentapeptide] complex there are two types of 31P couplings. Low-frequency cross-peaks, labeled as 14N, are also observed in 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 the 14N features in Figure 3 compared to Figure 5 is that the 14N cross-peaks in Figure 5 have nonzero intensity at the (+, -) quadrant. The frequency positions of the 14N features in the (+, +) and (+, -) in Figure 5 are similar, and this favors their assignment to one 14N coupling with hyperfine coupling close to the cancellation condition, i.e., when A(14N) ∼ 2νI(14N).8b,10b,c For example, this case has been previously verified both theoretically and experimentally by us10d,f and others.10b,c On the basis of eq 3a, we proceed with the calculation of the hyperfine couplings. To do this, we consider the possible 14N types, i.e., the peptide backbone nitrogens, NH, of the pentapeptide (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, setting νdq(+) ) 5.1 MHz from eq 3a, we estimate A ) 2.07 MHz (for NH), A ) 2.14 MHz (for NH2), or A ) 2.10 MHz (for N3′). This calculation shows that, irrespective of the type of the 14N atom, the estimated A values are comparable and all three are close to the cancellation condition. This has the immediate implication that the +2.2-2.5 MHz frequency should be a good approximation of ν+ ) K(3 + η), i.e., the double quantum frequency in the spin manifold where the nuclear Zeeman and the hyperfine terms have opposite signs. In this context, from the three sets of K, η values for NH, NH2, and N(3′), respectively, we estimate K(3 + η) values of 2.76 MHz for NH, 2.79 MHz for NH2, and 2.57 MHz for N3′. Among them the value for NH and NH2 are clearly higher than the uncertainty in the frequency position of the 2.2-2.5 MHz peak. On the other hand, the value for N(3′) fits reasonably with the experimentally observed value. Accordingly, we assign the 14N feature in Figure 5 to the N(3′) of the pyrimidine ring of HETPP. The 1H region in Figure 5 contains two types of cross-peaks, both located at the (+, +) quadrant. The first set, 1H(I), is characterized by strong anisotropy which leads to a considerable shift of ∼1.7 MHz, and this according to eq 2a corresponds to Aani ) 8.2 MHz. Further refinement by the simulation gives Aiso ) 6.4 MHz, Aani ) 8.0 MHz listed in Table 1 for 1H(I). The cross-peaks centered at [11.2 MHz, 17.8 MHz] and [17.7 MHz, 11.1 MHz] are characterized by small anisotropy. Their maximum shift is 0.1-0.2 MHz, which according to eq 2a gives Aani ) 1.8-2.6 MHz. Starting from this value, the simulation for this proton leads to Aiso ) 4.4 MHz, Aani ) 1.9 MHz, listed in Table 1 for 1H(II). In summary, in the three Cu2+ complexes studied here the HYSCORE data allowed the resolution, assignment, and

