Paramagnetic Susceptibility Tensor Research Articles

Influence of the Distal His in Imparting Imidazolate Character to the Proximal His in Heme Peroxidase: 1H 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.

Intramolecular Dynamics of Lanthanide(III) Tetraoxadiaza Macrocycle Complexes in Solution as Studied by NMR

1H and 13C NMR and 1H NMR relaxation spectroscopy (RS)measurements are reported for the CDCl3 and CD2Cl2 solutions of [La(NO3)3(diaza-18-crown-6)] ({bf I}), [Pr(NO3)3(diaza-18-crown-6)] ({bf II}) and [Nd(NO3)3(diaza-18-crown-6)] ({bf III}) complexes. Temperature dependencies of the 1H NMR spectra of II have been analyzed using the dynamic NMR methods for multi-site exchange. Enantiomeric isomer interconversion in II is characterized by Δ H‡ = 21.5 ± 4 kJ mol-1. Studies of the values of the lanthanide-induced shifts and the longitudinal relaxation rate enhancement revealed that the structure of complexes in solution is similar to that reported for the [La(NO3)3(18-crown-6)] complex in the crystal state. Nevertheless, it appears that the principal values of the molar paramagnetic susceptibility tensor (χi) significantly differ in complexes II and III. The possible reasons for the different characteristics of these complexes are 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.

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.

Intramolecular Dynamics of Asymmetric Lanthanide(III) Porphyrin Sandwich Complexes in Solution

Temperature dependencies of the 1H NMR spectra of asymmetric double-decker complexes of DyH(oep)(tpp) (I) and LuH(oep)(tpp) (II), where tpp is tetraphenylporphyrin and oep is octaethylporphyrin, have been analyzed. The kinetic and thermodynamic parameters for intramolecular phenyl ring rotations have been determined. The rotation in (I) is characterized by k(298 K) = 11.4 s−1, ΔH‡ = 48.0±6.0 kJ mol−1, while in (II) the derived parameters are found to be k(298 K) = 14.0 s−1, ΔH‡ = 65.5±7.1 kJ mol−1. The possible reasons for the different characteristics of these complexes are discussed. The structural parameters calculated for I and YbH(oep)(tpp) (III), indicate close similarities between the complexes. Nevertheless, it appears that the principal values of the molar paramagnetic susceptibility tensor (χi) differ significantly in these complexes.

The use of chemical shift temperature gradients to establish the paramagnetic susceptibility tensor orientation: implication for structure determination/refinement in paramagnetic metalloproteins.

The use of dipolar shifts as important constraints in refining molecular structure of paramagnetic metalloproteins by solution NMR is now well established. A crucial initial step in this procedure is the determination of the orientation. of the anisotropic paramagnetic susceptibility tensor in the molecular frame which is generated interactively with the structure refinement. The use of dipolar shifts as constraints demands knowledge of the diamagnetic shift. which, however, is very often not directly and easily accessible. We demonstrate that temperature gradients of dipolar shifts can serve as alternative constraints for determining the orientation of the magnetic axes, thereby eliminating the need to estimate the diamagnetic shifts. This approach is tested on low-spin, ferric sperm whale cyanometmyoglobin by determining the orientation, anisotropies and anisotropy temperature gradients by the alternate routes of using dipolar shifts and dipolar shift gradients as constraints. The alternate routes ultimately lead to very similar orientation of the magnetic axes, magnetic anisotropies and magnetic anisotropy temperature gradients which, by inference, would lead to an equally valid description of the molecular structure. It is expected that the use of the dipolar shift temperature gradients, rather than the dipolar shifts directly, as constraints will provide an accurate shortcut in a solution structure determination of a paramagnetic metalloprotein.

