A Multi-Frequency Pulse EPR and ENDOR Approach to Study

J. Phys. Chem. B 2008, 112, 3859-3870

3859

A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins M. Fittipaldi,†,§ I. Garcı´a-Rubio,‡ F. Trandafir,† I. Gromov,‡ A. Schweiger,‡,| A. Bouwen,† and S. Van Doorslaer*,† Department of Physics, UniVersity of Antwerp, UniVersiteitsplein 1, B-2610 Wilrijk-Antwerp, Belgium, and Laboratory of Physical Chemistry, ETH Zurich, CH- 8093 Zurich, Switzerland ReceiVed: October 9, 2007; In Final Form: December 4, 2007

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier singlecrystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Introduction The known versatility of the heme group to perform different functions in proteins has fascinated researchers for decades. Heme-containing proteins are involved in a variety of biological processes, whereby the function of many of the known heme proteins has not yet been identified. This is the case, for example, for two recently discovered proteinssneuroglobin and cytoglobinsbelonging to the vertebrate globin family.1,2 Since the function, reaction mechanism, and protein structure are intimately related, the characterization of the structural and electronic properties of the active site of the protein is of great interest. Some functional states of heme proteins contain the heme iron in the ferric form. The spin state of the Fe(III) center depends strongly on the nature of the axial ligands. In the case of globins, the proximal histidine ligand (F8His) is conserved in all cases. When the distal axial ligand is weak or missing, Fe(III) will be in a high-spin (HS) state, S ) 5/2, while a strong axial ligand leads to a low-spin (LS) state, S ) 1/2.3,4 Since the continuous-wave electron paramagnetic resonance (CW-EPR) spectra of both spin states differ considerably, this technique * Corresponding author. E-mail: [email protected]. † University of Antwerp. ‡ ETH Zurich. § Current address: Department of Chemistry, University of Florence, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Italy. | Arthur Schweiger passed away on January 4, 2006. He was involved in the first analyses of this work.

has traditionally been used to identify the spin state of ferric heme proteins. However, the interactions between the electron spin and the surrounding magnetic nuclei are not resolved in the CW-EPR spectra. These interactions can in principle be unraveled using CW and pulse electron nuclear double resonance (ENDOR) and pulse EPR techniques. X-band ESEEM (electron spin echo envelope modulation) and pulse ENDOR techniques have been successfully employed to study the LS Fe(III) center in proteins and in model complexes.5-8 In contrast, ENDOR and pulse EPR techniques have been only scarcely used to study HS ferric heme proteins. Predominantly, X-band CW-ENDOR experiments have been applied to study the hyperfine couplings of the directly coordinated nitrogens in HS ferric heme systems,9-11 although Q-band CW-ENDOR,12,13 X-band pulse ENDOR,14,15 and X-band three-pulse ESEEM15 studies of HS ferric systems have also been reported. Scholes et al. showed in their seminal study of aquometmyoglobin (metMb) that X-band CW ENDOR allows for a full determination of the hyperfine and nuclear-quadrupole tensors of the heme and histidine nitrogens bound to the Fe(III), provided single crystals are available.10 However, the amount of information that can be obtained with the standard ENDOR and ESEEM techniques reduces tremendously when no single crystals are at hand. In fact, all known ENDOR and ESEEM studies on frozen solutions of HS heme systems are limited to the analysis of the high-field part of the EPR spectrum (g|| extreme), and, consequently, the hyperfine and nuclear-quadrupole parameters

10.1021/jp709854x CCC: $40.75 © 2008 American Chemical Society Published on Web 03/06/2008

3860 J. Phys. Chem. B, Vol. 112, No. 12, 2008

Figure 1. Depiction of the heme plane with orientation of the axes (1, 2, and 3) of the hyperfine and nuclear quadrupole tensors of the porphyrin nitrogens as already determined by Scholes et al.10 The dashed line represents the direction of the gx axis. The solid line indicates the projection of the His plane in the heme plane.

of the surrounding magnetic nuclei are only partially determined.9,11-15 The need for characterizing HS ferric heme proteins for which no single crystals are available has prompted us to test the performance of more “advanced” pulse EPR methods. In our recent work, we already outlined the origin of the problems encountered when using the standard ENDOR and ESEEM techniques to characterize HS ferric systems and showed how X-band hyperfine sublevel correlation (HYSCORE) spectroscopy can be used to obtain some additional information.16,17 In this work, we probe the potential of a multitude of pulse ENDOR, ESEEM, and ELDOR (electron-electron double resonance)-detected NMR techniques at different microwave frequencies and outline a suitable multifrequency approach to study this kind of ferric center. Since single crystals of metMb have been extensively studied in the past, and, since the hyperfine and nuclear-quadrupole tensors of the directly coordinating heme nitrogens (Figure 1) and histidine nitrogens have been characterized,10 we chose this easy-to-handle protein as a model in the present study to evaluate the accuracy and performance of the outlined techniques. To complete the information on the spin distribution in the first coordination sphere of the ferric iron, the hyperfine interaction with the oxygen atom from the coordinating water is investigated for metMb in 17O-labeled water. It will be shown that the outlined multifrequency approach can be used as a canvas to investigate other frozen solutions of HS ferric heme proteins by pulse EPR and ENDOR spectroscopy. Materials and Methods Sample Preparation. Metmyoglobin from two sources was used for the experiments: from equine skeletal muscle (Aldrich) and from horse heart (Fluka). Lyophilized metmyoglobin (metMb) was dissolved in MOPS buffer (50 mM, pH 7) or in HEPES buffer (100 mM, pH 7) to a concentration of 5 mM. Glycerol (30%) was subsequently added to the solutions as a cryoprotector. No differences were found between the experimental results of samples using the different preparations and myoglobin sources, and therefore we will refer to the sample as metMb without further details. Different samples of metMb in D2O or H217O were prepared by dissolving the protein either in a buffer solution prepared with isotope-labeled water or in