7330 J. Phys. Chem. B, Vol. 105, No. 30, 2001 quantitative analysis of specific 1H, 31P, and 14N nuclei in the near vicinity of the copper atom. From the spectroscopic point of view, taking into account the remarkably complicated nature of the coordination environment of the present complexes, especially the [Cu2+-HETPP-pentapeptide] complex, the present work demonstrates the power of HYSCORE in addressing such structural problems. Discussion Assignment of Nuclear Couplings. In the preceding paragraphs, the resolved nitrogen couplings have been assigned based on the 14N quadrupole coupling parameters. It is found that in both Cu2+-[HETPP] and the Cu2+-[HETPP]-[pentapeptide] the observed 14N coupling is due to N(3′) of the pyrimidine ring. In the Cu2+-pentapeptide complex, no 14N modulation is resolved by ESEEM. Comparison of the experimental g and A(63Cu(I ) 3/2) values of the Cu2+-pentapeptide, estimated from CW EPR, with literature data19 suggests that the coordination environment of Cu2+ should contain at least two coordinated nitrogens and two oxygens. For the coordinated nitrogens their hyperfine coupling is expected to be strong, i.e., ∼40 MHz (for a detailed discussion, see ref 9). Thus the absence of 14N echo modulations from the coordinated nitrogens in the Cu-pentapeptide complex is due to the mismatch of their hyperfine coupling and the 14N 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 of particular interest since their proper understanding contributes important structural information. Therefore, it is worth discussing these 1H couplings in some detail. In this context it is pertinent to compare the measured hyperfine couplings, listed in Table 1, with reference values from the literature. These data come from pertinent 1H ENDOR measurements and are also included in Table 1. Cu2+-HETPP. The strong 1H coupling resolved for the Cu2+-HETPP complex at pH 7.5 (see Table 1) is unique among the cases studied here. Comparison of this coupling with the 1H coupling in Cu2+-H O20a,b leads to the conclusion that in 2 the case of Cu2+-HETPP this hyperfine coupling is not due to a H2O bound to Cu2+. Among the other candidates, from the structure of the HETPP molecule we consider the methyl protons at the 2′-position, the H(6′) proton, and the NH2 protons of the pyrimidine ring of HETPP. The CH3 hyperfine tensors are usually isotropic,20b,g and this disfavors the assignment of the observed anisotropic 1H hyperfine tensor to CH3. On the other hand, this anisotropic coupling is qualitatively compatible with 1H tensors reported for either NH protons20b or for the R-protons 2 of pyridine coordinated to copper.20c At this point, the observed paramagnetic broadening of the 1H NMR signals in the Cu2+[HETPP] system is helpful. These NMR data13 show that the C(6′)-H adjacent to N(1′) is the one most affected by the Cu2+ spin. This favors the assignment of the strong 1H coupling detected in the Cu2+-HETPP complex to the H(6′) proton of the pyrimidine ring. The assignment to this proton is also in agreement with the Cu-H(6′) distance limits imposed by the strong anisotropic coupling Aani(1H) ) 7.6 MHz: if this anisotropic hyperfine coupling is due to dipolar interaction with the copper, then eq 2b gives an effective Cu-H(6′) distance of R ∼ 2.1 Å. This distance estimate is a lower limit only, since local π-spin densities on the neighboring C(6′) atom might also contribute to the 1H dipolar interaction.20e Taking into account this effect, we estimate20e a Cu-H(′6) distance of R ∼ 2.5-2.8 Å. In conclusion this analysis poses the distance limits between

Malandrinos et al. the copper and the interacting H(6′) to be 2.1-2.8 Å. This can be envisaged only if the pyrimidine is coordinated to copper through the N(1′) atom. The measured Aiso(1H) of 6.5 MHz for the H(6′) corresponds to a s-spin density of ∼6.5/1420 ) 4.5‰20d delocalized on this proton. Thus the strong 1H coupling requires that a considerable amount of the unpaired electron spin is delocalized over the pyrimidine ring. In line with this is the observation of the N(3′) coupling (A ) 1.2 MHz) which requires that a fraction, equal to ∼1.2/1809 ) 0.6‰,20d of the electron spin density is delocalized through the pyrimidine ring to the N3′ atom. This amount of delocalized spin density is in agreement with the values reported in the literature for noncoordinated nitrogens of rings liganded to copper.10c,d,f,g Overall, the present data provide strong evidence that in Cu2+-HETPP the pyrimidine ring of HETPP is directly coordinated to the copper most likely through the N(1′) atom. The two 31P couplings observed in Cu2+-HETPP are assigned to the two phosphorus atoms of HETPP. By comparing the 31P couplings estimated for Cu2+-HETPP with pertinent data in the literature,18 we conclude that the two phosphorus atoms are coordinated to Cu2+ via the oxygens. These 31P hyperfine couplings in Cu2+-HETPP are inequivalent, and this indicates that the coordination modes of the two phosphate oxygen atoms to Cu2+ are different. In conclusion, the structural picture emerging from the HYSCORE data is that in the Cu2+-HETPP complex at pH 7.5 the coordination sphere of the Cu2+ atom contains two phosphate oxygens and the N(1′) atom of HETPP. This is corroborated by the CW EPR data, g| vs A| parameters, which is compatible with a 1N and 3O coordination.19b In this context, it is possible that an oxygen solvent atom completes the coordination sphere. Cu2+-Pentapeptide. The 1H couplings resolved for the Cu2+-pentapeptide complex at pH 7.2 can originate either from the protons of the solvent (H2O) or from the pentapeptide. Comparison of the hyperfine couplings in Table 1 with those reported for H2O bound to Cu2+ 20a,b renders the assignment of either coupling to solvent protons unlikely. On the other hand, we see that the hyperfine tensor I (see Table 1), which is characterized by relatively small anisotropy and small isotropic coupling, is similar to that reported for CH2 protons in Cuglycine complex.20b Accordingly, we suggest the assignment of this proton to a CH2 group of the pentapeptide. The strongly coupled proton II (see Table 1) has a hyperfine coupling tensor similar to that reported for the NH2 protons in the Cu-glycine complex.20b Interestingly the NH2 proton couplings in the Cuglycine complex are characterized by nonnegligible rhombicity, which is in agreement with the observation of horn-shaped features 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, we suggest that at pH 7.2 the pentapeptide is coordinated to Cu2+ via the terminal amino group and the β-carboxylate of aspartate residues. Cu2+-HETPP-Pentapeptide. The presence of the weak 1H coupling in the Cu2+-[HETPP]-[pentapeptide] system, assigned to the CH2 group of the aspartate, indicates that in this system the pentapeptide is also coordinated to the copper. It should be noticed at this point that the strong NH2 coupling observed in the Cu2+-[pentapeptide] (see Table 1) is absent in the Cu2+-[HETPP]-[pentapeptide] system. This indicates that in the tertiary system the NH2 coordination is modified, i.e., is

