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.     

Solution 1H NMR characterization of the distal H-bond network and the effective axial field in the resting-state, high-spin ferric, substrate-bound complex of heme oxygenase from N. meningitidis

The solution (1)H 1D and 2D NMR spectra of the high-spin ferric, resting-state, substrate-bound complex of heme oxygenase, HO, from the pathological bacterium N. meningitidis have been investigated to assess the prospects for definitive assignment of hyperfine shifted and relaxed residue protons and the interpretation of those shifts in terms of the anisotropy and orientation of the paramagnetic susceptibility tensor, chi. Appropriately tailored 1D/2D NMR data, together with analyses of paramagnetic relaxation and a preliminary estimate of the magnetic anisotropy, reveal a chi that is axially anisotropic and oriented along the Fe-His vector. Together with T(-)(2) dependence of the shifts, Deltachi(ax) yields a zero-field splitting constant, D = 9.1 cm(-)(1), which is expected to serve as a very sensitive probe of H-bond interactions between the iron-ligated water and a series of distal ordered water molecules implicated in the mechanism of HO action. The side chains, Gln49 and His53, involved in the stabilization of catalytically relevant water molecules, were found to exhibit orientations rotated by 180 degrees about the beta-gamma bonds in solution relative to those in the crystal. The implication of these reorientations on the details of the distal H-bond network is discussed. The H-bond donor strengths of Gln 49 and His53 were found to respond appropriately to H-bond donor (water) versus H-bond acceptor (cyanide) iron ligands. Very slow NH exchange for the N-terminal portion of the distal helix suggest that an intrinsically "unstable" distal helix may be valid only for the C-terminal portion.     

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.     

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.     

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.     

1H NMR detection of immobilized water molecules within a strong distal hydrogen-bonding network of substrate-bound human heme oxygenase-1

Solution 1H NMR is used to probe the environments of the donor protons of eight strong hydrogen bonds on the distal side of the heme substrate in the cyanide-inhibited, substrate-bound complex of human heme oxygenase, hHO. It is demonstrated that significant magnetization transfer from the bulk water signal to the eight labile protons does not result from chemical exchange, but from direct nuclear Overhauser effect due to the dipolar interaction of these labile protons with "ordered" water molecules. The enzyme labile proton to water proton distances are estimated at approximately 3 A. It is proposed that the role of the strong hydrogen-bonding network is to immobilize numerous water molecules which both stabilize the activated hydroperoxy species and funnel protons to the active site.     

The ferrous verdoheme-heme oxygenase complex is six-coordinate and low-spin

A biosynthetic and enzymatic method was developed for the preparation of 13C-labeled verdoheme, which permits the 13C NMR spectroscopic characterization of this elusive intermediate in the heme oxidation path catalyzed by the enzyme heme oxygenase. The 13C NMR data indicate that the ferrous verdoheme complex of Neisseria meningitides heme oxygenase is hexacoordinate and diamagnetic, with a proximal histidine and likely a distal hydroxide as axial ligands. The coordination number and spin state of the ferrous verdoheme-heme oxygenase complex is in stark contrast to the pentacoordinate and paramagnetic nature of the heme-heme oxygenase complex and heme centers in general.     

1H and 13C NMR investigation of the influence of nonligated residue contacts on the heme electronic structure in cyanometmyoglobin complexes reconstituted with centro- and pseudocentrosymmetric hemins

The 1H and 13C chemical shifts for the heme methyls of low-spin, ferric sperm whale cyanometmyoglobin reconstituted with a variety of centrosymmetric and pseudocentrosymmetric hemins have been recorded and analyzed to shed light on the nature of heme-protein contacts, other than that of the axial His, that modulate the rhombic perturbation to the heme's in-plane electronic asymmetry. The very similar 1H dipolar shifts for heme pocket residues in all complexes yield essentially the same magnetic axes as in wild type, and the resultant dipolar shifts allow the direct determination of the heme methyl proton and 13C contact shifts in all complexes. It is demonstrated that, even when the magnetic axes and anisotropies are known, the intrinsic uncertainties in the orientational parameters lead to a sufficiently large uncertainty in dipolar shift that the methyl proton contact shifts are inherently significantly less reliable indicators of the unpaired electron spin distribution than the methyl 13C contact shifts. The pattern of the noninversion symmetry in 13C contact shifts in the centro- or pseudocentrosymmetric hemes is shown to correlate with the positions of aromatic rings of Phe43(CD1) and His97(FG3) parallel to, and in contact with, the heme. These results indicate that such pi-pi interactions significantly perturb the in-plane asymmetry of the heme pi spin distribution and cannot be ignored in a quantitative interpretation of the heme methyl 13C contact shifts in terms of the axial His orientation in b-type hemoproteins.     