Fittipaldi et al. the pure isotope-labeled water. D2O was purchased from Cambridge Isotope Laboratory, Inc. and from Sigma-Aldrich (99.9% purity), and H217O was obtained from Isotec (7580.9%). In the case of the deuterated samples, 30% d8-glycerol (Cambridge Isotope Laboratory, Inc. or Sigma-Aldrich (98 atom % D)) was added as a cryoprotectant. Once prepared, the protein solutions were transferred to EPR quartz tubes, frozen, and conserved in liquid nitrogen until use. Methods. The X-band CW- and pulse-EPR and ENDOR spectra were recorded on Bruker ESP380E (University of Antwerp) and Bruker E580 (ETH Zurich) spectrometers operating at a microwave (mw) frequency of 9.76 GHz. Q-band pulse EPR measurements were carried out on a home-built spectrometer operational in the frequency range of 34.5-35.5 GHz,18 and the W-band measurements were performed on two Bruker E680 spectrometers (94 GHz) operational at the University of Antwerp and ETH Zurich, respectively. The latter spectrometer has a commercial mw power upgrade installed (Bruker Biospin). All of the EPR spectrometers were equipped with helium gasflow cryostats (Oxford, Inc.). Additionally the W-band E680 spectrometer at the University of Antwerp could be used with a custom-made CryoVac immersion cryostat (Troisdorf, Germany). The spectra were taken at temperatures ranging from 3.8 to 4.2 K at X-band, 5.5 K at Q-band, and 1.7-3.8 K at W-band. Generally, the repetition rate was taken in the range from 0.8 to 3.3 kHz. The HYSCORE, ENDOR, and ELDORdetected NMR experiments were carried out at different observer positions that correspond to different selections of orientations of the molecules with respect to the magnetic field (orientation selectivity). A. CW-EPR Experiments. The X-band CW-EPR spectrum was acquired at 9.7691 GHz at a temperature of 10 K, using a modulation amplitude of 5 G and a microwave power of 718 nW. B. Electron Spin Echo (ESE)-Detected EPR Experiments. The ESE-detected EPR spectra were detected using a π/2-τ-πτ-echo sequence. The X-band ESE-detected EPR spectra (4 K) shown in this manuscript were obtained using π/2 (π) pulse lengths of 16 (32) ns and τ ) 200 ns. The W-band ESE-detected EPR spectrum was obtained using π/2 (π) pulse lengths of 100 (200) ns and τ ) 348 ns. The latter spectrum was acquired at 1.85 K. C. HYSCORE Spectroscopy. Standard HYSCORE experiments19,20 were performed with the pulse sequence π/2-τ-π/ 2-t1-π-t2-π/2-τ-echo. Different pulse lengths and τ values are used as specified in the figure captions. The time intervals t1 and t2 were varied in steps of 8 or 16 ns starting from 96 ns. An eight-step phase cycle was used to eliminate unwanted echoes. For the matched HYSCORE experiments,19,21 the sequence π/2-τ-pM-t1-π-t2-pM-τ-echo was used (pM ) highturning angle (HTA) matched pulse). Depending on the strength of the interactions that were targeted to be matched, the matching-pulse field strength, ν1, was taken to be 15.625 MHz or 31.25 MHz. For each individual experiment, the length of the matching pulses was optimized using a matched 3-pulse ESEEM experiment.21 The optimal values are indicated in the figure captions as well as the used τ values and the values of the π/2 and π pulse lengths. An eight-step phase-cycle was performed. The SMART HYSCORE19,22 sequence is pM-t1-π-t2-pMτ-π-τ-echo. The power and length of the HTA pulses was adjusted as for the matched HYSCORE experiments. The length

Pulse EPR/ENDOR Study of HS Ferric Heme Proteins

J. Phys. Chem. B, Vol. 112, No. 12, 2008 3861

of the π pulses was taken to be 16 ns. A four-step phase cycle was used to eliminate unwanted echoes. All three variants of the HYSCORE experiment were performed at X-, Q- and W-band. The experimental time traces were baseline corrected, apodized with a Hamming or Gaussian window, and zero filled. After two-dimensional Fourier transformation, the absolute-value spectra were calculated. D. X-Band Pulse ENDOR Spectroscopy. DaVies ENDOR19,23 experiments were carried out with the mw pulse sequence π-T-π/2-τ-π-τ-echo, with mw pulse lengths of 48, 24, and 48 ns, respectively, and a τ value of 128 ns. A radio frequency (rf) pulse of variable frequency and length 2 µs was applied during time interval T (2.4 µs). The sequence used to perform HYEND19,24 spectroscopy is π-T0-π-T-π-T0-π/2-τ-π-τ-echo with π/2 and π pulses of 16 and 32 ns, respectively. The delays were T0 ) 2 µs and T ) 96 ns. During time intervals T0, two π/2 rf pulses of variable frequency were applied. E. ELDOR-Detected NMR Spectroscopy. The W-band ELDOR-detected NMR19,25 experiments were performed using the pulse sequence (HTA)mw2-τ-(π)mw1-FID. Two rectangular pulses were used. The pulse lengths were 4 µs for the first pulse with variable mw frequency (mw2), and 120 ns for the second pulse with fixed mw frequency mw1. The separation between the two pulses was τ ) 1.5 µs. The FID generated after the second pulse was integrated over a width of 420 ns. The real and imaginary parts were acquired and baseline shifted, and the absolute value was calculated. The ELDOR-detected NMR spectrum of the 17O-labeled metMb was acquired using either the above-reported detection scheme or one based on a primary echo instead of an FID: (HTA)mw2-t1-(π/2)mw1-τ-(π)mw1τ-echo with (π/2)mw1 and (π)mw1 pulses of 60 and 120 ns, respectively, for the measurements at an observer position near gz, and 80 and 160 ns for those taken at a magnetic field around g ) geff x,y. In all cases, an HTA pulse of 4 µs with variable frequency mw2 was used. t1 ) 332 ns and τ ) 216 ns for g ) gz, and t1 ) 1000 ns and τ ) 716 ns for g ) geff x,y were used. The different π/2 and π pulse lengths are due to the different levels of power available at the spectrometers at the time of the measurements. In particular, the spectrum acquired at gz was recorded at ETH Zurich (use of mw power upgrade). The spectra corresponding to the low-field observer positions were predominantly acquired with a detection scheme based on the FID. As a general rule, the FID detection has to be preferred when low mw power is available. On the other hand, the detection scheme based on the primary echo avoids the problems related to the frequency dependence of the phase, which require the above-reported treatment of the observed signals. The detection scheme based on the primary echo detection requires that a long τ is used in order to record a signal free from the FID. Otherwise, a phase cycle has to be introduced. Theory. The standard spin Hamiltonian of an HS Fe(III) (S ) 5/2) coupled to different nuclear spins Ii consists of several terms:

∑k

H ) S˜ DS + βeB ˜ 0gS/h - βn

gn,kB ˜ 0Ik/h +

∑k

S˜ AkIk +

∑ ˜IkPkIk

(1)

I > 1/2

Herein, we make use of the symbol ∼ to indicate the transpose of a vector or tensor. βe and βn are the Bohr and the nuclear magneton, respectively, and B0 is the static magnetic field vector. The first term in eq 1 reflects the zero-field splitting (ZFS), whereby D is the ZFS tensor. In the case of metMb, the

tetragonal zero-field splitting D ) 9.26 cm-1 and the rhombic zero-field splitting E ) 23.15 10-3 cm-1 10,26,27. The second and third terms represent the electron and nuclear Zeeman interactions, respectively. In the case of metMb, the electronic g tensor is approximately isotropic (gz ) 2.00, gx,y ) 1.98) and close to the one of the free electron.27 The fourth term takes into account the hyperfine interaction. If I > 1/2, as in the case of 14N (I ) 1) and 17O (I ) 5/2), the nuclear-quadrupole interaction has to be included (fifth term). Ak and Pk are the hyperfine and nuclearquadrupolar tensors, respectively. Note that, strictly spoken, the quantities g and A do not transform as tensors under rotation. Nevertheless, these parameters are termed g and A tensors throughout most of the EPR literature, and we will use this convention in this paper. Since the ZFS of metMb is much larger than the electronic Zeeman splitting at X-, Q-, and W-band, only transitions within the lowest Kramers doublet (mS ) (1/2) are observed,28 and it is therefore possible to describe the system in terms of an effective spin S′ ) 1/2. In a second-order approximation, the effective spin Hamiltonian is then10

