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J. Phys. Chem. A 2010, 114, 324–335
High-Field Solid-State 67Zn NMR Spectroscopy of Several Zinc-Amino Acid Complexes Kamal H. Mroue´ and William P. Power* Department of Chemistry, UniVersity of Waterloo, 200 UniVersity AVenue West, Waterloo, Ontario, N2L 3G1, Canada ReceiVed: August 28, 2009; ReVised Manuscript ReceiVed: October 22, 2009
We report the results of our solid-state 67Zn NMR study of the various zinc sites in four zinc-amino acid coordination complexes: bis(glycinato)zinc(II) monohydrate; bis(L-alaninato)zinc(II); bis(L-histidinato)zinc(II) dihydrate; and sodium bis(L-cysteinato)zincate(II) hexahydrate; as well as a related complex, bis(imidazole)zinc(II) chloride. We demonstrate the advantages of using high (21.1 T) applied magnetic fields for detecting 67 Zn directly at ambient temperatures using the quadrupolar Carr-Purcell Meiboom-Gill (QCPMG) pulse sequence. The stepped-frequency technique was employed in cases where the central-transition (CT) 67Zn NMR spectra were too broad to be uniformly excited. The parameters of the anisotropic zinc tensors were extracted by iterative simulations of the experimental spectra. In all cases, the quadrupolar interaction is found to dominate the central-transition 67Zn NMR spectra; no convincing effects from chemical shift anisotropy (CSA) on the NMR spectra of the five complexes could be reliably detected at this field strength. Analyses of the experimental NMR spectra reveal that the 67Zn quadrupolar coupling constants (CQ) range from 7.05 to 26.4 MHz, the isotropic chemical shifts (δiso) range from 140 to 265 ppm, and the quadrupolar asymmetry parameters (ηQ) range from 0.20 to 0.95. The first report of the NMR spectral features of pentacoordinated zinc sites is included for two complexes. Quantum chemical calculations of the electric field gradient (EFG) and magnetic shielding tensors reproduced the experimental results to a reasonable extent. Moreover, the computationally determined orientations of both tensors permit correlations between NMR tensor properties and zinc local environments to be understood. Introduction Zinc, the second-most abundant trace metal in the human body after iron, is indispensable for all forms of animal and plant life with an average adult human body containing 2.3 g of zinc.1-4 Moreover, zinc plays significant roles in a number of fundamental biological processes, as it is found in more than 300 enzymes covering all six classes of enzymes.5-7 This high occurrence of zinc in enzyme catalysis is due to the distinctive electronic and metallic properties displayed by this metal. First, the zinc ion is extremely stable in the +2 oxidation state and does not undergo redox activities, but rather acts as a strong Lewis acid to accept a pair of electrons. Second, zinc is an element of moderate polarizability (hard-soft character) reflected by its high affinity to nitrogen, oxygen, and sulfur ligands. In zinc enzymes, the common ligands are histidine, cysteine, aspartic acid, glutamic acid, and water/hydroxide, which provide nitrogen, oxygen, and sulfur donor atoms. Finally, the closed d10 electronic configuration of Zn2+ indicates that the coordination number and geometry of its complexes are only controlled by the ligand size and charge. This flexibility in coordination geometry enhances ligand exchange and facilitates the ability of zinc to regulate catalytic processes. In catalytic sites, the zinc ion is usually tetrahedrally bound to three amino acid residues, with a solvent molecule (water or hydroxide) completing the tetrahedral coordination sphere.1,8-10 However, five-coordinate distorted trigonal bipyramidal and square pyramidal geometries have been observed in some catalytic zinc binding sites.11-13 * To whom correspondence should be addressed. E-mail: wppower@ uwaterloo.ca. Telephone: 519-888-4567, ext 33626. Fax: 519-746-0435.
