Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated alpha-chain from human hemoglobin A

The solution electronic and molecular structure for the heme pocket of the cyanomet complex of the isolated alpha-chain of human adult hemoglobin (HbA) has been investigated by homonuclear two-dimensional 1H NMR in order to establish an assignment protocol for the dimeric chain that will guide similar assignments in the intact, heterotetrameric HbA complex, and to compare the structures of the alpha-chain with its subunit in HbA. The target residues are those that exhibit significant (>0.2 ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 97 target residues (approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than 0.2 ppm. The complete assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the alpha-chain in terms of the crystal coordinates of the alpha-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated alpha-chain relative to that in the intact HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in HbACO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature two-dimensional NMR spectra are crucial to effective assignments in the cyanomet alpha-chain and that this approach should be similarly effective in HbA.     

1H NMR investigation of reduced copper-cobalt superoxide dismutase

Human copper-cobalt superoxide dismutase in the reduced form has been investigated through ¹H NMR techniques. The aim is to monitor the structural properties of this derivative and to compare them with those of reduced and oxidized native superoxide dismutases. The observed signals of the cobalt ligands have been assigned as well as the signals of the histidines bound to copper(I). The latter signals experience little pseudocontact shifts which allow a rough orientation of the magnetic susceptibility tensor in the molecular frame. The connectivities indicate that, although the histidine bridge is broken in the reduced form, the interproton distances between ligands of both ions are essentially the same.Abbreviations WEFT water eliminated Fourier transform - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - SOD superoxide dismutase - E₂Co(II)SOD SOD with empty copper site (E=empty) and with cobalt(II) in the Zinc(II) site Offprint requests to: I. Bertini     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric beta-chain from human adult hemoglobin

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated beta-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D (1)H NMR in order to assess the extent of assignments allowed by (1)H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the beta-chain to the crystallographically characterized complexes that contains the beta-chain. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues ( approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the beta-chain in terms of the crystal coordinates of the beta-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated beta-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in the beta-subunit of HbCO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet beta-chain and that this approach should be similarly effective in HbA.     

Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

Nuclear Magnetic Resonance Characterization of a Paramagnetic DNA-drug Complex with High Spin Cobalt; Assignment of the 1H and 31P NMR Spectra, and Determination of Electronic, Spectroscopic and Molecular Properties

The proton NMR spectrum of the ternary complex between the octamer duplex d(TTGGCCAA)2, two molecules of the drug chromomycin-A3, and a divalent cobalt ion has been assigned. Assignment procedures used standard two-dimensional techniques and relied upon the expected NOE contacts observed in the equivalent diamagnetic complex containing zinc. The magnetic susceptibility tensor for the cobalt was determined and used to calculate shifts for all nuclei, aiding in the assignment process and verification. Relaxation, susceptibility, temperature and field dependence studies of the paramagnetic spectrum enabled determination of electronic properties of the octahedral cobalt complex. The electronic relaxation rate s was determined to be 2.5 ± 1.5 ps; the effective isotropic g value was found to be 2.6 ± 0.2, indicating strong spin-orbit coupling. The magnetic susceptibility tensor was determined to be xx = 8.9 * 10-3 cm3/mol, yy = 9.5 * 10-3 cm3/mol, zz = 12.8 * 10-3 cm3/mol. A tentative rotational correlation time of 8 ns was obtained for the complex. Both macroscopic and microscopic susceptibility measurements revealed deviations from Curie behavior over the temperature range accessible in the study. Non-selective relaxation rates were found to be inaccurate for defining distances from the metal center. However, pseudocontact shifts could be calculated with high accuracy using the dipolar shift equation. Isotropic hyperfine shifts were factored into contact and dipolar terms, revealing that the dipolar shift predominates and that contact shifts are relatively small.     

Application of the paramagnetic dipole field for solution NMR active site structure determination in low-spin, cyanide-inhibited ferric hemoproteins

The principles for the application of the paramagnetic dipolar field of low-spin, cyanide-inhibited ferrihemoproteins for determining active site structure are briefly described. The ubiquitous dipolar shifts for assigned residues, together with crystal coordinates of some appropriate structural homolog, allow determination of the orientation and anisotropies of the paramagnetic dipolar tensor. The orientation of chi uniquely defines the orientation of the Fe-CN unit, which is tilted variably and sensitively monitors distal steric and H-bond interactions. The mapped dipolar field, in turn, can be used to determine the orientation of mutated residues. Case studies involving unusual genetic variants and point mutants of myoglobins, human hemoglobins, horseradish peroxidase and its substrate complex of heme oxygenase are presented as examples.     

