Modulation of the axial water hydrogen-bonding properties by chemical modification of the substrate in resting state, substrate-bound heme oxygenase from Neisseria meningitidis; coupling to the distal H-bond network via ordered water molecules

The hydrogen bonding of ligated water in ferric, high-spin, resting-state substrate complexes of heme oxygenase from Neisseria meningitidis has been systematically perturbed by variable electron-withdrawing substituents on the hemin periphery. The pattern of 1H NMR-detected dipolar shifts due to the paramagnetic anisotropy is strongly conserved among the four complexes, with the magnitude of dipolar shifts or anisotropy increasing in the order of substituent formyl < vinyl < methyl. The magnetic anisotropy is axial and oriented by the axial Fe-His23 bond, and while individual anisotropies have uncertainties of approximately 5%, the relative values of deltachi (and the zero-field splitting constant, D proportional, variant deltachi(ax)) are defined to 1%. The unique changes in the axial field strength implied by the variable zero-field splitting are in accord with expectations for the axial water serving as a stronger H-bond donor in the order of hemin substituents formyl > vinyl > methyl. These results establish the axial anisotropy (and D) as a sensitive probe of the H-bonding properties of a ligated water in resting-state, substrate complexes of heme oxygenase. Correction of observed labile proton chemical shifts for paramagnetic influences indicates that Gln49 and His53, some approximately 10 angstroms from the iron, sense the change in the ligated water H-bonding to the three nonligated ordered water molecules that link the two side chains to the iron ligand. The present results augur well for detecting and characterizing changes in distal water H-bonding upon mutagenesis of residues in the distal network of ordered water molecules and strong H-bonds.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

Solution 1H NMR characterization of the axial bonding of the two His in oxidized human cytoglobin

Solution 1H NMR spectroscopy has been used to determine the relative strengths (covalency) of the two axial His-Fe bonds in paramagnetic, S = 1/2, human met-cytoglobin. The sequence specific assignments of crucial portions of the proximal and distal helices, together with the magnitude of hyperfine shifts and paramagnetic relaxation, establish that His81 and His113, at the canonical positions E7 and F8 in the myoglobin fold, respectively, are ligated to the iron. The characterized complex (approximately 90%) in solution has protohemin oriented as in crystals, with the remaining approximately 10% exhibiting the hemin orientation rotated 180 degrees about the alpha-, gamma-meso axis. No evidence could be obtained for any five-coordinate complex (<1%) in equilibrium with the six-coordinate complexes. Extensive sequence-specific assignments on other dipolar shifted helical fragments and loops, together with available alternate crystal coordinates for the complex, allowed the robust determination of the orientation and anisotropies of the paramagnetic susceptibility tensor. The tilt of the major axis is controlled by the His-Fe-His vector, and the rhombic axes are controlled by the mean of the imidazole orientations for the two His. The anisotropy of the paramagnetic susceptibility tensor allowed the quantitative factoring of the hyperfine shifts for the two axial His to reveal an indistinguishable pattern and magnitudes of the contact shifts or pi spin densities, and hence, indistinguishable Fe-imidazole covalency for both Fe-His bonds.     

Linear correlation between 1H and 13C chemical shifts of ferriheme proteins and model ferrihemes

The (1)H{(13)C} HMQC experiment at natural-abundance (13)C provides a very useful way of determining not only (1)H but also (13)C chemical shifts of most heme substituents, without isotopic labeling of the hemin. This is true both in model low-spin ferriheme complexes and in low-spin ferriheme proteins, even when the proton resonances are buried in the protein diamagnetic region, because the carbon shifts are much larger than the proton shifts. In addition, in many cases, the protohemin methyl cross peaks are fairly linearly related to each other, with the slope of the correlation, δ(C)/δ(H), being approximately -2.0 for most low-spin ferriheme proteins. The reasons why this should be the case, and when it is not, are discussed.     