∑k B˜ 0gn,keff Ik/h + ∑k S˜ ′Aeffk Ik + ˜IkPeff ∑ k Ik I>1/2

Heff ) βeB ˜ 0geffS′/h - βn

(2)

In the limit D . hν, the g tensor in the effective spin representation, geff, is not isotropic but axial symmetric (and coaxial with the ZFS tensor) and is strongly deviating from ge17

(

geff x ) gx 3 -

12E eff 12E eff ; gy ) gy 3 + ; gz ) gz D D

)

(

)

(3)

In the case of metMb, the principal effective g values have been eff eff eff 10,29 reported to be geff x ) 5.87 gy ) 5.98 and gz ) g|| ) 2. In case the hyperfine and g tensors have the same principal axes, the principal values of the effective hyperfine tensors 17 (Aeff k ) are given by

(

eff ) Ak,x 3 Ak,x

12E eff 12E eff ; Ak,y ) Ak,y 3 + ; Ak,z ) Ak,z D D 2Ak,xAk,y (4) D

)

(

)

This equation basically means that the effective hyperfine components in the x and y directions (i.e., in the heme plane) are the components of the hyperfine tensor in eq 1 multiplied by a factor of approximately three. Furthermore, there will be a large pseudo-nuclear contribution to the nuclear gn tensor of the form17,30 eff ) gn + gn,k,x

2gxβeAk,x eff 2gyβeAk,y eff ; gn,k,y ) gn + ; gn,k,z ) gn β nD βnD (5)

Finally, the effective nuclear-quadrupole tensors also differ slightly from Pk, although these changes are usually too small to be revealed experimentally.17 Simulations of the HYSCORE Spectra. The HYSCORE simulation programs and their limitations to treat HS ferric heme centers have been reported and discussed in reference 17. In the present work, we systematically calculated the HYSCORE patterns using the EasySpin Matlab toolbox programmed for EPR simulations31 (free download at www.easyspin.ethz.ch). The nuclear frequencies were computed using the Hamiltonian given in eq 1, whereby the orientation selection of the


Fittipaldi et al. Simulations of the ENDOR and ELDOR-Detected NMR Spectra. The simulations of the ENDOR and ELDOR-detected NMR spectra have been performed using the ENDOR-simulation utilities in EasySpin. The full spin Hamiltonian of an electron spin S ) 5/2 coupled with the various nuclei was used (eq 1). However, the intensities of the double-quantum (dq) peaks (i.e., peaks corresponding with |∆mI| ) 2 transitions) observed in the ELDOR-detected NMR spectrum were not reproduced correctly by the simulations. This is due to the different transition probabilities and relaxation mechanisms involved in ELDOR-detected NMR and ENDOR experiments. Therefore the simulated ENDOR dq peaks have been amplified to allow comparison with the experimental ELDOR-detected NMR spectra. Results and Discussion

Figure 2. (A) X-band CW-EPR spectrum of a frozen solution of metMb taken at a temperature of 10 K. The asterisk indicates nonheme iron.; (B,C) X-band ESE-detected EPR spectra of a frozen solution of metMb in H2O (B) and in D2O (C). (D) W-band ESE-detected EPR spectrum of a frozen solution of metMb. The experimental parameters are given in the Methods section.

experiment was taken into account. From these frequencies, the position and shape of the HYSCORE correlation ridges can be deduced, but no information is obtained about the intensity of the cross-peaks. Therefore, a second approach was used in parallel, whereby the HYSCORE simulations were done using a simulation package developed at ETH Zurich,32 hereafter referred to as GAMMA-HYSCORE. This package allows a simulation of the HYSCORE time-domain spectra, which gives the peak intensities after Fourier transformation, but it has the drawback that it is restricted to the treatment of S ) 1/2, Ii g 1/2 systems. In our earlier work, we showed that the approximation of the HS system to a S′ ) 1/2, Ii g 1/2 system works well for the simulation of the HYSCORE spectra at the canonical eff orientations17 (i.e., geff ⊥ and g|| ). Therefore, we have simulated the HYSCORE spectrum at these orientations using both abovementioned simulation approaches, while, at intermediate orientations, only the correlation pattern has been calculated with the EasySpin-based simulation package.

A. CW- and ESE-Detected EPR. The X-band CW-EPR spectrum of a frozen solution of metMb shows the axial EPR signal typical of a HS ferric heme protein (Figure 2A). Earlier experiments showed that the geff z axis is approximately normal to the heme plane.10 The feature observed around g ) 4.3 is due to extra-heme iron and is not relevant for this study. Figure 2B shows a corresponding X-band ESE-detected EPR spectrum. As expected, the form of the spectrum depends strongly on the interpulse distance, τ, due to strong echo-modulation effects, and it does not match the absorption EPR spectrum that would be obtained by a simple integration of the spectrum in Figure 2A. The effect of the echo modulation is nicely illustrated by the comparison with the X-band ESE-detected EPR spectrum of metMb in D2O recorded under the same conditions (Figure 2C). The exchange of protons by deuterons causes a change in the nuclear modulations that has a profound effect on the shape of the ESE-detected EPR spectrum. At higher mw frequencies, the echo modulations are generally shallower as a consequence of the field dependence of the modulation depth parameter19 and the W-band ESE-detected EPR spectrum therefore resembles more the standard absorption EPR spectrum (Figure 2D). The gain in resolution achieved at W-band allows one to distinguish a small non-axiality in the effective g tensor, although the resolution gain is hampered by g-strain mechanisms that make the lines much broader at this mw frequency.33 The effective g values obtained from the simulations are geff x ) eff ) 5.95, and g ) 1.99. Note that from eq 3 and the 5.85, geff y z eff known values of E/D, gx, and gy,10,26,27 geff g is expected to y x be 0.12 for metMb. In Figure 2D the echo intensity at observer positions near g ≈ geff z is very weak, because the flip angle of the pulses has been optimized at a field value corresponding to g ≈ geff y . Indeed, for a given pulse length, tp, and mw field B1, the flip angle β0 of a pulse driving the transition between the states mS T mS+1 depends directly on the spin Hamiltonian parameters as is shown in the simple expression for the case of an isotropic g:19

β0 ) xS(S + 1) - mS(mS + 1)

gβeB1 t p p

(6)