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.     

Ligand effects toward the modulation of magnetic anisotropy and design of magnetic systems with desired anisotropy characteristics

Magnetic anisotropy of a set of octahedral Cr(III) complexes is studied theoretically. The magnetic anisotropy is quantified in terms of zero-field splitting (ZFS) parameter D, which appeared sensitive toward ligand substitution. The increased π-donation capacity of the ligand enhances the magnetic anisotropy of the complexes. The axial π-donor ligand of a complex is found to produce an easy-plane type (D > 0) magnetic anisotropy, while the replacement of the axial ligands with π-acceptors entails the inversion of magnetic anisotropy into the easy-axis type (D < 0). This observation enables one to fabricate a single molecule magnet for which easy-axis type magnetic anisotropy is an indispensable criterion. The equatorial ligands are also found to play a role in tuning the magnetic anisotropy. The magnetic anisotropy property is also correlated with the nonlinear optical (NLO) response. The value of the first hyperpolarizability varies proportionately with the magnitude of the ZFS parameter. Finally, it has also been shown that a rational design of simple octahedral complexes with desired anisotropy characteristics is possible through the proper ligand selection.     

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.     

Large magnetic anisotropy in pentacoordinate Ni(II) complexes

Pentacoordinate complexes in which Ni(II) is chelated by the tridentate macrocyclic ligand 1,4,7-triisopropyl-1,4,7-triazacyclononane (iPrtacn) of formula [Ni(iPrtacn)X(2)] (X=Cl, Br, NCS) have relatively large magnetic anisotropies, revealed by the large zero-field splitting (zfs) axial parameters |D| of around 15 cm(-1) measured by frequency-domain magnetic resonance spectroscopy (FDMRS) and high-field high-frequency electron paramagnetic resonance (HF-HFEPR). The spin Hamiltonian parameters for the three complexes were determined by analyzing the FDMRS spectra at different temperatures in zero applied magnetic field in an energy window between 0 and 40 cm(-1). The same parameters were determined from analysis of HF-HFEPR data measured at different frequencies (285, 380, and 475 GHz) and at 7 and 17 K. The spin Hamiltonian parameters D (axial) and E (rhombic) were calculated for the three complexes in the framework of the angular overlap model (AOM). The nature and magnitude of the magnetic anisotropy of the three complexes and the origin of the influence of the X atoms were analyzed by performing systematic calculations on model complexes.     

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.     

The highest D value for a Mn(II) ion: investigation of a manganese(II) polyoxometalate complex by high-field electron paramagnetic resonance

Using magnetization measurements and multifrequency high-field electron paramagnetic resonance, the largest zero-field splitting for any individual isolated Mn(II) ion has been found in a polyoxometalate complex, suggesting that the inorganic ligand induces large Ising-type magnetic anisotropy.     

A Hemin-Analogous Corrphycene Derivative: Suppression of Heme Oxygenase and Reconstitution with Apomyoglobin

Summary. A closely hemin-analogous corrphycene derivative was prepared in good overall yield. By spectroscopic measurements it was shown that it complexes with the stress protein heme oxygenase and apomyoglobin in a similar way as hemin. However, due to its molecular structure it is not attacked by heme oxygenase, but is able to block this enzyme to some degree. In addition, the complex with apomyoglobin displays oxygen and carbon monoxide ligation comparable to myoglobin. These properties make this novel corrphycene derivative a candidate to be used as heme oxygenase blocker or otherwise as a blood pigment substitute.Received July 15, 2003; accepted September 5, 2003 Published online October 23, 2003     

Distance measurements in Au nanoparticles functionalized with nitroxide radicals and Gd(3+)-DTPA chelate complexes

Nanosized gold particles were functionalised with two types of paramagnetic surface tags, one having a nitroxide radical and the other one carrying a DTPA complex loaded with Gd(3+). Selective measurements of nitroxide-nitroxide, Gd(3+)-nitroxide and Gd(3+)-Gd(3+) distances were performed on this system and information on the distance distribution in the three types of spin pairs was obtained. A numerical analysis of the dipolar frequency distributions is presented for Gd(3+) centres with moderate magnitudes of zero-field splitting, in the range of detection frequencies and resonance fields where the high-field approximation is only roughly valid. The dipolar frequency analysis confirms the applicability of DEER for distance measurements in such complexes and gives an estimate for the magnitudes of possible systematic errors due to the non-ideality of the measurement of the dipole-dipole interaction.     