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System SCHEME 3

severely weakened. Most likely the NH2 group occupies an axial position. This is based on the observation of a ca. 9 nm blue shift of the d-d transition of the Cu2+-[HETPP]-[pentapeptide] compared to those of Cu2+-[pentapeptide], which can be attributed to the coordination of a N atom in an axial position resulting in an elongated Cu-N bond.21 Proceeding in a similar way as in the case of the Cu2+HETPP, we assign the strong 1H coupling observed in the Cu2+-[HETPP]-[pentapeptide] system to the H(6′) of HETPP. This H(6′) interaction together with the observed N(3′) coupling implies a coordination of the HETPP via the N(1′). The two inequivalent 31P couplings resolved in Cu2+-HETPP are consistently resolved in the Cu2+-[HETPP]-[pentapeptide] system as well. Accordingly, they are assigned to the two phosphorus atoms of HETPP. In Cu2+-[HETPP]-[pentapeptide], the 31P couplings are only slightly modified when compared to the Cu2+-HETPP complex. This small difference indicates that the main characteristics of the coordination mode of the phosphate’s oxygen atoms to Cu2+ are not perturbed by the presence of the pentapeptide. Overall these data show that in the Cu2+-[HETPP][pentapeptide] complex the pentapeptide is coordinated via the aspartate β-carboxylate(s) and possibly via the terminal NH2 at an axial position, while the HETPP molecule is coordinated to the copper via two phosphate oxygen atoms and the N(1′) of the pyrimidine. Based on this structural information, a more complete structural model of the Cu2+-[HETPP]-[pentapeptide] in solution can be proposed; see Scheme 3. In this structure, both the peptide and the thiamin binding must impose a distorted geometry. The coordination of thiamin pyrophosphate group and N(1′) is supported due to formation of a macrochelate ring. On the other hand, bidentate coordination of peptide in Cu2+-[HETPP]-[pentapeptide] would involve binding of terminal NH2-Asp1 group, of -OOCβ-Asp1 or -OOCβ-Asp2 carboxylates, and finally of a deprotonated peptide nitrogen N- (Scheme 3). The d-d transition of this complex (centered at 621 nm) and the CW EPR parameters (g| ) 2.22, A| ) 203 G) are compatible with 2N coordination into the equatorial plane as in Cu2+-[pentapeptide] (g| ) 2.22, A| ) 198 G).13 Conclusion Cu2+-[HETPP]-[pen-