This has important consequences for the measurements and interpretation of the ESE-detected EPR spectra of systems characterized by a large (effective) g anisotropy. B. Basic Problems Encountered When Measuring HS Ferric Heme Systems Using Standard ENDOR and ESEEM Techniques. As we explained in references 16 and 17, the heart of the problems encountered when studying HS ferric heme



proteins with standard ESEEM techniques lies in eq 4. The inplane effective hyperfine values are about 3 times bigger than Ak,x and Ak,y. Typically, the effective hyperfine values of the heme and histidine nitrogen can easily amount to 30 MHz, which is not accessible with standard ESEEM techniques. The ENDOR and ESEEM spectral interpretation is further complicated, because the nuclear Zeeman frequencies no longer agree with the tabulated values (eq 5). In contrast to the single-crystal case, where individual molecular orientations are selected by the magnetic field setting, each spectrum of a “powder-like” sample taken at a given observer position will reflect the sum of the spectra belonging to a set of different orientations, adding to the spectral complexity. This explains why only a limited amount of ESEEM and ENDOR studies have been reported on HS ferric heme proteins and why these studies focus on the single-crystal-like observer positions at g ≈ geff z . In the following section, we will show how (i) to maximize the information obtained at the latter observer position and (ii) to determine the hyperfine and nuclear-quadrupole values at the other observer positions. C. Obtaining the Hyperfine and Nuclear-Quadrupole Interactions of the Directly Coordinated Nitrogens. Figure 3A shows a standard X-band HYSCORE spectrum of metMb acquired at an observer position corresponding to g ) geff z . Many correlation peaks due to several magnetic nuclei in the heme pocket are observed in the spectrum. The most intense pair of correlation peaks in the (-,+) quadrant appear at (-5.5, +9.9) and (-9.9, 5.5) MHz (Peak 6 in Figure 3A). They have a slightly elongated shape that runs almost parallel to the antidiagonal and are separated by approximately 4νN (νN is the nitrogen Zeeman frequency). The first-order equation for the dq nuclear frequencies eff νR,β dq ≈ |A - 2νN|

(7)

suggests that they can be associated with the dq nuclear frequencies of a strongly coupled nitrogen nucleus (i.e., the hyperfine coupling is larger than twice the nuclear Zeeman frequency). Using eq 7, the hyperfine coupling at this observer position is estimated to be around 7.7 MHz. On the basis of previous studies and on the single-crystal CW-ENDOR analysis,9,10,16,27,34 these nuclear frequencies can be assigned to the porphyrin nitrogens. In the same quadrant, the cross-peaks between the single-quantum (sq) nuclear frequencies of the porphyrin nitrogens can be identified (cross-peaks around (-5.25, 2.35) MHz, (-4.4, 3.1) MHz, (-3.1, 4.4) MHz and (-2.35, 5.25) MHz, indicated as 1, 2, 3, and 4 in Figure 3A). Additionally, cross-peaks are observed at (-10, 2.4) MHz and (-2.4, 10) MHz, correlating one of the dq frequencies with one of the sq frequencies of the porphyrin nitrogens. Weaker cross-peaks can be observed at (-6.4, 5.1) MHz, (-5.1, 6.4) MHz, (-2.8, 8.5) MHz, and (-8.5, 2.8) MHz that can be ascribed to sq frequencies of the directly coordinated nitrogen of the proximal histidine ligand with Aeff ≈ 11 MHz (Peaks 7 and 8 in Figure 3A). As already observed by Scholes et al.,10 the hyperfine value of this His nitrogen is larger than that of the porphyrin nitrogens for observer positions near g ≈ geff z . The cross-peaks correlating the dq frequencies are expected to appear at (-14.9, 7.9) MHz and (-7.9, 14.9) MHz, but they are not visible in the spectrum in Figure 3A. Several other cross-peaks can be identified in the (-,+) quadrant (see Table 1 for full assignment). The signals around (-19, 5.5) and (-5.5, 19) MHz correspond to the correlations between twice the dqβ nuclear frequency and the dqR frequency

Figure 3. X-band HYSCORE and HYEND spectra of a frozen solution of metMb taken at 3.8 K and B0 ) 349 mT corresponding with g ) geff z . (A) X-band standard HYSCORE spectrum of metMb. π/2 and π pulse lengths of 16 ns and τ ) 80 ns were taken. The labeling of the peaks corresponds with the notation in Table 1. (B) X-band HYEND spectrum. Microwave π/2 and π pulses of 16 and 32 ns, respectively, were used, and the π/2 rf pulses were taken to be 2 µs. The dotted lines have a slope of 0.5 and are displaced vertically ( νN ( Qzz, with Qzz ) 1.2 MHz, a value corresponding to the histidine nitrogen for this field position. (C) X-band matched HYSCORE spectrum of metMb. The unmatched π/2 and π pulses had a length of 16 ns, and the matched second and fourth mw pulses were taken to be 48 ns. τ ) 80 ns. The correlation peaks highlighted with circles are the combination peaks between dqP and νH.

of the porphyrin nitrogens (cross-peak 12). It is worth noting that these peaks are doubled, as well as the earlier mentioned (sqP, sqP) cross-peaks, which means that two sets of porphyrin nitrogens with slightly different hyperfine couplings are present (Table 1). In addition, many cross-peaks appear in the (+,+) quadrant of Figure 3A. The ridge on the diagonal situated at the proton Zeeman frequency (marked with 19) is due to proton nuclei with small hyperfine couplings. The peak at (12, 18) MHz (cross-peak 18) has been assigned to the protons of the water molecule coordinating the iron center.16,17 Moreover, a combination of νH with the dq of the porphyrin nitrogens of the kind


Fittipaldi et al.

TABLE 1: Assignment of Cross-Peaks Observed in the HYSCORE Spectra of Figure 3 Nitrogen Cross-Peaks (MHz) (-5.4, 2.4) (-5.1, 2.3) (-4.5, 3.2) (-4.3, 3.0) (-9.9, 5.5)a (-9.4, 5.3) (-6.4, 5.1) (-8.5, 2.8) (-9.9, 2.4) (-9.4, 2.3) (-19.3, 5.2) (-18.9, 5.5) (-14.5, 2.4) (-13.7, 3.3) (-14.8, 7.8) (-14.2, 8.7) (-19.4, 11.0)

(-sqP1β sqP1R) (-sqP2β, sqP2R) (-sqP1β, sqP1R) (-sqP2β, sqP2R) (-dqP1β, dqP1R) (-dqP2β, dqP2R) (-sqHisβ, sqHis R) (-sqHisβ, sqHis R) (-dqP1β, sqP1R) (-dqP2β, sqP2R) (-dqP2β- dqP1β, dqP2R) (-2dqP2β, dqP1R) (-dqP1β - sqP2β, sqP1R) (-dqP2β- sqP2β, sqP1R) (-dqHisβ, dqHisR) (-dqP1β -sqP1β, dqP1R + sqP1R) (-2dqP1β, 2dqP1R)

Hydrogen Cross-Peaks (MHz) (18, 12) (14.9, 14.9)

water (ν1wβ, ν2wR) (νH, νH)

Combinations N and H (5.6, 14.9) (5.0, 14.9) (14.9, 24.2) (14.9, 24.8) (5.7, 20.0) (24.8, 9.3) (8.5, 17.3) (27.8, 6.4) a

(νH - dqP2β, νH) (νH - dqP1β, νH) (νH, νH + dqP2 β) (νH, νH + dqP1 β) (νH - dqP2β, νH + dqP2R) (νH + dqP2β, νH - dqP1R) (ν1wβ - dqP2β, ν2wR + dqP2R) (ν1wβ + dqP2β, ν2wR - dqP1R)

Label in Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Label in Figure 3 18 19 Label in Figure 3 20 21 22 23 24 25 26 27

Not observed in the spectrum.