1H NMR study of the magnetic properties and electronic structure of the hydroxide complex of substrate-bound heme oxygenase from Neisseria meningitidis: influence of the axial water deprotonation on the distal H-bond network

The substrate and active site residues of the low-spin hydroxide complex of the protohemin complex of Neisseria meningitidis heme oxygenase (NmHO) have been assigned by saturation transfer between the hydroxide and previously characterized aquo complex. The available dipolar shifts allowed the quantitation of both the orientation and anisotropy of the paramagnetic susceptibility tensor. The resulting positive sign, and reduced magnitude of the axial anisotropy relative to the cyanide complex, dictate that the orbital ground state is the conventional "d(pi)" (d(2)(xy)(d(xz), d(yz))(3)); and not the unusual "d(xy)" (d(2)(xz)d(2)(yz)d(xy)) orbital ground state reported for the hydroxide complex of the homologous heme oxygenase (HO) from Pseudomonas aeruginosa (Caignan, G.; Deshmukh, R.; Zeng, Y.; Wilks, A.; Bunce, R. A.; Rivera, M. J. Am. Chem. Soc. 2003, 125, 11842-11852) and proposed as a signature of the HO distal cavity. The conservation of slow labile proton exchange with solvent from pH 7.0 to 10.8 confirms the extraordinary dynamic stability of NmHO complexes. Comparison of the diamagnetic contribution to the labile proton chemical shifts in the aquo and hydroxide complexes reveals strongly conserved bond strengths in the distal H-bond network, with the exception of the distal His53 N(epsilon)(1)H. The iron-ligated water is linked to His53 primarily by a pair of nonligated, ordered water molecules that transmit the conversion of the ligated H-bond donor (H(2)O) to a H-bond acceptor (OH(-)), thereby increasing the H-bond donor strength of the His53 side chain.     

The magnetic properties of myoglobin as studied by NMR spectroscopy

Deoxymyoglobin has been investigated by NMR spectroscopy to determine the magnetic anisotropy through pseudocontact shifts and the total magnetic susceptibility through Evans measurements. The magnetic anisotropy values were found to be Deltachi(ax)=-2.03+/-0.08 x 10(-32) m(3) and Deltachi(rh)=-1.02+/-0.09 x 10(-32) m(3). The negative value of the axial susceptibility anisotropy originates from the z tensor axis lying in the heme plane, unlike all other heme systems investigated so far. This magnetic axis is almost exactly orthogonal to the axial histidine plane. The other two axes lie essentially in the histidine plane, the closest to the heme normal being tilted by about 36 degrees from it, towards pyrrole A on the side of the proximal histidine. From the comparison with cytochrome c' it clearly appears that the position of the one axis lying in the heme plane is related to the axial histidine orientation. Irrespective of the directions, the magnetic anisotropy is smaller than that of the analogous reduced cytochrome c' and of the order of that of low-spin iron(III). The magnetic anisotropy of the system permits the measurement of residual dipolar couplings, which, together with pseudocontact shifts, prove that the solution structure is very similar to that in the crystalline state. Magnetic measurements, at variance with previous data, demonstrate that there is an orbital contribution to the magnetic moment, micro(eff)=5.5 micro(B). Finally, from the magnetic anisotropy data, the hyperfine shifts of iron ligands could be separated in pseudocontact and contact components, and hints are provided to understand the spin-delocalisation mechanism in S=2 systems by keeping in mind the delocalisation patterns in low-spin S=1/2 and high-spin S= 5/2 iron(III) systems.     

Solution 1H NMR characterization of axial interactions of the proximal and distal His in the cyanomet complexes of the isolated chains and the 65 kDa intact tetramer of human hemoglobin A.