In addition to its catalytic role, zinc plays a structural role in proteins that may involve lowering the folding enthalpy of a protein to induce a particular conformation or to stabilize the tertiary structure.2,3,14 In structural sites, the zinc ion is usually coordinated to four amino acid residues, in a tetrahedral geometry, so that the solvent is excluded as a first-coordination sphere ligand.7,15 The most important class of proteins in which zinc plays mainly a structural role is the zinc finger family, which are involved in DNA binding and gene regulation. In zinc finger proteins, zinc is tetrahedrally coordinated to cysteines and/or histidines that form various types of zinc-coordination environments, the most common of which are the Cys2His2Zn, Cys3His1Zn, and Cys4Zn polyhedra, thus providing ZnS2N2, ZnS3N, and ZnS4 coordination motifs, respectively.14,16 Hence, another avenue toward a complete understanding of the structure and function of zinc fingers is provided by a detailed knowledge about the local structure of the zinc binding sites in the different protein environments in which they exist. This can be achieved by the investigation of small and stable zinc-coordination complexes that mimic the structure of the zinc binding sites in zinc finger domains. Unfortunately, direct observation of the zinc ion in biologically relevant systems by routine electronic or EPR spectroscopy is difficult, if not impossible, because the ion is colorless and diamagnetic. A potentially more powerful tool for providing new physical insights into the structure about the zinc ions in these systems is direct nuclear magnetic resonance (NMR) spectroscopy of zinc nuclei. From this perspective, zinc has only one NMR-active isotope, namely the 67Zn isotope. Zinc-67 is one of the so-called low-γ nuclides (those with Larmor frequencies smaller than 10% of that of 1H) that are quite problematic to study by NMR spectroscopy. The inherent
10.1021/jp908325n 2010 American Chemical Society Published on Web 11/17/2009
67Zn
NMR Spectroscopy of Zinc-Amino Acid Complexes
difficulty of 67Zn NMR spectroscopy is due to a combination of many of its unfavorable NMR properties: 67Zn is a halfinteger quadrupolar nucleus of spin I ) 5/2 with a fairly large nuclear quadrupole moment Q ) 15.0 ( 1.5 fm2,17 a small magnetic dipole moment (Ξ ) 6.256 803%),18 and a low natural abundance of 4.10%.18 The quadrupolar nature of 67Zn, combined with its quite large quadrupole moment, result in significant second-order quadrupolar broadening of the powder line shapes in solid-state NMR (SSNMR). In solution NMR, rapid quadrupolar relaxation of 67Zn nuclei in isotropic solutions results in broad resonances, in addition to the intrinsic weak signals due to their low natural abundance, thus making 67Zn NMR in solutions practically uninformative. The low natural abundance of 67Zn translates directly into low sensitivity unless isotopic enrichment and large sample volumes are employed. These deleterious effects are drastically intensified by the small magnetic moment of 67Zn (i.e., a small gyromagnetic ratio), which makes 67Zn an insensitive nucleus in NMR. Despite all the aforementioned difficulties associated with 67 Zn NMR studies, significant developments in its application have been achieved in the past decade and have rendered 67Zn NMR, as well as other low-γ nuclei (such as 25Mg, 33S, 35/37Cl, 43 Ca, 53Cr, and 95Mo), more accessible in the solid state. These developments involve the increasing availability of high magnetic field NMR spectrometers (g18.8 T) equipped with adequate hardware and software necessary for SSNMR spectroscopy, as well as the introduction of new sensitivity enhancement techniques that can improve the signal-to-noise ratio (S/ N) of NMR spectra for low-γ half-integer quadrupolar nuclei. In addition to the intrinsic gain in sensitivity acquired by all nuclei at high magnetic fields, performing NMR experiments of a half-integer quadrupolar nucleus at the highest possible magnetic field strength is beneficial for two reasons. One is that the second-order quadrupolar broadening of the central (+1/2 T -1/2) transition is substantially reduced because it scales inversely with the applied magnetic field, B0, meaning that narrower line shapes are obtained at higher applied magnetic fields. The other is the more precise and complete detection of chemical shift anisotropy (CSA) at higher fields due to the direct scaling of the breadth (in Hz) of the chemical shift interaction with B0.19-21 The most popular signal enhancement method makes use of the quadrupolar Carr-Purcell Meiboom-Gill (QCPMG) pulse sequence22,23 as a robust technique for increasing S/N and reducing experimental times in SSNMR experiments on unreceptive nuclei. The QCPMG sequence, which consists of a train of π refocusing pulses following an initial π/2 pulse, produces a series of spin echoes in the time domain. Fourier