(νHR, νHβ ( vdqβ) are observed (cross-peaks 20-23). Peaks 2023 strongly reflect the shape of the proton matrix ridge and the doubling of these combination ridges reveals again the inequivalence of the porphyrin nitrogens. Note as well that the porphyrin dq cross-peaks appearing at approximately (5.5, 9.9) MHz in the (+,+) quadrant are also doubled. The two nonequivalent sets of porphyrin nitrogens can also be revealed using X-band HYEND spectroscopy (Figure 3B). This 2D technique renders the ENDOR frequencies versus the hyperfine interaction.19,24 Its increased resolution with respect to the standard Davies-ENDOR experiment (similar to the skyline projection in the figure) not only allows complete separation of the contributions of the His nitrogens (|AHis zz | ) 11.5 MHz) from those of the porphyrin nitrogens, but also resolves the inequivalence of the porphyrin nitrogens, one of them with a hyperfine coupling constant |AP2| ) 7.1 MHz and another one with |AP1| ) 7.7 MHz (see Table 2). These porphyrin hyperfine values are not eigenvalues of the hyperfine tensors because of the tilt of the z axis of the hyperfine-tensors with respect to the Fe-NHis direction10 which coincides with the z axis of the g tensor (Table 2). The corresponding nuclearquadrupole couplings along the heme normal can also be easily P1 derived from the HYEND spectra (|QHis zz | ) 1.28 MHz, |Qzz | ) P2 0.33 MHz, |Qzz | ) 0.23 MHz). Note that the g and A strain are much stronger in frozen solutions than in single crystals. As a matter of fact, the signals in the HYEND spectra are elongated, indicating a significant distribution of A values. In the Supporting Information, the simulation of the HYSCORE spectrum

in Figure 3A using the hyperfine and nuclear-quadrupole values matching the HYEND data are shown. Although the inequivalence between the different nitrogen interactions could nicely be resolved in the X-band HYEND at observer position g ) geff z (Figure 3B), the technique’s performance at lower magnetic field settings was very poor. This is inherent to the method, which is known to perform well for single crystals with nuclei of arbitrary spin I and for disordered systems with I ) 1/2 nuclei.19 For disordered systems with I > 1/2 the hyperfine lines become broad and often no HYEND signal can be detected. The X-band HYEND in Figure 3B could only be detected because the observer position is in this case a “single-crystal-like” position. Finally, matched HYSCORE experiments allow obtaining even more information at observer position g ) geff z (Figure 3C and Table 1 for assignment of the peaks). In matched HYSCORE, the second and third π/2 pulse are replaced by HTA pulses in order to enhance specific interactions.19,21 We now observe the cross-peaks between the dq frequencies of the histidine nitrogen ((-14.8, 7.8) MHz and (-7.8, 14.8) MHz, peaks 15) that remained undetected with standard HYSCORE (Figure 3A versus Figure 3C). Interestingly, the peaks highlighted with circles in the figure correspond to a combination between the water-proton nuclear frequencies and the dq frequencies of the heme nitrogens (26 and 27 in Table 1). Since only combinations of nuclear frequencies in the same electron spin manifold are possible and gn(1H) and gn(14N) have the same sign, the observed combinations immediately reveal that the hyperfine values have the same sign in this observer position. Because the hyperfine coupling of the water protons is predominantly dipolar in character,9 the hyperfine constant of the water protons is positive along the heme normal. Consequently, the hyperfine-coupling constant of the porphyrin nitrogens is also positive. This gives experimental proof to the earlier assumption of a positive sign for the nitrogen hyperfine values based on ligand-field theory.10 It also explains our earlier assignment of nuclear frequencies to the R or β manifold. It should be stressed at this point that, for the observer position g ) geff z , the nitrogen hyperfine and nuclear quadrupole couplings can be obtained from the X-band HYSCORE and/or HYEND experiments of the frozen metglobin solution without prior knowledge from the single-crystal CW-ENDOR data.10 This was demonstrated in our HYSCORE analysis of ferric E7Q neuroglobin.16 The interpretation of the HYEND spectrum (Figure 3B) is straightforward. The elucidation of the HYSCORE spectra (Figures 3A,C) may seem less obvious, but cross-peaks 1, 2, 3, 4, 6, 12, and 13 are clearly recognizable as belonging to a set of at least two equivalent nitrogens (thus the heme nitrogens), and this gives the key to fully interpret these spectra. Furthermore, the HYSCORE spectra allow obtaining information about the relative sign of the hyperfine couplings via the combination frequencies. This information does not follow from ENDOR-type experiments. Figure 3A shows that the standard HYSCORE experiment at g )geff z is very informative as far as the nitrogens of the first coordination sphere are concerned. However, the intensity of the correlation peaks of these nitrogens decreases dramatically when the observer position is moved to lower field. In the close vicinity of the g ) geff z magnetic field setting, this problem can be solved by the use of matched HYSCORE. Nevertheless, at the field settings close to geff x,y no signal could be observed neither with standard HYSCORE nor with its matched variant. Indeed, the effective hyperfine of the coordinating nitrogens in the perpendicular plane are of the order of 21-30 MHz,10 which



Figure 4. X-band SMART HYSCORE of metMb in 2H2O. The high turning angle (HTA) pulses lengths were tuned to optimize the modulation depth of the coupled nitrogens. Experimental spectrum acquired at (A) B0 ) 117 mT g ≈ geff y , HTA pulse length was 52 ns, and τ ) 132 ns, and (B) B0 ) 240 mT, HTA pulse length was 24 ns, and τ ) 96 ns. (C,D) Simulations of the correlation ridges of the porphyrin and histidine nitrogens using the parameters collected in Table 2.

TABLE 2: Hyperfine and Nuclear-Quadrupole Parameters Used for Simulating the Spectral Contributions of Nuclei Directly Surrounding the Fe(III) Center in metMba

17 O-water His N Nporphyrin

P1 P2

Axx (MHz)

Ayy (MHz)

Azz (MHz)

γ (°)

β (°)

R (°)

Qxx (MHz)

Qyy (MHz)

Qzz (MHz)

-11.5 8.30 10.00 9.60

-11.8 8.03 6.90 6.70

-17 11.50 7.42 7.10

0 0 30 -60

0 0 1 5

0 11 0 0

-0.44 0.06 -0.75 -0.78

0.29 1.21 1.08 1.01

0.15 -1.28 -0.33 -0.23

a The Euler angles relating the hyperfine tensors with the g tensor. The orientation of the hyperfine tensors and nuclear quadrupole tensors of the nitrogens was taken from ref 10.