Calculation of z-coordinates and orientational restraints using a metal binding tag

We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings. Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.     

NMR study of Galeorhinus japonicus myoglobin. 1H-NMR study of molecular structure of the heme cavity

The molecular structure of the active site of myoglobin from the shark, Galeorhinus japonicus, has been studied by 1H-NMR. Some hyperfine-shifted amino acid proton resonances in the met-cyano form of G. japonicus myoglobin have been unambiguously assigned by the combined use of various two-dimensional NMR techniques; they were compared with the corresponding resonances in Physter catodon myoglobin. The orientations of ThrE10 and IleFG5 residues relative to the heme in G. japonicus met-cyano myoglobin were semiquantitatively estimated from the analysis of their shifts using the magnetic susceptibility tensor determined by a method called MATDUHM (magnetic anisotropy tensor determination utilizing heme methyls) [Yamamoto, Y., Nanai, N. & Ch?j?, R. (1990) J. Chem. Soc., Chem. Commun., 1556-1557] and the results were compared with the crystal structure of P. catodon carbonmonoxy myoglobin [Hanson, J. C. & Schoenborn, B. P. (1981) J. Mol. Biol. 153, 117-124]. In spite of a substantial difference in shift between the corresponding amino acid proton resonances for the two proteins, the orientations of these amino acid residues relative to the heme in the active site of both myoglobins were found to be highly alike.     

An NMR structural study of nickel-substituted rubredoxin

The Ni(II) and Zn(II) derivatives of Desulfovibrio vulgaris rubredoxin (DvRd) have been studied by NMR spectroscopy to probe the structure at the metal centre. The βCH₂ proton pairs from the cysteines that bind the Ni(II) atom have been identified using 1D nuclear Overhauser enhancement (NOE) difference spectra and sequence specifically assigned via NOE correlations to neighbouring protons and by comparison with the published X-ray crystal structure of a Ni(II) derivative of Clostridium pasteurianum rubredoxin. The solution structures of DvRd(Zn) and DvRd(Ni) have been determined and the paramagnetic form refined using pseudocontact shifts. The determination of the magnetic susceptibility anisotropy tensor allowed the contact and pseudocontact contributions to the observed chemical shifts to be obtained. Analysis of the pseudocontact and contact chemical shifts of the cysteine Hβ protons and backbone protons close to the metal centre allowed conclusions to be drawn as to the geometry and hydrogen-bonding pattern at the metal binding site. The importance of NH–S hydrogen bonds at the metal centre for the delocalization of electron spin density is confirmed for rubredoxins and can be extrapolated to metal centres in Cu proteins: amicyanin, plastocyanin, stellacyanin, azurin and pseudoazurin.     

1H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2-avid hemoglobin from the trematode Paramphistomum epiclitum.

The solution molecular and electronic structures of the active site in the extremely O2-avid hemoglobin from the trematode Paramphistomum epiclitum have been investigated by 1H NMR on the cyanomet form in order to elucidate the distal hydrogen-bonding to a ligated H-bond acceptor ligand. Comparison of the strengths of dipolar interactions in solution with the alternate crystal structures of methemoglobin establish that the solution structure of wild-type Hb more closely resembles the crystal structure of the recombinant wild-type than the true wild-type met-hemoglobin. The distal Tyr66(E7) is found oriented out of the heme pocket in solution as found in both crystal structures. Analysis of dipolar contacts, dipolar shift and paramagnetic relaxation establishes that the Tyr32(B10) hydrogen proton adopts an orientation that allows it to make a strong H-bond to the bound cyanide. The observation of a significant isotope effect on the heme methyl contact shifts confirms a strong contact between the Tyr32(B10) OH and the ligated cyanide. The quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that the cyanide is tilted approximately 10 degrees from the heme normal so as to avoid van der Waals overlap with the Tyr32(B10) Oeta. The pattern of heme contact shifts with large low-field shifts for 7-CH3 and 18-CH3 is shown to arise not from the 180 degrees rotation about the alpha-gamma-meso axis, but due to the approximately 45 degrees rotation of the axial His imidazole ring, relative to that in mammalian globins.     