Solution 1H NMR has been used to investigate the axial bonding of the proximal His and the hydrogen-bonding of the distal His to the bound ligand in the isolated chains as well as the subunits of intact, tetrameric, cyanomet human hemoglobin A. The complete proximal His, including all ring protons necessary to monitor bonding in each subunit, could be definitively assigned by 1D/2D methods despite the large size (approximately 65 kDa) and severe relaxation (to T(1) approximately 3 ms, line width approximately 1.5 kHz) of two of the protons. The complete distal His E7 ring was assigned in the alpha-chain and alpha-subunit of HbA, and the dipolar shifts and relaxation were analyzed to reveal a disposition intermediate between the positions adopted in HbCO and HbO2 that is optimal for forming a hydrogen bond with bound cyanide. The lability of the alpha-subunit His E7 NepsilonH is found to be similar to that in sperm whale cyanomet myoglobin. The orientation of the distal His E7 in the beta-subunit is found to be consistent with that seen in either HbCO or HbO2. While the His E7 labile NepsilonH proton signal could not be detected in either the beta-chain or subunit, it is concluded that this more likely reflects increased lability over that of the alpha-subunit, and not the absence of a hydrogen bond to the bound ligand. Analysis of the heme mean methyl hyperfine shift, which has been shown to be very sensitive to the presence of distal hydrogen bonds to bound cyanide (Nguyen, B. D.; Xia, Z.; Cutruzzolá, F.; Travaglini Allocatelli, C.; Brancaccio, A.; Brunori, M.; La Mar, G. N. J. Biol. Chem. 2000, 275, 742-751), directly supports the presence of a distal His E7 hydrogen bond to cyanide in the beta-chain and beta-subunit which is weaker than the same hydrogen bond in the alpha-subunit. The potential for the proximal His hyperfine shifts in serving as indicators of axial strain in the allosteric transition of HbA is discussed.     

Solution 1H NMR of the molecular and electronic structure of the heme cavity and substrate binding pocket of high-spin ferric horseradish peroxidase: effect of His42Ala mutation.

Solution 1H NMR has been used to assign a major portion of the heme environment and the substrate-binding pocket of resting state horseradish peroxidase, HRP, despite the high-spin iron(III) paramagnetism, and a quantitative interpretive basis of the hyperfine shifts is established. The effective assignment protocol included 2D NMR over a wide range of temperatures to locate residues shifted by paramagnetism, relaxation analysis, and use of dipolar shifts predicted from the crystal structure by an axial paramagnetic susceptibility tensor normal to the heme. The most effective use of the dipolar shifts, however, is in the form of their temperature gradients, rather than by their direct estimation as the difference of observed and diamagnetic shifts. The extensive assignments allowed the quantitative determination of the axial magnetic anisotropy, Deltachi(ax) = -2.50 x 10(-8) m(3)/mol, oriented essentially normal to the heme. The value of Deltachi(ax) together with the confirmed T(-2) dependence allow an estimate of the zero-field splitting constant D = 15.3 cm(-1), which is consistent with pentacoordination of HRP. The solution structure was generally indistinguishable from that in the crystal (Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nature Structural Biology 1997, 4, 1032-1038) except for Phe68 of the substrate-binding pocket, which was found turned into the pocket as found in the crystal only upon substrate binding (Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054-8060). The reorientation of several rings in the aromatic cluster adjacent to the proximal His170 is found to be slow on the NMR time scale, confirming a dense, closely packed, and dynamically stable proximal side up to 55 degrees C. Similar assignments on the H42A-HRP mutant reveal conserved orientations for the majority of residues, and only a very small decrease in Deltachi(ax) or D, which dictates that five-coordination is retained in the mutant. The two residues adjacent to residue 42, Ile53 and Leu138, reorient slightly in the mutant H42A protein. It is concluded that effective and very informative 1H NMR studies of the effect of either substrate binding or mutation can be carried out on resting state heme peroxidases.     

SOLID-STATE HIGH RESOLUTION NMR STUDY ON POLY (2, 6-DIMETHYL-1,4-PHENYLENE OXIDE)

Experiments including ~(13)C spin-lattice relaxation, ~(13)C heteronuclear dipolar dephasing and~1H spin diffusion are performed on poly (2, 6-dimethyl-1, 4-phenylene oxide) (PPO). Theresults show that the rotation of the methyl groups in solid PPO is partially restricted, whichresults in a surprisingly efficient spin diffusion between the aromatic proton and methyl protoncharacterized by a diffusion time of 150μs. The results also show that the aromatic ring insolid PPO is rigid and twisted, which causes all aromatic carbons to be chemicallyunequivalent.     