makes detection not trivial. In contrast, the SMART HYSCORE scheme19,22 allows for detection of coordinating nitrogen signals also for g values close to geff x,y (see Figure 4). This detection scheme combines the enhanced sensitivity due to the use of matched pulses with the lack of blind spots. The SMART HYSCORE spectrum of metMb in D2O taken at geff y (Figure 4A) shows correlation peaks between the dq frequencies of the His nitrogen. These peaks are separated by 4 times the effective nitrogen Zeeman frequency, 4νN,eff, which is larger than the tabulated νN as a result of the pseudo-nuclear correction (eq 5). From this observation and the knowledge that D is positive,10,26,27 the positive sign of hyperfine value at this observer position is obtained (eq 5). This gives experimental proof for earlier assumptions about the sign of the hyperfine values.10 The ridges linking the dq frequencies of the porphyrin nitrogens are also observed, although the overall intensity is lower because in-plane anisotropy of the hyperfine matrix is larger for these nitrogens than for the histidine nitrogen (Table 2). Moreover, the correlation peaks that appear further away from the diagonal,

are combination peaks involving the dq frequencies of the His nitrogen and νH (see labels in Figure 4A). The ridges around (-13,13) MHz on the diagonal are due to correlations between the sq frequencies of the porphyrin nitrogens, and the crosspeaks appearing further away from the diagonal are correlating the sqP frequencies and νH (see label in Figure 4A). Figure 4B displays the SMART HYSCORE spectrum taken at B0 ) 240 mT, in the intermediate region of the spectrum. Both, the dqdq correlations of the His and porphyrin nitrogens and the sq cross-ridges of the porphyrin nitrogens can be followed to this field range, although no signal is obtained with standard or matched HYSCORE. Figure 4C,D shows the simulations of the correlation ridges for the porphyrin and histidine nitrogens based on the parameters in Table 2. In the Supporting Information, the simulations are shown overlain with the experimental spectra. The orientation of the hyperfine and nuclear quadrupole principal axes are found to match those given by Scholes et al.,10 and for the porphyrin nitrogens they are depicted in Figure 1. The β Euler angle reported in Table 2 for the porphyrin


Figure 5. Simulations of the X-band HYSCORE spectra at g ≈ geff y (B0 ) 117.2 mT) performed using the GAMMA HYSCORE program. The simulations are shown with the same contour levels. (A) Simulation of the N(His) contribution and a water deuteron (the deuteron nuclear quadrupole interaction was ignored), B0 ) 117.2 mT. (B) Simulation assuming the interactions with the N(His) and a water proton.

nitrogens accounts for the fact that the iron atom is slightly displaced out of the porphyrin plane.10 The parameters found are in agreement with those previously reported.10 Both spectra shown in Figure 4A,B were recorded on metMb in deuterated water. We observed experimentally that the detection of the dq cross-peaks of the directly coordinating nitrogens is facilitated in a deuterated solvent. First of all, the phase memory time increases by a factor of 13 in D2O versus normal water.19 Furthermore, the simulations in Figure 5 show that the cross-suppression effect recently reported for other systems35 also plays a role in this case. The simulations of the HYSCORE spectra in Figure 5 were performed with GAMMAHYSCORE. The effective spin Hamiltonian (eq 2) was considered taking into account the effective hyperfine tensors (eq 4) and the Zeeman frequency of the different nuclei was corrected according to eq 5. As we showed earlier, this simulation approach works well at the canonical orientations.17 The simulations were obtained assuming the interaction with the axial His nitrogen and one water deuteron (Figure 5A) and the contribution of the N(His) and one water proton (Figure 5B). Although the contour levels are the same for both simulations the correlation peaks of the His nitrogen are not seen in Figure 5B, that is, the echo modulation due to protons of the coordinating water suppresses the modulations of the coordinating nitrogen. Consequently, we can derive that the use of deuterated water eliminates the strong modulations of the water protons that are responsible for the suppression of the nitrogen modulations. In the Supporting Information, the simulation of only the correlation peaks of the His nitrogen is shown. Furthermore, our simulations using GAMMAHYSCORE show that the proton cross-suppression effect is less pronounced in the Q-band (not shown). This can be easily

Fittipaldi et al. understood, since the proton modulation depth decreases dramatically at the Q-band. Indeed, it is experimentally observed that the use of higher mw frequencies facilitates the detection of coordinating nitrogen signals, even at magnetic fields corresponding to geff y (Figure 6). Correlation peaks between the sq frequencies of the porphyrin nitrogens can already be observed in the Q-band standard HYSCORE spectrum at observer position g ≈ geff y (Figure 6A) and identified by the simulation of their nuclear transitions according to the parameters of Table 2 (Figure 6C and Supporting Information). Furthermore, the HYSCORE spectrum can be obtained at a temperature of 5.5 K, which, at X-band, is impossible, because even the intensity of the two-pulse echo is very low at this temperature. This illustrates the mw dependence of the populations of the energy levels and the consequent influence on the different relaxation mechanisms. Until now, no W-band HYSCORE spectra have been reported in the literature, because the standard available W-band pulse EPR spectrometers, such as the commercial Bruker spectrometers used in this work, do not allow one to generate a hard enough inversion π pulse (i.e., a narrow pulse with sufficient excitation width) to transfer the nuclear coherence from one mS manifold to the other at these high mw frequencies. However, this situation is different for the HS ferric system at hand. Here, the observer positions near geff x,y correspond to “low” magnetic fields (around 1.15 T), which are similar fields as needed to study an organic radical at Q-band and the earlier mentioned spin-system dependence of the pulse flip angle β0 (eq 6) implies that, for a given (instrument determined) maximum microwave field strength B1, a π/2 pulse can be obtained at a smaller pulse length than would be needed for an S ) 1/2 system.36 Therefore, it becomes feasible to record W-band HYSCORE for these HS ferric systems. Figure 6B shows the first HYSCORE spectrum recorded at W-band microwave frequencies. The spectrum again reveals the correlations between the sq frequencies of the porphyrin nitrogens. Figure 6D shows the corresponding simulation of the correlation ridges using the values from Table 2. This is shown overlain with the experimental spectra in the Supporting Information. It is worth noting that the shape of the ridges in Figure 6A,B again reflects a small difference in two sets of porphyrin nitrogens with slightly different parameters (see Supporting Information). Although this inequivalence of the heme nitrogens is known from the single-crystal study,10 it has never been resolved in frozen solutions of HS ferric heme systems at this field position. Figure 7 displays the W-band ELDOR-detected NMR spectrum of a frozen solution of metMb acquired at an observer position corresponding to g ≈ geff y . In ELDOR-detected NMR, the spin system is prepared by a pulse sequence of mw frequency 1 (e.g., inversion of population on a selected EPR transition via a π pulse). This system is then observed while the microwave frequency mw2 of the ELDOR pulse is swept. Each time mw frequency 2 equals a forbidden or allowed EPR transition, the population in the observed transition will change. Observation of this change as a function of the difference between the two mw frequencies results in a spectrum reflecting the nuclear frequencies. In Figure 7, the sq transitions of the strongly coupled nitrogens of the heme and of the His contribute mainly in the frequency region between 5 and 25 MHz. The bands between 25 and 40 MHz are attributed to dq transitions of the same nitrogens. The dq transitions also contribute around 15 MHz. The band around 6.5 MHz can partially be due to remote nitrogens. The simulations of the spectrum were



Figure 6. (A) Q-band standard HYSCORE spectrum of a frozen solution of metMb taken close to g ≈ geff y , B0 ) 462 mT, T ) 5.5 K. The π/2 pulse lengths were 24 ns, and the length of the inversion π pulse was 16 ns. τ ) 80 ns. (B) W-band standard HYSCORE spectrum of a frozen solution of metMb at g ) geff y , B0 ) 1.146 mT, T ) 3.8 K. The π/2 pulse lengths and the length of the inversion π pulse were 16 ns. The spectrum is the sum of three spectra with τ ) 80, 128, and 192 ns. (C,D) Simulations of the correlation ridges calculated for the porphyrin and histidine nitrogens for spectra A and B using the parameters collected in Table 2.