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

J. Phys. Chem. B, Vol. 105, No. 30, 2001 7331 metal binding site [Asp185-Asp186-Asn187-Lys188-Ile189] of transketolase, offers three coordination sites to the metal ion, i.e., the terminal amino group, the side chain of one Asp, and one main chain nitrogen. The coordination sphere is completed by two phosphate oxygens of the coenzyme as in the crystal structures of the enzymes,3 and instead of a water molecule, the N(1′) of the pyrimidine ring is bound. This system includes a polypeptide plus the HETPP moiety, which is a covalent adduct of cofactor TPP with the substrate, common to all TPPcatalyzed reactions, and metal ions. Thus, the present study constitutes the first example of the synthesis and characterization of a reliable model of the active site of thiamin-dependent enzymes in solution. The main mechanistic implications of these findings are as follows: (i) In solution, the high basicity of N(1′) results in coordination of this site. (ii) This N(1′) coordination does not cause a major change at the metal coordination environment when compared to published crystal structures of TPP-dependent enzymes. The main difference is that the N(1′) replaces one water molecule. (iii) As already mentioned, recent studies showed that HETPP-metal complexes, which present a direct N(1′)-metal bond, are able to release acetaldehyde.6 All these observations indicate that N(1′) is a potential metal-cofactor binding site in the thiamin catalytic cycle. Moreover, they substantiate our previous proposal2a that if N(1′) is to be used as a metal coordination site during the catalytic cycle, then the coordination to N(1′) should happen after the formation of the “active aldehyde” intermediates. The main structural information of the system Cu2+[HETPP]-[pentapeptide] studied here was obtained based on HYSCORE, and this demonstrates that this technique can be used very efficiently for the structural study of particularly complicated coordination spheres. In this context the analytical expressions derived for the HYSCORE line shapes for I ) 1/2 in the case of axial g-tensor offer a facile tool for analyzing the spectra. Appendix In the principal axis system of the axially symmetric g-tensor, the vector of the external magnetic field H is defined by its direction cosines h ≡ sin θ0 cos φ0, h2 ≡ sin θ0 sin φ0, h3 ≡ cos θ0 and vector R by n1 ≡ cos θI, n2 ≡ 0, h3 ≡ sin θI; see Figure 1. Having defined the vectors in this way, and assuming the point-dipole approximation,22 then the matrix of the hyperfine coupling tensor A can be written in the form

where we define

|T| ≡

( )

µ0 gnβnβe 4π R3

(A2)

Then, the nuclear transition frequencies νR, νβ for one I ) 1/2

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

Malandrinos et al.

nucleus can be written22

ν2R,β ) (MsA1 - νIh1)2 + (MsA2 - νIh2)2 + (MsA3 - νIh3)2 (A3) where R and β refer to the two Ms ) (1/2 electron spin manifolds, respectively, and

A1 ≡

T (g h A + g|h3A13) geff ⊥ 1 11

(A4)

T (g h A ) geff ⊥ 2 22

(A5)

T (g h A + g|h3A33) geff ⊥ 1 31

(A6)

A2 ≡ A3 ≡

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

θ0 ) 0°: ν2|(R,β) ) (MsTA33 - νI)2 + (MsTA13)2 θ0 ) 90°:

(A7)

ν2⊥(R,β) ) (MsTA11 - νI)2 cos2 φ0 + (MsTA22 - νI)2 sin2 φ0 + (MsTA31)2 cos2 φ0 (A8)

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

ν2R(β) )

[( )

( ) ( )

]

g| 2 2 g⊥ 2 2 ν | R(β) ν ⊥ R(β) cos2 θ0 + geff geff g⊥ 2 2 ν ⊥ R(β) + [MsC1 + Ms2C2] (A9) geff

where

[

(

)

(

)

g⊥ g⊥ T A11h12g⊥ 1 + A22h22g⊥ 1 + C1 ≡ 2 geff geff geff g| + h1h3νI(A13g| + A31g⊥) (A10) A33h32g| 1 geff

(

C2 ≡ 2

( )

)

T 2 h h g g [A A + A11A31] geff 1 3 | ⊥ 13 33

]

(A11)

Equation A9 can then be rearranged in the following form:

1 1 1 1 ν2R(β) ) ν2β(R)QR(β) + GR(β) C1 - C2 + C1 + C2 2 4 2 4 (A12)

[

] [

]

where Q and G are defined as

QR(β) ≡

GR(β) ≡

ν2| R(β) - ν2⊥ R(β) ν2| β(R) - ν2⊥ β(R)

ν2⊥ β(R)ν2| R(β) - ν2⊥R(β)ν2| β(R) ν2| β(R) - ν2⊥ β(R)

(A13)