A justification for using NMR model-free methods when investigating the solution structures of rhombic paramagnetic lanthanide complexes

The detailed analysis of the 1H NMR hyperfine shifts according to the model-free methods shows that the semi-rigid monometallic complexes [Ln(L)(NO3)3] (Ln = Eu-Yb) are isostructural in solution. The associated separation of contact and pseudo-contact contributions to the hyperfine NMR shifts in each rhombic lanthanide complex at room temperature provides paramagnetic susceptibility tensors whose principal magnetic axes match the expected symmetry requirements. Moreover, both axial (Delta chi(ax)) and rhombic (Delta chi(rh)) paramagnetic anisotropies display satisfactory linear dependence on Bleaney's factors, a correlation predicted by the approximate high-temperature expansion of the magnetic susceptibility limited to T(-2). Consequently, the simple, and chemically attracting NMR model-free methods are not limited to axial systems, and can be safely used for the investigation of the solution structures of any lanthanide complexes. Molecular-based structural criteria for the reliable estimation of paramagnetic susceptibility tensors by NMR are discussed, together with the assignment of the labels of the crystal-field and magnetic axes within Bleaney's approach.     

Study of electron spin crossover in iron(III) tris-dithiocarbamate complexes by carbon-13 NMR spectroscopy

¹³C and ¹H isotropic shifts have been measured for a series of Fe(III) tris-dithiocarbamate complexes. The ¹³C isotropic shifts may be interpreted as arising solely from contact hyperfine coupling and demonstrate that as the low-spin state of the metal is favoured there is an increase in metal-ligand π-bonding. σ-delocalization of unpaired spin density is more important in determining the ¹³C isotropic shifts than those of the contiguous proton.     

Heme axial methionine fluxion in Pseudomonas aeruginosa Asn64Gln cytochrome c551

Heme axial methionine ligands in ferricytochromes c552 from Hydrogenobacter thermophilus (HT) and Nitrosomonas europaea, both members of the cyt c8 family, display fluxional behavior. The ligand motion, proposed to be inversion at sulfur, results in an unusually small range of hyperfine shifts for heme substituents in these proteins. Herein, heme axial Met fluxion is induced in a structurally homologous cytochrome c551 from Pseudomonas aeruginosa (PA) by substituting heme pocket residue Asn64 with Gln. The mutant, PA-N64Q, displays a highly compressed range of heme substituent hyperfine shifts, temperature-dependent heme methyl resonance line broadening, low rhombic magnetic anisotropy, and a magnetic axes orientation consistent with Met orientational averaging. Analysis of NMR properties of PA-N64Q demonstrates that the heme pocket of the mutant resembles that of HT. This result confirms the importance of peripheral interactions and, in particular, residue 64 in determining axial Met orientation and heme electronic structure in proteins in the cyt c8 family.     

C-13 magnetic relaxation rates and H-1 and C-13 paramagnetic shifts of Ni(II) complex of dopamine

The ¹H and ¹³C isotropic contact shifts and the ¹³C relaxation times of dopamine in aqueous solution have been measured in the presence of the Ni(II) ion. The pD dependence of the ¹H and ¹³C paramagnetic shifts was also investigated. From the analysis of the shifts at pD = 6.5 and from the INDO MO calculations on selected models of dopamine radicals, a dominant σ delocalization mechanism of the spin density is proposed. From the spin distribution on the ligand carbon atoms, the metal centered as well as the ligand centered dipolar contributions of the modified Solomon—Bloembergen equation were calculated and an estimate of the correlation time τ_c was given.     