Evaluation of dipolar shifts and paramagnetic anisotropy in penta coordinated cobalt(II) complexes

The isotropic proton shifts for the pyridine N-oxide and γ-picoline N-oxide protons have been observed in the penta coordinated adducts of these bases with bis[di(p-tolyl)dithiophosphinato] cobalt(II). The contribution to the observed shifts due to dipolar interaction has been calculated. From the dipolar shifts, it was ascertained that the pyridine N-oxide complexes have a bent structure in solution with a Co-O-N angle of 125°. An estimate of the paramagnetic anisotropy of the cobalt complex yields K_?-K_⊥ = 4244 × 10^?6 cm³/mole.     

13C and 15N NMR studies of iron-bound cyanides of heme proteins and related model complexes: sensitive probe for detecting hydrogen-bonding interactions at the proximal and distal sides

Studies of the 13C and 15N NMR paramagnetic shifts of the iron-bound cyanides in the ferric cyanide forms of various heme proteins containing the proximal histidine and related model complexes are reported. The paramagnetic shifts of the 13C and 15N NMR signals of the iron-bound cyanide are not significantly affected by the substitution of the porphyrin side chains. On the other hand, the paramagnetic shifts of both the 13C and 15N NMR signals decrease with an increase in the donor effect of the proximal ligand, and the 13C NMR signal is more sensitive to a modification of the donor effect of the proximal ligand than the 15N NMR signal. With the tilt of the iron-imidazole bond, the paramagnetic shift of the 13C NMR signal increases, whereas that of the 15N NMR signal decreases. The hydrogen-bonding interaction of the iron-bound cyanide with a solvent decreases the paramagnetic shift of both 13C and 15N NMR signals, and the effect is more pronounced for the 15N NMR signal. Data on the 13C and 15N NMR signals of iron-bound cyanide for various heme proteins are also reported and analyzed in detail. Substantial differences in the 13C and 15N NMR shifts for the heme proteins can be explained on the basis of the results for the model complexes and structures around the heme in the heme proteins. The findings herein show that the paramagnetic shift of the 13C NMR signal of the iron-bound cyanide is a good probe to estimate the donor effect of the proximal imidazole and that the ratio of 15N/13C NMR shifts allows the hydrogen-bonding interaction on the distal side to be estimated.     

Azide-inhibited bacterial heme oxygenases exhibit an S = 3/2 (dxz,dyz)3(dxy)1(dz2)1 spin state: mechanistic implications for heme oxidation

The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningitidis (nm-HO) have been studied with the aid of (1)H and (13)C NMR spectroscopy. These complexes have been shown to exist as an equilibrium mixture of two populations, one exhibiting an S = (1)/(2), (d(xy))(2)(d(xz), d(yz))(3) electron configuration and planar heme and a second with a novel S = (3)/(2), (d(xz), d(yz))(3)(d(xy))(1)(d(z)(2))(1) spin state and nonplanar heme. At physiologically relevant temperatures, the equilibrium shifts in the direction of the population exhibiting the latter electron configuration and nonplanar heme, whereas at temperatures approaching the freezing point of water, the equilibrium shifts in the direction of the population with the former electronic structure and planar heme. These findings indicate that the microenvironment of the distal pocket in heme oxygenase is unique among heme-containing proteins in that it lowers the sigma-donating (field strength) ability of the distal ligand and, therefore, promotes the attainment of heme electronic structures thus far only observed in heme oxygenase. When the field strength of the distal ligand is slightly lower than that of azide, such as OH(-) (J. Am. Chem. Soc. 2003, 125, 11842), the corresponding complex exists as a mixture of populations with nonplanar hemes and electronic structures that place significant spin density at the meso positions. The ease with which these unusual heme electronic structures are attained by heme oxygenase is likely related to activation of meso carbon reactivity which, in turn, facilitates hydroxylation of a meso carbon by the obligatory ferric hydroperoxide intermediate.