(A14)

and ν2| and ν2⊥ are defined by expressions A7 and A8, respectively. Equation A12 describes the correlation between the nuclear frequencies in opposite electron spin manifolds R and β. In a νR vs νβ plot, keeping angle φ0 as a constant and varying θ0, eq

A12 leads to arc-shaped cross-peaks. Different φ0 values lead to different arcs; all these arcs have a common point at θ0 ) 90°. Thus in a powder spectrum the line shapes of a HYSCORE spectrum with an axial effective g-tensor are horn-shaped ridges. The details of the line shapes depend on the orientation selection, the hyperfine coupling, and the nuclear disposition. The forms of eqs A12, A13, and A14 bear similarities to expressions 4 and 5 of Dikanov and Bowmann which were derived for the isotropic g-tensor.16a It can be easily proven that relations A12A14 are simplified to those of Dikanov and Bowmann for an isotropic g-tensor.17a The extra terms C1 and C2 appearing in A12, are due to the nonisotropic g-tensor. From eqs A9 and A10 it is seen that C1 and C2 are zeroed at the canonical orientations, i.e., when the magnetic field becomes parallel to any of the X, Y, or Z axes. However, at intermediate angles the extra terms C1 and C2 are in general nonzero, and this influences the accumulation of intensity of the cross-peaks.16b The analytical expressions have been used to generate representative HYSCORE spectra, displayed in Figure 2. To generate these spectra, we have calculated the various parameters in eqs A9-A14 by using the spreadsheet software QuartoPro 6.0. At each position the intensity of the cross-peak was weighted by the transition probabilities of the allowed and the semiforbidden transitions, respectively:16a

I ∼ 4s2c2 ) |νI2 - (1/4)(νR + νβ)2||νI2 (1/4)(νR - νβ)2|/( νRνβ)2 (A15) The analytical methodology described here is simpler than the calculation of the time-domain signal and its Fourier transformation. The latter allows additional factors, i.e., apart from spin-Hamiltonian parameters, to be taken into account, which influence the appearance of real spectra. These are the time-domain effects, i.e., τ-suppression, windowing, and dead time. For comparison we have also calculated the time-domain HYSCORE spectra for the cases described in Figure 2 (the computational details of our program have been described in ref 10d,e). This comparison (not shown) shows that, in practical terms, the analytical expressions allow to a good approximation a credible determination of the coupling parameters. Thus, among the aims of the present work is to point out that the analytical approach, although less sound than the time-domain calculations, gives an easy way for generation of the HYSCORE spectra and that this is of practical use even by non EPR specialists. 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. Acta 1998, 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. Science 1993, 259, 965. (b) Dyda, F.; Furey, W.; Swaminathan, S.; Sax, M.; Farrenkopf, B.; Jordan, F. Biochemistry 1993, 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. Biochemistry 1998, 37, 9918. (4) Kern, D.; Kern, G.; Neef, H.; Tittmann, K.; Killenberg-Jabs, M.; Wikner, C.; Schneider, G.; Hu¨bner, G. Science 1997, 275, 67.