Fulvene,VII. Analyse der 13C- und 1H-NMR.-Spektren

Carbon-13 and proton NMR. spectra of pentafulvene and of a series of 6-substituted fulvenes have been analysed and assigned by homo- and heteronuclear double resonance and with the aid of iterative computation. ¹³C and ¹H chemical shifts are interpreted in terms of substituent effects and compared with π-electron charges calculated for the unsubstituted fulvene. From ¹³C shifts a 10 percent contribution of dipolar structures to the electronic configuration of fulvene may be estimated. All long-range proton-proton coupling constants including relative signs and some proton-carbon couplings in the fulvene spin system have been determined and assigned.     

Determination of natural abundance 15N-1H and 13C-1H dipolar couplings of molecules in a strongly orienting media using two-dimensional inverse experiments

NMR spectra of molecules oriented in liquid crystals provide homo- and heteronuclear dipolar couplings and thereby the geometry of the molecules. Several inequivalent dilute spins such as 13C and 15N coupled to protons form different coupled spin systems in their natural abundance and appear as satellites in the proton spectra. Identification of transitions belonging to each spin system is essential to determine heteronuclear dipolar couplings, which is a formidable task. In the present study, using 15N-1H and 13C-1H HSQC, and HMQC experiments we have selectively detected spectra of each rare spin coupled to protons. The 15N-1H and 13C-1H dipolar couplings have been determined in the natural abundance of 13C and 15N for the molecules pyrazine, pyrimidine and pyridazine oriented in a thermotropic liquid crystal.     

Active site environment of heme-bound amyloid β peptide associated with Alzheimer's disease

Recent reports show that there is a large increase in heme in the temporal brain of Alzheimer's disease (AD) patients, as heme, biosynthesized in brain cells, binds to amyloid β (Aβ), forming heme-Aβ complexes. This leads to the development of symptoms that are characteristic pathological features of AD, e.g., abnormal iron homeostasis, decay of iron regulatory proteins, dysfunction in mitochondrial complex IV, oxidative stress, etc. However, the active site resulting from heme binding to Aβ is not well characterized. For example, the coordinating residue, relevant second-sphere residues, and spin state of the Fe center are not known. In this study we have used wild-type and mutated Aβ peptides and investigated their interaction with naturally occurring heme. Our results show that, out of several possible binding sites, His(13) and His(14) residues can both bind heme under physiological conditions, resulting in an axial high-spin active site with a trans axial water-derived ligand. Peroxidase assays of these heme-peptide complexes along with pH perturbations indicate that Arg(5) is a key second-sphere residue that H-bonds to the trans axial ligand and is responsible for the peroxidase activity of the heme-Aβ complexes. The His(13) and Arg(5) residues identified in this study are both absent in rodents, which do not show AD, implicating the significance of these residues as well as heme in the pathology of AD.     

Magnetic susceptibility tensor and heme contact shifts determinations in the Rhodobacter capsulatus ferricytochrome c': NMR and magnetic susceptibility studies.

The 1H and 15N resonances of the carbon monoxide complex of ferrocytochrome c' of Rhodobacter capsulatus, a ferrous diamagnetic heme protein, have been extensively assigned by TOCSY-HSQC, NOESY-HSQC, and HSQC-NOESY-HSQC 3D heteronuclear experiments performed on a 7 mM sample labeled with 15N. Based on short-range and medium-range NOEs and H(N) exchange rates, the secondary structure consists of four helices: helix 1 (3-29), helix 2 (33-48), helix 3 (78-101), and helix 4 (103-125). The 15N, 1HN, and 1H(alpha) chemical shifts of the CO complex form are compared to those of the previously assigned oxidized (or ferric) state. From the chemical shift differences between these redox states, the orientation and the anisotropy of the paramagnetic susceptibility tensor have been determined using the crystallographic coordinates of the ferric state. The chi-tensor is axial, and the orientation of the z-axis is approximately perpendicular to the heme plane. The paramagnetic chemical shifts of the protons of the heme ligand have been determined and decomposed into the Fermi shift and dipolar shift contributions. Magnetic susceptibility studies in frozen solutions have been performed. Fits of the susceptibility data using the model of Maltempo (Maltempo, M. M. J. Chem. Phys. 1974, 61, 2540-2547) are consistent with a rather low contribution of the S = 3/2 spin state over the range of temperatures and confirm the value of the axial anisotropy. Values in the range 10.4-12.5 cm(-1) have been inferred for the axial zero-field splitting parameter (D). Analysis of the contact shift and the susceptibility data suggests that cytochrome c' of Rb. capsulatus exhibits a predominant high-spin character of the iron in the oxidized state at room temperature.     