Figure 7. Experimental W-band ELDOR-detected NMR spectrum of metMb acquired at B0 ) 1.17 T, corresponding to geff y , T ) 1.7 K (top). Simulations of the spectrum using the parameters of the directly coordinated nitrogens collected in Table 2 (bottom). The peaks around 50 MHz are due to different protons.

performed as described in the Methods section. In the simulation, a Lorentzian response of the resonator has been considered, which enhances the bands at low frequencies. The signals between 40 and 60 MHz stem from the surrounding protons and were not included in the simulation. Note that the resolution of the W-band ELDOR-detected NMR spectrum is lower than that obtained with the multi-frequency HYSCORE approach. However, the technique has the advantage that it is fast (this spectrum was taken with one scan). This hugely contrasts the conditions required for detection of strongly coupled nitrogens using W-band Davies ENDOR, requiring thousands of scans if detection is at all possible. Finally, it is important to evaluate what information about the in-plane nitrogen hyperfine and nuclear-quadrupole couplings can be obtained from the above-mentioned experiments

without making use of the prior knowledge of the single-crystal data. It is clear that, since the effective g tensor is quasi-axial, the orientation of the in-plane principal hyperfine and nuclear quadrupole axes cannot be obtained from a study on a frozen solution, independent of the used method. However, the largest nuclear quadrupole value (absolute value) of the heme nitrogens is expected to lie in plane perpendicular to the iron-N bond, as is found for other metal porphyrin complexes.6 By taking this into account, the orientation of the in-plane principal values of the heme nitrogen hyperfine interactions follows from the experimental data. In the particular case at hand, the X-band SMART HYSCORE spectra allow the full determination of the in-plane hyperfine and nuclear quadrupole principal values of the heme nitrogens without prior knowledge of the single-crystal ENDOR data. It should be noted, however, that, at the geff x,y observer position, the HYSCORE dq cross-peaks of the heme nitrogens are much broader (and hence weaker) than the corresponding dq cross-peaks of the His nitrogen (Figure 4A). In cases of low-concentrated samples, the former peaks may become undetectable, as we showed for the ferric E7Q neuroglobin case.17 In this case, the X-band HYSCORE experiments should be extended with either Q- or W-band HYSCORE in which the (sqP, sqP) correlations are very well resolved or with W-band ELDOR-detected NMR. The latter technique gives an ENDOR-like spectrum, albeit with less resolution than the HYSCORE method, which may make an accurate determination of the nuclear quadrupole values more difficult. The dq crosspeaks of the metal-bound His nitrogen can be easily observed in the X-band SMART HYSCORE spectra at an observer position near g ) geff x,y and therefore allow for an accurate


Fittipaldi et al.

Figure 8. (A) Solid lines: X-band Davies ENDOR signals of metMb in 17O-labeled water at various observer positions (corresponding g values are indicated for each spectrum). The bottom spectrum shows the corresponding X-band Davies-ENDOR spectrum of metMb in normal water at a magnetic-field setting corresponding with g ) 2. Simulations of the 17O contributions are shown as dashed lines. (B) X-band standard HYSCORE spectrum of metMb labeled with 17O-water at B0 ) 320 mT measured at 3.8 K. π/2 and π pulse lengths of 16 ns and τ ) 128 ns were used. (C) The simulation of the 17O correlation ridges for spectrum B using the parameters given in Table 2. (D) W-band ELDOR-detected NMR spectra at various observer positions (the corresponding g values are indicated). The simulations of the 17O contribution using the parameters in Table 2 are shown (dashed lines). (E) W-band ELDOR-detected NMR spectra at B0 ) 1.15 T of the 17O-labeled metMb (continuous line), of the metMb (dashed-dotted line). The simulation of the 17O contributions is shown as a dashed line. The experimental parameters are given in the Methods section.

determination of the in-plane hyperfine value of this nitrogen. Since the dq frequencies depend to the second order on the nuclear quadrupole coupling and since the sq cross-peaks of the His nitrogen can sometimes be masked by the sq crosspeaks of the porphyrin nitrogens, the experimental error on the in-plane nuclear quadrupole values of the His nitrogen may be quite large. D. Obtaining the Hyperfine and Nuclear-Quadrupole Interactions of the 17O Nucleus of the Coordinating Water. Only partial information has been reported up till now about the spin density on the distal ligand in metMb.13 To complete the spin-density map on the first coordination sphere of the iron,

we address here the pulse EPR/ENDOR investigation of metMb in 17O-labeled water. Figure 8A shows a set of X-band Davies-ENDOR spectra of metMb in 17O-labeled water acquired at different magnetic fields ranging from g ) 2.005 to g ) 2.488. The bottom spectrum shows the ENDOR spectrum of metMb in normal water. Upon labeling of the coordinating oxygen with 17O (I ) 5/2), we clearly observe two additional bands in the ENDOR spectra, with no resolved nuclear-quadrupole structure, separated by approximately twice νO () 1.96 MHz at B0 ) 340 mT). These bands are centered at |Aeff/2|. The simulations of these spectra obtained using the parameters reported in Table 2 are also shown

Pulse EPR/ENDOR Study of HS Ferric Heme Proteins in Figure 8A. At lower magnetic-field settings, no resolved 17O Davies-ENDOR spectra could be recorded. The comparison between the X-band HYSCORE of metMb in natural abundance and 17O-labeled water allows assigning the 17O signals at g ) geff z . Moreover, it is possible to follow these signals for a few lower fields (see Figure 8B). The correlations around (-11.5, 9) MHz and (-9, 11.5) MHz are centered at |Aeff/2| and separated by approximately twice the nuclear Zeeman frequency of 17O (2νO). These ridges are therefore assigned to (sqΟ,sqΟ), and they partially overlap the low-frequency ends of the heme (dq, dq) correlations (see nuclear frequency calculations in Supporting Information, Figure 5). The large width of each (sqΟ,sqΟ) ridge is due to the unresolved 17O nuclear quadrupole interaction. Figure 8C shows the calculations of the 17O contributions to the spectrum, which are sensitive to the nuclear quadrupole parameters. 17 From the experiments taken close to geff z , the Azz( O) can be determined quite accurately while the parameters at g⊥ are underdetermined. The attempts to record the X-band ENDOR or HYSCORE signals of 17O at g⊥ failed at all mw frequencies even using the matched and SMART HYSCORE pulse sequences. The W-band ELDOR-detected NMR experiment on the 17O metMb was initially performed at geff x,y at 3.8 K. At this temperature, no signal of 17O could be detected due to the low resolution of the experiment, which is determined by the relaxation times. Tm and T1 are longer for magnetic-field settings near g ) geff z than for the lower field positions and hence the ELDOR-detected NMR spectrum at g )geff z revealed a clear 17O signal at 3.8 K (see Figure 8D). By going to lower temperatures (1.7 K), the relaxation times could be sufficiently slowed down in order to observe a 17O signal at observer positions near g ) geff x,y (see Figure 8D,E). The W-band ELDOR-detected NMR thus provides a means to determine the full 17O hyperfine tensor where other EPR and ENDOR experiments fail. From the simulations of all the pulse experiments on 17Olabeled metMb, the spin-Hamiltonian parameters were deduced and are listed in Table 2. Given the fact that the spin density on the porphyrin and histidine nitrogens is positive and that the gn(17O) value has a sign opposite to that of gn(14N), negative values of the 17O hyperfine eigenvalues were used in the simulations. The dependence of the effective nuclear Zeeman frequency on the sign of the hyperfine interaction (eq 5) and the reasonable good agreement between the different experiments and their simulations also at orientations different from that of the single-crystal-like position g ) geff z give confidence on the sign of the hyperfine values of 17O. The Azz(17O) found in this work is 0.5 MHz smaller than the one reported previously13 based on a study focused only on geff z . Since the nuclear-quadrupole interaction could not be resolved in the spectra, the nuclear-quadrupole parameters given in Table 2 present maximum values for this splitting derived from the linewidths. The 17O nuclear-quadrupole couplings reported for a gadolinium(III)-bound water37 were used as starting values for the spectral simulations. Note that a somewhat lower nuclearquadrupole coupling constant (e2qQ/h ) 6.6 MHz) and larger asymmetry parameter (η ) 0.95) was reported for the low-spin ferric form in 17O-labeled cytochrome P450cam.38 The isotropic hyperfine parameter is proportional to the unpaired electron density, F2s, in the 2s oxygen orbital by10


