Hyperfine shifts

NMR spectra have been reported for the Rieske-type ferredoxins from Xanthobacter strain Py2 (88) and of toluene 4-monooxygenase from Pseudomonas mendocina (T4MOC) (88a) as well as for the water-soluble Rieske fragment from the bci complex of Paracoccus deni-trificans (ISFpd) (89). The spectra of these proteins are similar, which is consistent with the close structural relationship between the three proteins. In the reduced (paramagnetic) state, all three proteins show several hyperfine-shifted resonances between +83 and -16 ppm at 400 MHz or between 110 and +25 ppm at 300 MHz (Table X). 

With these assignments at hand the analysis of the hyperfine shifts became possible. An Fe(III) in tetrahedral structures of iron-sulfur proteins has a high-spin electronic structure, with negligible magnetic anisotropy. The hyperfine shifts of the protons influenced by the Fe(III) are essentially Fermi contact in origin 21, 22). An Fe(II), on the other hand, has four unpaired electrons and there may be some magnetic anisotropy, giving rise to pseudo-contact shifts. In addition, there is a quintet state at a few hundred cm which may complicate the analysis of hyperfine shifts, but the main contribution to hyperfine shifts is still from the contact shifts 21, 22). 

Contact shifts give information on the electronic structure of the iron atoms, particularly on the valence distribution and on the magnetic coupling within polymetallic systems. The magnetic coupling scheme, which is considered later, fully accounts for the variety of observed hyperfine shifts and the temperature dependence. Thus, through the analysis of the hyperfine shifts, NMR provides detailed information on the metal site(s) of iron-sulfur proteins, and, thanks to the progress in NMR spectroscopy, also the solution structure 23, 24 ). 

By assuming that the hyperfine shifts are contact shifts in origin, it is possible to evaluate the hyperfine coupling constant from the following equation (50) ... 

Fig. 4. Top Theoretical temperature dependence of the hyperfine shift of the H/3 protons of reduced spinach [Fe2S2] ferredoxin 151). The solid line corresponds to the situation where only one species exists in solution, whereas the dashed line corresponds to a situation where there is fast equilibrium between two species (in a 20/80 ratio) differing for the location of the extra electron 151). Bottom.-. Experimental temperature dependence of the H NMR shifts. The signals are labeled as in Fig. 2B.

In addition to the standard constraints introduced previously, structural constraints obtainable from the effects of the paramagnetic center(s) on the NMR properties of the nuclei of the protein can be used (24, 103). In iron-sulfur proteins, both nuclear relaxation rates and hyperfine shifts can be employed for this purpose. The paramagnetic enhancement of nuclear relaxation rates [Eqs. (1) and (2)] depends on the sixth power of the nucleus-metal distance (note that this is analogous to the case of NOEs, where there is a dependence on the sixth power of the nucleus-nucleus distance). It is thus possible to estimate such distances from nuclear relaxation rate measurements, which can be converted into upper (and lower) distance limits. When there is more than one metal ion, the individual contributions of all metal ions must be summed up (101, 104-108). If all the metal ions are equivalent (as in reduced HiPIPs), the global paramagnetic contribution to the 7th nuclear relaxation rate is given by... 

As mentioned in the Introduction, in iron—sulfur proteins, the hyperfine shifts of the nuclei of the coordinating cysteines are essentially contact in origin (21, 22). In the case of [Fe4S4l (17) and [FegS4] (112) cluster, it has been shown that the hyperfine shift of the cysteinyl H/3 and Ca nuclei can be related to the value of the Fe-Sy-C/S-H/S/Ca dihedral angle (6) through a Karplus-type relationship of the form... 

H NMR spectroscopy studies of iron(IIl) a-alkyl and o-aryl porphyrins have been very important in elucidating spin states. Alkyl and most aryl complexes with simple porphyrin ligands (OEP, TPP, or TTP) are low spin, S — I /2 species. NMR spectra for the tetraarylporphyrin derivatives show upheld resonances for the porphyrin pyrrole protons (ca. — 18 to —35 ppm), and alternating upfield and downfield hyperfine shifts for the axial alkyl or aryl resonances. For -alkyl complexes, the a-protons show dramatic downfield shifts (to ca. 600 ppm), upfield shifts for the /3-protons (—25 to — 160 ppm) and downfield shifts for the y-protons (12 ppm). The cr-protons of alkyliron porphyrins are not usually detected as a result of their large downfield shift and broad resonance. These protons were first detected by deuterium NMR in the dcuterated complexes Fe(TPP)CD3 (532 ppm) and Fe(TPP)CD2CDi (562, -117 ppm). ... 