Unusual Contact Shifts and Magnetic Tensor Orientation in Rhodobacter capsulatus Ferrocytochrome c‘: NMR, Magnetic Susceptibility, and EPR Studies

In contrast to high-spin ferrous paramagnetic heme proteins, the chemical shifts of the heme protons are very unusual in the ferrocytochromes c‘. Magnetic susceptibility studies of Rhodobacter capsulatus ferrocytochrome c‘ in frozen solutions have been performed and indicate an S = 2 spin state and a large negative axial (D) zero-field splitting parameter (−18.3 cm-1) as well as a significant rhombic (E) value (−4.9 cm-1). The 1H and 15N resonances have been extensively assigned by TOCSY−HSQC, NOESY−HSQC, and HSQC−NOESY−HSQC 3-D heteronuclear experiments performed on a 8 mM sample labeled with 15N. Based on short-range and medium-range NOEs and HN exchange rates, the secondary structure consists of four helices: helix-1 (3−30), helix-2 (34−49), helix-3 (78−97), and helix-4 (103−117). The 15N, HN, and Hα chemical shifts of the reduced (or ferro) state are compared to those previously assigned for the diamagnetic carbon monoxide complex form. 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. Values of −23 and −3 cm-1 have been inferred for D and E, and the z-axis of the tensor is tilted approximately 30° from the normal to the heme. The paramagnetic chemical shifts of the heme protons have been determined and split up into Fermi shift and the dipolar shift contributions. The pattern of the contact shifts is very unusual, exhibiting a 2-fold symmetry, and is discussed in terms of molecular orbital interactions between the porphyrin macrocycle and the imidazole ring.

Solution NMR Determination of the Anisotropy and Orientation of the Paramagnetic Susceptibility Tensor as a Function of Temperature for Metmyoglobin Cyanide: Implications for the Population of Excited Electronic States

Comprehensive 1H NMR assignments of the heme cavity proton resonances of sperm whale metmyoglobin cyanide have provided the dipolar shifts for nonligated residues which, together with the crystal coordinates of carbonyl myoglobin, allow accurate determination of both the anisotropies and orientation of the paramagnetic susceptibility tensor, χ, in the molecular framework. The resulting axial, Δχax = 2.48 × 10-8 m3/mol, and rhombic anisotropy, Δχrh = −0.58 × 10-8 m3/mol, values at 25 °C determined from the most complete set of dipolar shifts are determined to 2% and 6% uncertainty, respectively, and agree well with theoretical estimates (Horrocks, W. D., Jr. and Greenberg, E. S. Mol. Phys. 1974, 27, 993−999). Numerically and spatially restricted input data sets lead to larger uncertainties in Δχax and Δχrh, but do not systematically bias the orientation of the tensor. Determination of the anisotropies and orientation over the temperature range 5−50 °C shows that the susceptibility tensor orientation is minimally influenced, with both anisotropies well-behaved, and with Δχax exhibiting a temperature behavior close to that predicted for the system. The quantitative determination of the magnetic anisotropies over temperature allows the quantitative separation of contact and dipolar shifts for the iron ligands. The heme contact shifts reflect the expected dominant π spin density at pyrrole positions, but the meso-protons exhibit low-field contact shifts indicative of unpaired spin in a σ orbital. Such delocalized σ spin density could arise from either deformation of the heme from planarity or the loss of σ/π separation for the dxz, dyz orbitals when the major magnetic axis is tilted strongly from the heme normal as is experimentally observed. The observed anomalous temperature dependencies of the heme methyl and axial His ring contact shifts, as well as that of the rhombic anisotropy, are all consistent with thermal population of the excited orbital state. The limitations for quanitatively determining the excited orbital state energy separation from the available NMR data are discussed.

Solution 1H NMR Study of the Electronic Structure and Magnetic Properties of High-Spin Ferrous or Deoxy Myoglobins