F2s )

5‚Aiso(17O) Ath iso

(8)

Ath iso is the theoretical value calculated for a unitary spin density in the 2s oxygen orbital for an electron spin 1/2,39 the factor 5 in eq 8 takes into account that in this case the electron spin is 5/2. Here a 2s spin density of 1.45% is derived using a value 39 of -4622.83 MHz for Ath iso. Furthermore, our study reveals an almost axial hyperfine tensor for the 17O nucleus of the Aiso + (-Texp, -Texp, 2 Texp) with Texp ) -1.68 MHz. The dipolar interaction between the 17O nuclear spin and the spin density on the iron atom will contribute to the anisotropic part of the hyperfine tensor. The Fe-O distance is 2.15 Å40 (PDB code:1mbn). Assuming an unpaired spin density on the Fe of ∼70%,10 TFe results to be -0.75 MHz, which accounts for less than half of the experimentally observed value. Therefore, the spin density in the oxygen p orbitals clearly contributes to the anisotropic part of the hyperfine tensor. The symmetry of the hyperfine tensor indicates that the contribution from the pσ oxygen orbital is dominating. Therefore, the spin-density in the pσ oxygen orbital (Fpσ) of 3.12% is predicted using the formula

Fpσ )

5‚(Texp - TFe) Tth‚2/5

(9)

where the factor 5 in the numerator takes into account that the electron spin is 5/2, the factor 2/5 at the denominator is the angular factor for a p orbital,41 and the value of Tth ) -372.18 MHz39 was used. As is also the case for the coordinating nitrogens, the spin density in the pσ orbital is larger than that in the s orbital. The small rhombic character of the hyperfine tensor indicates minor π bonding. Conclusions Here, we investigated the performance of a number of pulse EPR and ENDOR techniques to study frozen solutions of highspin ferric heme proteins. It is obvious that the resolution that can be obtained from a single-crystal analysis will always be superior to the one obtained on disordered systems, no matter what the used technique is. Nevertheless, the here presented pulse EPR and ENDOR data show that a lot of information can be obtained from disordered HS ferric heme systems and that even small inequivalences between the heme nitrogens can be revealed, provided suitable methods are used. The study proves that X-band HYSCORE methods can be used to determine the principal hyperfine and nuclear-quadrupole values of the directly coordinating heme and histidine nitrogens. At observer positions deviating from the single-crystal-like position (g )geff z ), matched and SMART HYSCORE schemes need to be applied. Furthermore, the use of deuterated solvents has an advantageous effect on the detection of the HYSCORE spectra, because of the increase of the phase memory time and the disappearance of the pronounced proton-related cross-suppression. Note that X-band HYEND at g ) geff z can pinpoint small inequivalences between the heme nitrogens, although this information can also be obtained from the HYSCORE analysis. Compared to the classical one-dimensional ENDOR techniques, the HYSCORE and HYEND methods offer the advantage of a second correlation dimension, which allows unraveling spectral features that are strongly overlapping (and are hence not interpretable) in the classical ENDOR spectra. In cases when

3870 J. Phys. Chem. B, Vol. 112, No. 12, 2008 only small sample quantities are available, HYSCORE and ELDOR-detected NMR at higher mw frequencies may provide a valuable alternative. Because the proton modulation depth is quasi zero at these mw frequencies, HYSCORE spectra can be detected without the need for deuterated solvents. In this context, the first W-band HYSCORE spectrum was reported in this study. Moreover, the W-band ELDOR-detected NMR experiments turned out to be an easy method by which the screening of the interaction of the coordinating nitrogens and 17O-nucleus can be accomplished, although a low temperature is desired in order to achieve a good resolution in the spectrum. At W-band frequencies, this method is superior in signal-to-noise to Davies ENDOR for the detection of interactions with high nuclear spins. The present study thus provides a blueprint for future EPR analyses of HS ferric heme proteins. A set of advanced pulse EPR techniques has been applied to frozen solutions of metMb in order to construct a “road map” for obtaining complete spectroscopic data with accuracy close to single-crystal conditions. Acknowledgment. M.F. acknowledges financial support by the Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR), Italy and by the COST Action P15. This work was further supported by the Swiss National Science Foundation, by the Fund of Scientific Research-Flanders (FWO) Grant G.03468.03 (to S.V.D.) and by a “New Research Initiative” (NOI-BOF) Ph.D. Grant of the University of Antwerp (to F.T.). Supporting Information Available: X-band and Q-band HYSCORE spectra of metMb under various conditions. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Ascenzi, P.; Bocedi, A.; de Sanctis, D.; Pesce, A.; Bolognesi, M.; Marden, M. C.; Dewilde, S.; Moens, L.; Hankeln, T.; Burmester, T. Biochem. Mol. Biol. Educ. 2004, 32, 305-313. (2) Pesce, A.; Bolognesi, M.; Bocedi, A.; Ascenzi, P.; Dewilde, S.; Moens, L.; Hankeln, T.; Burmester, T. EMBO Rep. 2002, 3, 1146-1151. (3) Smith, D. T.; Pilbrow, J. R. ESR of iron proteins. In Biological Magnetic Resonance; Berliner, L. J., Reuben, J., Eds.; Plenum: New York, 1980; p 85. (4) Walker, F. A. Inorg. Chem. 2003, 42, 4526-4544. (5) Garcia-Rubio, I.; Martinez, J. I.; Picorel, R.; Yruela, I. L.; Alonso, P. J. J. Am. Chem. Soc. 2003, 125, 15846-15854. (6) Vinck, E.; Van Doorslaer, S. Phys. Chem. Chem. Phys. 2004, 6, 5324-5330. (7) Vinck, E.; Van Doorslaer, S.; Dewilde, S.; Mitrikas, G.; Schweiger, A.; Moens, L. J. Biol. Inorg. Chem. 2006, 11, 467-475.

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A Multi-Frequency Pulse EPR and ENDOR Approach to Study

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