FIGURE 5.7 Second-order hyperfine shift in the X-band EPR of the Cu(II)-Tris complex. The thin solid line is the experimental spectrum of 1.5 mM CuS04 in 200 mM Tris-HCl buffer, pH 8.0 taken at v = 9420 MHz and T = 61 K. Tris is tris-(hydroxymethyl)aminomethane or 2-amino-2-hydroxymethyl-l,3-propanediol. The broken lines are simulations using the parameters g = 2.047, gN = 2.228, Atl = 185 gauss. In the lower trace the second-order correction has been omitted. 

The solution structure of Aspl3Cys, a thermostable mutant of Fd, has been solved by H-NMR and compared to that of the wild-type (WT) protein. The overall folding of the WT protein was maintained in the mutant, except for the immediate vicinity of the new cysteine. The geometry of the new cluster was a typical 4Fe-4S cubane, as monitored by the hyperfine shifts of the co-ordinated cysteines. Conformational heterogeneity, which was partly abolished by heat treatment, was observed and ascribed to a kinetic phenomenon. 

Figure 5. Li MAS NMR spectrum of the Mn(IV) spinel (Lio.5Zno.5)tet(Lio.5Mni 5)oct04 and the typical hyperfine shifts observed for lithium in a series of local environments. Hyperfine shifts are given next to the two isotropic resonances in the Li spectrum all other peaks are spinning sidebands, which are predominantly caused by the electron—nuclear dipolar coupling.

Figure 6. Li MAS NMR spectrum of the layered compound Li2MnOs acquired at a MAS frequency, Vr, of 35 kHz. Spinning sidebands are marked with asterisks. The local environment in the Mn +/Li+ layers that gives rise to the isotropic resonance at 1500 ppm is shown. Spin density may be transferred to the 2s orbital of Li via the interaction with (b) a half-filled t2g orbital and (c) an empty d/ Mn orbital to produce the hyperfine shifts seen in the spectrum of Li2MnOs. The large arrows represent the magnetic moments of the electrons in the t2g and p orbitals, while the smaller arrows indicate the sign of the spin density that is transferred to the Li 2s and transition-metal d orbitals.

Table 1. Comparison of the Hyperfine Shifts (in ppm) Observed for Lithium Coordinated to a Single Mn + Ion via an Intervening Oxygen Ion for a Series of Local Environments ...

The effective distances obtained by Nordenskiold et al. (40) are compared with the internuclear distances in Table I. Clearly, the point dipole approximation is reasonable for the hydrogen nuclei in these complexes, while substantial deviations are observed for the oxygen nuclei. The findings of these early quantum chemical studies were confirmed by Sahoo and Das (41-43). Wilkens et al. have reported DFT calculations using Eq. (16) for a 104 atom model for high-spin Fe(III) rubredoxin (44). Large discrepancies between the effective distances and the input distances for the calculations were found for the hyperfine-shifted nitrogen-15 resonances, as well as for proton and carbon-13 nuclei in cysteines bound to the iron center. 

The observed hyperfine shifts could come from contact coupling or pseudocontact interactions between the electrons and the protons. Contact shifts arise when a finite amount of unpaired electron density is transferred to the observed protons. The contact shifts of the proton resonances for isotropic systems are given by Bloembergen s (9) expression... 

The NMR spectra of paramagnetic hemoproteins contain resolved hyperfine-shifted lines in addition to the ring-current shifted resonances (Kowalsky (62) Kurland et al. (64) Wiithrich et al. (112)). Between DSS and ca. +5 ppm the spectra can then contain resolved ring current-shifted... 

The hyperfine shifts in the proton NMR spectra of paramagnetic hemes and hemoproteins are closely related to the electronic structures of these molecules. At present the most extensive NMR studies of the electronic spin distribution in the heme groups have been done with low spin ferric compounds, which will be discussed in this section. Procedures similar to those described here would apply to the analysis of the NMR spectra of hemoproteins in the other paramagnetic states (Table 1). 

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