Solution 1D and 2D NMR, together with limited isotope labeling, have led to the assignment of the heme, axial His, and numerous heme contact residues in sperm whale, horse, and human deoxy myoglobin. The paramagnetic relaxivity leads to increased line widths and shorter T1s with little compensation in increased dispersion due to dipolar shifts. Hence only limited, but crucial F helix standard backbone sequence specific assignment could be made for heme cavity residues. Numerous other residues with significant dipolar shifts could be assigned from the characteristic scalar connectivities and dipolar contacts to the heme predicted by the crystal structure. It is concluded that the complete and unambiguous assignment of the heme pyrrole substituent signals is not attainable by 2D NMR alone without either partial deuterium labeling of the heme or parallel assignment of key residues in dipolar contact with the heme; hence the present study revises some earlier assignments. The resulting dipolar shifts for nonligated residues, together with the crystal coordinates of deoxy myoglobin, were used to determine the orientation relative to the heme and the anisotropy of the paramagnetic susceptibility tensor. The significant anisotropy, |Δχ| ∼1 × 10-9 m3/mol, however, is shown to result in dipolar shift with reciprocal square, rather than just reciprocal, absolute temperature dependence, which is indicative of large zero field splitting rather than g-tensor anisotropy. The appropriate equation for a 5B2 ground state allows an estimate of the zero-field splitting, D ∼−10 cm-1, which is in good agreement with earlier results. The present NMR data favor a spin-only magnetic moment with S = 2 and D ∼−10 cm-1 over a ground state with S < 2 and significant orbital contribution (Hendrich and Debrunner, 1989).

A myoglobin mutant designed to mimic the oxygen-avid Ascaris suum hemoglobin: elucidation of the distal hydrogen bonding network by solution NMR

The solution 1H NMR structure of the active site and ligand dissociation rate for the cyanomet complex have been determined for a sperm whale myoglobin triple mutant Leu29(B10)-->Tyr, His64(E7)-->Gln, Thr67(E10)-->Arg that mimics the distal residue configuration of the oxygen-avid hemoglobin from Ascaris suum. A double mutant that retains Leu29(B10) was similarly investigated. Two-dimensional NMR analysis of the iron-induced dipolar shifts, together with the conserved proximal side structure for the two mutants, allowed the determination of the orientations of the paramagnetic susceptibility tensor for each complex. The resulting magnetic axes, together with paramagnetic relaxation and steady-state NOEs, led to a quantitative description of the distal residue orientations. The distal Tyr29(B10) in the triple mutant provides a strong hydrogen bond to the bound cyanide comparable to that provided by His64(E7) in wild-type myoglobin. The distal Gln64(E7) in the triple mutant is sufficiently close to the bound cyanide to severe as a hydrogen bond donor, but the angle is not consistent with a strong hydrogen bond. Dipolar contacts between the Arg67(E10) guanidinium group and the Gln64(E7) side chain in both mutants support a hydrogen-bond to the Gln64(E7) carbonyl group. The much lower oxygen affinity of this triple mutant relative to that of Ascaris hemoglobin is concluded to arise from side-chain orientations that do not allow hydrogen bonds between the Gln64(E7) side-chain NHs and both the ligand and Tyr29(B10) hydroxyl oxygen. Cyanide dissociation rates for the reduced cyanide complexes are virtually unaffected by the mutations and are consistent with a model of the rate-determining step as the intrinsically slow Fe-C bond breaking that is largely independent of any hydrogen bonds to the cyanide nitrogen.

Characterization of a site-directed mutant of cytochrome b5 designed to alter axial imidazole ligand plane orientation.

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.

Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-ferricytochrome c free in solution and bound to cytochrome c peroxidase.

Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase. Complete heme proton resonance assignments were made for free iso-1-ferricytochrome c, while for CcP-complexed iso-1-ferricytochrome c, 70% of heme proton assignments were made. Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme. These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes. Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c. Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts. Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-ferricytochrome c were elucidated. The results indicated that the iso-1-ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation. The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes. However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.

Proton-NMR investigation of the heme cavity in the cyanomet derivative of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis

The active-site structure of the paramagnetic cyanomet complex of the cooperative homodimeric hemoglobin from Scapharca inaequivalvis has been investigated by solution homonuclear NMR. In spite of the large size (32 kDa), the residues on the key proximal F- and distal E-helices could be sequence-specifically assigned and placed in the heme pocket in a manner common to diamagnetic systems. These backbone assignments were greatly facilitated by the significant dispersion of backbone chemical shifts by the highly anisotropic paramagnetic susceptibility tensor of the low-spin ferric state. The remainder of the residues in contact with the heme are assigned based on unique contacts to the heme predicted by the crystal structure and the observations of scalar connectivities diagnostic for the residues. The magnitude of the dipolar shifts for non-ligated residues was used to determine the anisotropy and orientation of the paramagnetic susceptibilty tensor, and the major axis found tilted from the normal in a manner similar to that found for the FeCO unit in the crystal structure. The combination of NOESY inter-residue and heme-residue contacts, paramagnetic-induced relaxation and correlation between observed and dipolar shifts provide a description of the heme cavity in cyanomet Hb that is essentially the same as found in the carbonmonoxy Hb crystal structure. The pattern of both the heme methyl dominant contact shifts and the heme meso-proton dominant dipolar shifts are shown to be consistent with the orientation of the axial His. It is concluded that the present homonuclear NMR methods allow effective solution structure determination in the cyanomet form for dimeric Hb and suggest profitable extension to the tetrameric vertebrate hemoglobins.

Ab initio calculation of atomic axial tensors and vibrational rotational strengths using density functional theory

We document a new density functional theory (DFT) methodology for the ab initio calculation of atomic axial tensors (AATs) using direct, analytical derivative methods and gauge-invariant atomic orbital (GIAO) basis sets. AATs are calculated for ethylene oxide. Their accuracy is assessed using AAT sum rules and the experimental electric dipole moment and paramagnetic susceptibility tensor. AATs calculated for the chiral molecule 6,8-dioxabicyclo[3.2.1]octane, in combination with DFT harmonic force fields and atomic polar tensors, are used to predict vibrational rotational strengths and vibrational circular dichroism (VCD) spectra. Their accuracy is assessed by comparison with the experimental rotational strengths and VCD spectrum.The dependence of calculated results on density functional and basis set is explored. The DFT calculations are compared to SCF (Hartree-Fock) calculations carried out in parallel.

Solution 1H‐NMR Structure of the Heme Cavity in the Low‐Affinity State for the Allosteric Monomeric Cyano‐Met Hemoglobins from Chironomus thummi thummi

Solution 1H-NMR studies of the heme cavity were performed for the cyanomet complexes of monomeric hemoglobins III and IV from the insect Chironomus thummi thummi, each of which exhibit marked Bohr effects. The low pH 5, paramagnetic (S = 1/2) derivatives were selected for study because the large dipolar shifts provide improved resolution over diamagnetic forms and allow distinction between the two isomeric heme orientations [Peyton, D. H., La Mar, G. N. & Gersonde, K. (1988) Biochim. Biophys. Acta 954, 82-94]. The crystal structure for the low-pH form of the hemoglobin III derivative, moreover, has been reported and showed that the functionally implicated distal His58 side chain adopts alternative orientation, either in or out of the pocket [Steigemann, W. & Weber, E. (1979) J. Mol. Biol. 127, 309-338]. All heme pocket residues for the low-pH forms of the two hemoglobins were located, at least in part, and positioned in the heme cavity on the basis of nuclear Overhauser effects to the heme and each other, dipolar shifts, and paramagnetic-induced relaxation. The resulting structure yielded the orientation of the major axis of the paramagnetic susceptibility tensor. The heme pocket structure of the cyanomet hemoglobins III and IV were found to be indistinguishable, with both exhibiting a distal His58 oriented solely into the heme cavity and in contact with the ligand, and with two residues, Phe100 and Phe38, exhibiting small but significant displacements in solution relative to hemoglobin III in the crystal.

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.

Solution structure determination of the heme cavity in the E7 His-->Val cyano-met myoglobin point mutant based on the 1H NMR detected dipolar field of the iron: evidence for contraction of the heme pocket.