Spectroscopy of Cu+-[Pentapeptide]-[HETPP] System (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. (10) (a) Ho¨fer, P.; Grupp, A.; Nebenfu¨hr, H.; Mehring, M. Chem. Phys. Lett. 1986, 132, 279. (b) Kofman, V.; Shane, J.; Dikanov, S. A.; Bowman, M. K.; Libman, J.; Shanzer, A.; Goldfarb, D. J. Am. Chem. Soc. 1995, 117, 12771. (c) Dikanov, S. A.; Xun, L.; Karpiel, A. B.; Tyryshkin, A. M.; Bowman, M. K. J. Am. Chem. Soc. 1996, 118, 8408. (d) Deligiannakis, Y.; Astrakas, L.; Kordas, G.; Smith, R. A. Phys. ReV. B 1998, 58, 11420. (e) Astrakas, L.; Deligiannakis, Y.; Kordas, G. J. Chem. Phys. 1998, 109, 8612. (f) Deligiannakis, Y.; Hanley, J.; Rutherford, A. W. J. Am. Chem. Soc. 1999, 121, 7653. (g) Deligiannakis, Y.; Hanley, J.; Rutherford, A. W. J. Am. Chem. Soc. 2000, 122, 400. (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. Acta Chem. Scand. 1991, 45, 1047. (13) Malandrinos, G.; Louloudi, M.; Deligiannakis, Y.; Hadjiliadis, N. Inorg. Chem., in press. (14) (a) Lee, H.-I.; Doan, P. E.; Hoffman, B. M. J. Magn. Reson. 1999, 140, 91. (b) Louloudi, M.; Deligiannakis, Y.; Tuchagues, J.-P.; Donnadieu, B.; Hadjiliadis, N. Inorg. Chem. 1997, 36, 6335. (c) Schempp, E.; Bray, P. J. J. Magn. Reson. 1971, 5, 78. (15) (a) Gemperle, G.; Aebli, G.; Schweiger, A.; Ernst, R. R. J. Magn. Reson. 1990, 88, 241. (b) Ho¨ffer, P. J. Magn. Reson. A 1994, 111, 77. (16) (a) Dikanov, S. A.; Bowman, M. K. J. Magn. Reson. A 1995, 116, 125. (b) Dikanov, S. A.; Bowman, M. K. J. Biol. Inorg. Chem. 1998, 3, 18. (c) Dikanov, S. A.; Tyryshkin, A. M.; Bowman, M. K. J. Magn. Reson. 2000, 144, 228. (d) Deligiannakis, Y.; Rutherford, A. W. J. Am. Chem. Soc. 1997, 119, 4471. (e) Po¨ppl, A.; Kevan, L. J. Phys. Chem. 1996, 100, 3387. (17) In the ideal case of narrow excitation only the Cu2+ complexes with their g⊥ axes parallel to external magnetic field contribute to the HYSCORE spectrum. In real experiments, however, a broad range of

J. Phys. Chem. B, Vol. 105, No. 30, 2001 7333 orientations contribute to the spectrum. In the case of our experimens, based on the CW EPR spectrum, the orientation selection estimated as described previously14b is 65° < θ0 < 90° within (5°. For simplicity hereafter we refer to the experimental spectra recorded in the field of 3345-3355 G as the g⊥ position. (18) (a) Buy, C.; Matsui, T.; Adrianambinitsoa, S.; Sigalat, C.; Girault, G.; Zimmerman, J.-L. Biochemistry 1996, 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 Figure 2A,B have been performed by using a negative isotropic coupling. This negative value means that the unpaired spin density at the phosphorus is generated via spin polarization. It should be pointed out, however, that the choice of the sign of Aiso cannot be dictated by the HYSCORE spectra only. For example, acceptable simulations of the experimental 31P features can be obtained by choosing positive Aiso of +6.3-6.6 MHz and larger anisotropy, e.g., 3.6-3.8 MHz. We favor the negative sign of Aiso/smaller anisotropy, i.e., the values listed in Table 1, for two reasons: One is that this is in good agreement with the earlier observation of negative Aiso in ref 18d. Second, the dipolar anisotropy of 3.6-3.8 MHz would require a Cu-31P distance of e.g. 2 Å, and this would imply a rather unusual geometry of 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. Bonding 1982, 51, 1. (c) Po¨ppl, A.; Hartmann, M.; Bo¨hlmann, W.; Bo¨ttcher, R. J. Phys. Chem. 1998, 102, 3599. (d) Morton, J. R.; Preston, K. F. J. Magn. Reson. 1978, 30, 577. (e) According to the McConnell spin-polarization mechanism,20f an isotropic coupling at the R-proton H(6′) can be generated by π-spin density at the C(6′) carbon of the pyrimidine. Application of the McConnell equation allows an estimation of the π-spin density at the neighboring carbon, i.e., F[C(6′)] ) Aiso[1H(6′)]/Q, where Q is the McConnell constant. If we assume a Q value of 70 (see Chapter 3 in ref 20g), then the Aiso[1H(6′)] of 6.5 MHz would imply F[C(6′)] ) 6.5/70 ) 9%. A unit π-spin density at the carbon would generate an anisotropic coupling of Aani ∼ 30-40 MHz (see Chapter 5 in ref 20g) at the (C)-H proton. Thus the F[C(6′)] ) 9% would account for Aani ) 2.7-3.6 MHz. In this limit, the part of the observed Aani ) 7.6 MHz due to dipolar coupling is 4.9-3.7 MHz which corresponds to R(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.