1H NMR study of the effect of variable ligand on heme oxygenase electronic and molecular structure

Heme oxygenase carries out stereospecific catabolism of protohemin to yield iron, CO and biliverdin. Instability of the physiological oxy complex has necessitated the use of model ligands, of which cyanide and azide are amenable to solution NMR characterization. Since cyanide and azide are contrasting models for bound oxygen, it is of interest to characterize differences in their molecular and/or electronic structures. We report on detailed 2D NMR comparison of the azide and cyanide substrate complexes of heme oxygenase from Neisseria meningitidis, which reveals significant and widespread differences in chemical shifts between the two complexes. To differentiate molecular from electronic structural changes between the two complexes, the anisotropy and orientation of the paramagnetic susceptibility tensor were determined for the azide complex for comparison with those for the cyanide complex. Comparison of the predicted and observed dipolar shifts reveals that shift differences are strongly dominated by differences in electronic structure and do not provide any evidence for detectable differences in molecular structure or hydrogen bonding except in the immediate vicinity of the distal ligand. The readily cleaved C-terminus interacts with the active site and saturation-transfer allows difficult heme assignments in the high-spin aquo complex.     

Improving the Accuracy of NMR Structures of Large Proteins Using Pseudocontact Shifts as Long-Range Restraints

We demonstrate improved accuracy in protein structure determination for large (>/=30 kDa), deuterated proteins (e.g. STAT4(NT)) via the combination of pseudocontact shifts for amide and methyl protons with the available NOEs in methyl-protonated proteins. The improved accuracy is cross validated by Q-factors determined from residual dipolar couplings measured as a result of magnetic susceptibility alignment. The paramagnet is introduced via binding to thiol-reactive EDTA, and multiple sites can be serially engineered to obtain data from alternative orientations of the paramagnetic anisotropic susceptibility tensor. The technique is advantageous for systems where the target protein has strong interactions with known alignment media.     

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.     

An analysis of the Co2+-induced nuclear magnetic resonance perturbations of hen egg white lysozyme

A general methodology is presented for analyzing dipolar shifts induced by paramagnetic ions in the nuclear magnetic resonance (NMR) spectra of ligand molecules. The method is applied to the shift perturbations induced by Co2+ in the spectrum of hen egg white lysozyme. A hypothesis testing scheme is employed to evaluate statistically the relative precision with which the axially symmetric and non-axially symmetric forms of the dipolar shift equation fit the observed data. The assumption of axial symmetry for the magnetic susceptibility tensor of Co2+ is rejected at the confidence level of 99%. Since the results presented here are similar to those reached in our analysis of lanthanide-induced shifts, we suggest that the assumption of axial symmetry may, in general, not hold. Similar conclusions have been reached by other investigators in studies of paramagnetic metal binding to model systems. We have included the three Co2+ coordinates in an eight-parameter fit of the Co2+ shift data. The Co2+ position obtained from this fit is in statistical agreement with the position inferred from x-ray data. Thus, the analysis of shift data may furnish a means for determining the site of metal complexation in macromolecules whose structure has been determined by x-ray crystallography.     

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic ¹³C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.     

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]     

Solution H NMR characterization of substrate-free C. diphtheriae heme oxygenase: Pertinence for determining magnetic axes in paramagnetic substrate complexes

Proton 2D NMR was used to confirm in solution a highly conserved portion of the molecular structure upon substrate loss for the heme oxygenase from the pathogenic bacterium Corynebacterium diphtheriae, HmuO. The chemical shifts for the conserved portion of the structure are assessed as references for the dipolar shifts needed to determine the orientation of the paramagnetic susceptibility tensor, χ, in paramagnetic substrate complexes of HmuO. It is shown that the chemical shifts for the structurally conserved portion of substrate-free HmuO serve as excellent references for residues with only small to moderate sized dipolar shifts in the cyanide-inhibited substrate complex of HmuO, yielding an orientation of χ that is essentially the same as conventionally obtained from large dipolar shifts based on empirical estimates of the diamagnetic reference. The implications of these diamagnetic chemical shifts for characterizing the hydrogen bonding in the physiologically relevant, resting-state, high-spin aquo complex are discussed. The pattern of labile proton exchange in the distal H-bond network of substrate-free HmuO allowed comparison of changes in dynamic stability of tertiary contacts in the substrate-free and substrate-bound HmuO and with the same complexes of human heme oxygenase.