The 1H NMR spectrum of the cyanomet complex of the sperm whale His[E7]Val myoglobin (Mb) point mutant has been analyzed by 2D methods to yield the assignments for the active site residues, including the substituted Val E7. The dipolar shifted proximal residues are used to quantitatively locate the magnetic axes for the paramagnetic susceptibility tensor in the molecular framework. The orientation of the major axis, which correlates with the ligand tilt, is approximately 15 degrees from the heme normal, as found in wild-type (WT) Mb, but is tilted in a direction rotated approximately 40 degrees toward the heme gamma-meso position with respect to WT and similar to that in the His[E7]Gly mutant [Rajarathnam, K., La Mar, G. N., Chiu, M., & Sligar, S. G. (1992) J. Am. Chem. Soc. 114, 9048-9058]. The altered direction of an unchanged tilt angle for the Fe+3-CN unit is shown to be qualitatively consistent with earlier computations of the potential energy surface for MbCO [Kuriyan, J., Wilz, S., Karplus, M., & Petsko, G. A. (1986) J. Mol. Biol. 192, 133-154]. It is concluded that His E7 does not significantly contribute to the ligand tilt but strongly influences the direction of tilt. Deviations between observed and predicted dipolar shifts for the E-helix backbone protons and perturbed patterns of their respective nuclear Overhauser effect between the E-helix and the heme 1,8-methyls are separately analyzed for movement of the E-helix and agree on a translation of the E-helix of the order of 0.8 A in a direction toward the iron. The discrepancy between observed and predicted dipolar shifts for Phe CD1 indicates a approximately 0.5-A movement by the ring parallel to the heme and towards the E-helix. The E-helix and Phe CD1 movements are consistent with a contraction of the pocket to fill the space created by the His-->Val substitution. The correlation between the observed dipolar shifts of the substituted Val E7 side chain and those calculated as a function of rotation of the residue with and without movement of the E-helix confirm the movement of the E-helix and allow a quantitative description of the Val orientation. It is concluded that the dipolar field of the paramagnetic susceptibility tensor provides an important quantitative constraint for defining the heme cavity structure in cyanomet complexes of distal point mutants of myoglobin and hemoglobin.

Determination of the orientation of the magnetic axes of the cyano-MetMb complexes of point mutants of myoglobin by solution 1H NMR: influence of his E7 .fwdarw. Gly and Arg CD3 .fwdarw. Gly substitutions

A method is described for obtaining the axes of the diagonal paramagnetic susceptibility tensor for the low-spin cyanide complexes of distal point mutants of ferric sperm whale myoglobin (metMbCN). It relies on using the crystal coordinates of the wild-type (WT) protein for that portion of the molecule unperturbed by the point mutation, together with the experimental dipolar shifts, to search for the Euler rotation that correctly converts the crystal coordinates to the magnetic axes

Evaluation of theories of vibrational magnetic dipole transition moments using atomic axial tensor sum rules and paramagnetic susceptibilities

Vibrational magnetic dipole transition moments, and hence rotational strengths and vibrational circular dichroism intensities, are determined by the atomic axial tensors. The electronic contributions to the latter, I λ αβ), are related via a sum rule to the paramagnetic susceptibility tensor, χ ϱ αβ. Their accuracy can be gauged by comparison of χ ϱ αβ tensors calculated using this sum rule to experimental data. This procedure is used to evaluate the atomic polar tensor (APT) and localized molecular orbital (LMO) theories of vibrational magnetic dipole transition moments, implemented ab initio. Our conclusion in regard to the LMO theory is not in agreement with that recently arrived at by Polavarapu.

Magnetic anisotropy of the Trenton limestone revisited

Previous studies have shown clear differences between the anisotropies of low‐field magnetic susceptibility (LFS) and of anhysteretic remanent magnetization (ARM) in samples of the Ordovician Trenton limestone from Trenton Falls, NY. ARM was found to be significantly more anisotropic than LFS, and to exhibit a well‐defined east‐northeasterly lineation which was not apparent in anisotropy of magnetic susceptibility (AMS). Because of significant local variation in mean LFS (by approximately a factor of two) at this locality, it has been possible to reanalyze the AMS of these samples by the method of Henry [1983], which in some cases allows mathematical differentiation of the ferromagnetic and paramagnetic susceptibility tensors. The ferromagnetic susceptibility tensor obtained in this way shows the same tectonic shortening apparent in the ARM anisotropy, but surprisingly, it shows no evidence of horizontal foliation. The paramagnetic tensor is horizontally foliated, with no lineation. The difference between the ferromagnetic susceptibility and ARM tensors suggests different ferromagnetic sources of ARM and LFS.