transformation of this echo train yields an NMR spectrum composed of equally separated spikelets that mimic the NMR spectrum of a stationary sample in the solid state. When combined with other signal enhancement techniques such as cross-polarization (CP),24,25 rotor-assisted population transfer (RAPT),26,27 double-frequency sweep (DFS),27,28 hyperbolic secant pulses (HS),29-31 or wide-band uniform-rate smooth truncation (WURST) adiabatic pulses,32-36 the QCPMG experiment is capable of increasing the sensitivity by an order of magnitude or greater in comparison to classical qudrupolar echo experiments. In the case of 67Zn NMR, only the QCPMG method has been reported, either alone or in combination with CP (vide infra). Although recent advances in SSNMR techniques and instrumentation, as well as the availability of very high magnetic fields, have provided new opportunities for NMR of lowfrequency quadrupolar nuclei like 67Zn in the solid state, the
J. Phys. Chem. A, Vol. 114, No. 1, 2010 325 number of zinc systems that have been studied so far is still limited in comparison to other nuclei. Table 1 summarizes all 67 Zn SSNMR data that have been reported to date. These data include NMR studies on metallic zinc,37-39 numerous simple inorganic compounds,40-48 19 small organozinc complexes,43,49-59 and five zinc-containing macromolecules.55,60-62 The data in Table 1 have been classified according to the zinc coordination geometry in each system. Aside from zinc metal, which has a hexagonal close-packed structure, all other zinc atoms are found in sites of tetrahedral or octahedral coordination, with the former being more abundant. A quick inspection of Table 1 reveals two separate ranges of isotropic chemical shifts for the tetrahedral and octahedral coordinations, albeit with a few exceptions. Octahedral chemical shifts range from -27 to 112 ppm, whereas tetrahedral ones are less shielded and cover a wider range from 119 to 495 ppm. These two ranges are disturbed by the shift values of ZnCl2 and ZnBr2 in the octahedral region, and by those of ZnTe, [TpBut,Me]ZnBr, and Zn[S2CN(CH3)2]2 in the tetrahedral region. The first application of QCPMG on zinc compounds was reported by Ellis and co-workers,52 in which the 67Zn quadrupolar coupling parameters and the isotropic chemical shifts in 67Zn-enriched powdered Zn(imidazole)2(acetate)2 and Zn(formate)2 · 2H2O were extracted from static QCPMG NMR spectra collected at 9.4 and 11.7 T. They have since developed a general methodology to directly observe low-γ half-integer quadrupolar nuclei (such as 67Zn, 25Mg, and 43Ca) in dilute environments such as proteins via low-temperature solid-state NMR experiments.63,64 This method involves the utilization of low temperatures (as low as 10 K) to enhance the Boltzmann factor and reduce the ambient noise of the NMR probe, in combination with cross-polarization (CP) from 1H to the dilute spin (67Zn for example), followed by signal averaging through a quadrupolar echo or QCPMG pulse sequence. The combination of these techniques has allowed the application of 67Zn NMR spectroscopy to the zinc binding sites in metalloproteins, such as the minimal DNA binding domain of xeroderma pigmentosum A (XPA-MBD),55 human carbonic anhydrase isozyme (CAII),60 Zn2+-substituted Pyrococcus furiosis rubredoxin (Pf Zn-Rd),61 and both the wild type and the H265A mutant of Aquifex aeolicus LpxC.62 This method was also applied to small systems, as synthetic analogues of zinccontaining enzymes, with naturally abundant zinc (at 4.10% 67 Zn) such as the tris(pyrazolyl)hydroborato zinc complex [TpBut,Me]ZnOH along with its chloride and bromide derivatives.58 In the present study, five small zinc-coordination complexes (with imidazole, L-alanine, L-histidine, L-cysteine, and glycine) previously uncharacterized by SSNMR, are investigated using 67 Zn NMR spectroscopy and complementary single-crystal X-ray diffraction, as well as quantum chemical calculations. These complexes are: (1) bis(imidazole)zinc(II) chloride; (2) bis(Lalaninato)zinc(II); (3) bis(L-histidinato)zinc(II) dihydrate; (4) sodium bis(L-cysteinato)zincate(II) hexahydrate; and (5) bis(glycinato)zinc(II) monohydrate (Figure 1). A summary of relevant crystallographic information for the five complexes is given in Table 2. These complexes were chosen because they contain ligands that are representatives of the zinc binding sites in zinc-containing biological materials. For example, 1 is implicated in the binding sites of two polymorphs of the zinc-insulin hexamers.65-67 Histidine and cysteine are key amino acids in the binding site of the zinc finger domains, as mentioned earlier. The alaninate (2) and glycinate (5) zinc salts, on the other hand, both represent the first report of 67Zn SSNMR
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Mroue´ and Power
TABLE 1: Survey of 67Zn NMR Parameters Available in the Solid State zinc system
δisoa (ppm)
zinc coord.