A new approach in 1D and 2D 13C high-resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning

Novel 1D and multidimensional solid-state NMR (SSNMR) methods using very fast magic-angle spinning (VFMAS) (spinning speed > 20 kHz) for performing 13C high-resolution SSNMR of paramagnetic organometallic complexes are discussed. VFMAS removes a majority of 13C-1H and 1H-1H dipolar couplings, which are often difficult to remove by RF pulse techniques in paramagnetic complexes because of large paramagnetic shifts. In the first systematic approach using the unique feature of VFMAS for paramagnetic complexes, we demonstrate a means of obtaining well-resolved 1D and multidimensional 13C SSNMR spectra, sensitivity enhancements via cross polarization, and signal assignments, and applications of dipolar recoupling methods for nonlabeled paramagnetic organometallic complexes of moderate paramagnetic shifts ( approximately 800 ppm). Experimental results for powder samples of small nonlabeled coordination complexes at 1H frequencies of 400.2-400.3 MHz show that highly resolved 13C SSNMR spectra can be obtained under VFMAS, without requirements of 1H decoupling. Sensitivity enhancement in 13C SSNMR via cross polarization from 1H spins was demonstrated with an amplitude-sweep high-power CP sequence using strong RF fields ( approximately 100 kHz) available in the VFMAS probe. 13C CPMAS spectra of nonlabeled Cu(II)(dl-alanine)2.(H2O) and V(III)(acetylacetonate)3 (V(acac)3) show that it is possible to obtain high-resolution spectra for a small quantity ( approximately 15 mg) of nonlabeled paramagnetic organometal complexes within a few minutes under VFMAS. Experiments on Cu(II)(dl-alanine)2.(H2O) demonstrated that 1H-13C dipolar recoupling for paramagnetic organometal complexes can be performed under VFMAS by application of rotor-synchronous pi-pulses to 1H and 13C spins. The results also showed that signal assignments for 13CH, 13CH3, and 13CO groups in paramagnetic complexes are possible on the basis of the amount of 13C-1H dipolar dephasing induced by dipolar recoupling. Furthermore, the experimental 2D 13C/1H chemical-shift correlation NMR spectrum obtained for nonlabeled V(acac)3 exhibits well-resolved lines, which overlap in 1D 13C and 1H spectra. Signals for different chemical groups in the 2D spectrum are distinguished by the 13C-1H dipolar dephasing method combined with the 2D 13C/1H correlation NMR. The assignments offer information on the existence of nonequivalent ligands in the coordination complex in solids, without requiring a single-crystal sample.     

Proton NMR Studies of the Electronic Effect of Substituents at the Azomethine Carbon of N,N′-Bisethylene (Salicylideniminato) Cobalt (II) Complex

The proton paramagnetic shifts of low spin N,N′-ethylenebis(salicylideniminato)cobalt(II) and its derivatives were studied to understand the effect of substituents at the azomethine carbon on the electronic structure of the Schiff-base cobalt complexes. Analysis of the ¹H paramagnetic shifts of H and CH₃ bonded to the azomethine carbon reveals that spin delocalization through a interaction is responsible for the contact contribution to the paramagnetic shift. When the phenyl group is bonded to the azomethine carbon, the plane of the phenyl group is perpendicular to the plane of the complex and the phenyl group makes a negligible contact contribution to the paramagnetic shift.