[Tp]2Znd [TpMe2]2Znd
Octahedral Systems and Higher 1000 1500 1776 2000 ZnLi4Zn4 ZnF6 -20 ZnCl6 300 ZnBr6 240 ZnI6 70 ZnO6 15 ZnO6 13 10 ZnO6 25 ZnO6 -3.0 ZnO6 0 -123c -125 c 2 ZnO6 -9 -27 -24 0 ZnO6 -15 -26 ZnN6 112 ZnN6 92
ZnO (wurtzite)
ZnO4
ZnS (wurtzite)
ZnS4
ZnS (cubic)
ZnS4
ZnSe (cubic)
ZnSe4
ZnTe (cubic)
ZnTe4
Rb2ZnCl4 K2Zn(CN)4 Zn(imidazole)4(ClO4)2 Zn(thiourea)4(NO3)2
ZnCl4 ZnC4 ZnN4 ZnS4
Zn(acetate)2
ZnO4
Zn(imidazole)2(acetate)2 [Bp]2Znd [BpMe2]2 Znd Zn[S2CN(CH3)2]2 Zn[SPh]4(Me4N)2 Zn[SPhMe]4(Me4N)2
ZnN2O2 ZnN4 ZnN4 ZnN4 ZnN4 ZnN4 ZnN4 ZnN3O ZnN3Cl ZnN3Br ZnS4 ZnS4 ZnN3O ZnN3O ZnS4 ZnS4 ZnN2O2 ZnN2O2 ZnN2O2 ZnN2O2
Zinc metal
ZnZn12
LiZn ZnF2 γ-ZnCl2 ZnBr2 ZnI2 ZnSO4 ZnSO4 · 7H2O ZnSO4 · xH2O Zn(ClO4)2 · 6H2O Zn(acetate)2 · 2H2O
Zn(formate)2 · 2H2O
site 1 site 2
But,Me
]ZnOH [Tp [TpBut,Me]ZnCld But,Me ]ZnBrd [Tp [TmPh]ZnSPhd
site 1 site 2
d
But,Me
]}Zn(OH2)}[HOB(C6F5)3] {[Tp Human CAIIe XPA-MBDf Pf Zn-Rdg WT Aa LpxCh, pH 6
site 1 site 2 d
site 1 site 2
WT Aa LpxC, pH 9 H265A Aa LpxCi, pH 6.3 and 8.7 Zn(L-alaninate)2 · H2O
site 1 site 2
Tetrahedral Systems 240.1 240 238 365 364.5 360 380.5 381.9 378 378 276.3 276 273 87.6 85 350 291 291 359 325 337.1 245 67 155 119 158 70.0 333.0 339.0 340.0 166.5 311.3 57.7 400 495 327.6 429 133 170 284 Unclassified Systems 110 273
CQ (MHz)
ηQ
NMR methodb
ref
QE QE SE static SE SE SE SE SE SE static SE MAS QE SC SC QCPMG QCPMG QE SC QCPMG QE SC CP/QCPMG CP/QCPMG
37 38 39 40 41 41 41 41 41 41 41 42 43 49 50 50 51 52 53 53 52 53 53 54 54 44 42 45 46 44 42 45 44 42 45 47 44 43 47 44 47 41 48 43 43 55 56 43 57 52 54 54 56 56 56
51
12.0 12.73 11.983
0 0 0
7.87 2.50 1.95 2.70 1.75 4.50 1.7 4.65
