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J Biol Chem, Vol. 274, Issue 45, 31819-31826, November 5, 1999

¹H NMR Investigation of the Distal Hydrogen Bonding Network and Ligand Tilt in the Cyanomet Complex of Oxygen-avid Ascaris suum Hemoglobin^*

Zhicheng Xia, Wei Zhang, Bao D. Nguyen, and Gerd N. La Mar

From the Department of Chemistry, University of California, Davis, California 95616

Andrew P. Kloek, and Daniel E. Goldberg^§

From the Howard Hughes Medical Institute and the Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The O₂-avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O₂ off rates. Solution ¹H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO₂ crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4224-4228) with the exception that the heme in solution is rotated by 180 ° about the ,-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO₂, provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented ~30 ° away from the heme normal and indicates significant steric tilt because of interaction with Tyr³⁰(B10). The three side chain labile protons for the distal residues Tyr³⁰(B10) and Gln⁶⁴(E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO₂ crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O₂. The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr³⁰(B10) side chain supports a crowded and constrained distal pocket.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The O₂ binding globins, myoglobin (Mb)¹ and hemoglobin (Hb), despite highly varied sequences throughout phylogeny, possess a highly conserved folding topology of 7-8 helices (A-H), with the heme wedged in between the E and F helices and ligated by one, His F8 (proximal), of only two (the other is Phe CD1) completely conserved residues (1-3). Despite this strong structural homology, the O₂ ligation rates and O₂ affinities vary over a remarkably wide range (by ~10⁵), depending on the exact nature of several distal residues at the key positions B10, E7, and E10 (4-8). The most important distal interaction for stabilizing bound O₂ is hydrogen bonding to the ligand, for which the donor is generally His E7 (1, 7, 9, 10) and is Gln E7 in a few cases (2). In several invertebrates, such as Aplysia and Dolbella Mbs that possess a Val E7, the distal H bond to the ligand is provided by an Arg at position E10 (11-13).

A particularly noteworthy class of globins is that of parasitic nematodes that possess, in addition to a H bond donor at position E7, a Tyr at position B10 that is also capable of H bonding to the ligand (4, 5, 8, 14-17). In the case of the Hb from Ascaris suum, the extraordinarily high O₂ affinity and extremely low O₂ off-rates have been attributed to a distal H bonding interactions for the Tyr³⁰(B10) and Gln⁶⁴(E7) side chains with bound O₂. Stabilizing H bond interactions of Gln⁶⁴(E7) and Tyr³⁰(B10) with ligated O₂ are supported by the observations of enhanced O₂ off-rates upon mutating either residue (5, 15). The positions of these two key residues are clearly defined in the crystal structure of A. suum HbO₂, in which the two residues are appropriately poised to serve as H bond donors to bound O₂, with the Gln⁶⁴(E7) additionally providing an H bond to the Tyr³⁰(B10) side chain O that stabilizes the optimal dispositions of these two residues (8). Resonance Raman spectroscopy has confirmed the role of Tyr³⁰(B10) as a H bond donor (18, 19), and flash photolysis experiments (19) have indicated that A. suum Hb possesses a very compact and constrained distal pocket when compared with other globins.

Resonance Raman spectroscopy has shown that A. suum HbCO exhibits the lowest reported CO stretching frequency (19). The strong modulation of the CO stretching frequency by globin distal environment has been discussed in the context of both steric tilt/bending of the Fe-CO unit from the heme normal and pocket dielectric effects (20-23), and the currently accepted interpretive basis is that the latter effect is the major determinant of _CO (7, 24). Nevertheless, crystal structures of myoglobins invariably find the carbonyl oxygen placed off-axis from the heme normal, indicating that the Fe-CO unit is bent/tilted from the heme normal (7, 25-27).

The cyanomet derivatives of globins can serve as valuable structural (but not functional) (28, 29) models for both O₂ and CO binding, in that Fe^IIICN, like Fe^IIO₂, is polar and is a good H bond acceptor (30) and, like Fe^IICO, prefers to bind normal to the heme in the absence of distal steric interactions (31). In the one case where the crystal structures of both the carbonyl and cyanomet globins have been reported, there is a good correlation in the degree and direction of the off-axis placement of the terminal atoms (26). Theoretical considerations have indicated that distal ligand tilt could be modulated by tilt of the proximal His (32). In the crystallographically and NMR characterized globins to date, the axial His is essentially normal to the heme. The crystal structures of A. suum HbO₂, on the other hand, show that the axial His imidazole plane is tilted some ~8 ° in the direction of pyrrole C with respect to the heme plane (8). Thus a determination of orientation of the Fe^IIICN or Fe^IICO units relative to the heme in A. suum Hb would indicate whether the axial His could contribute to distal ligand tilt and provide some insight as to whether there is likely to be a large Fe-CO tilt that could contribute to the reduced value for _co.

Solution ¹H NMR of the paramagnetic cyanomet Hb derivatives can provide significant structural details on the distal pocket in relation to both stabilizing H bonding and destabilizing steric interactions with the bound ligand (30, 33, 34). On the one hand, the dipolar shifts and moderate relaxation imparted to distal residues and their labile protons facilitate their detection, identification, and detailed placement relative to the bound ligand (35, 36). On the other hand, the sizable dipolar shifts for active site residues allow the quantitative determination of the orientation of the paramagnetic susceptibility tensor, for which the major magnetic axis can be correlated to the degree of Fe-CN tilt from the heme normal (34). There is generally very good agreement in the magnitude and direction of Fe-CN tilt observed in crystal structure and the orientation of the major magnetic axes determined by solution ¹H NMR (26, 27, 38-41). Lastly, the expanded chemical shift scale for heme pocket residues because of the hyperfine interaction increases the prospect for measuring rapid dynamic processes, such as ring orientation, that can constitute probes for the constraints in the heme pocket (42).

We report herein on the solution ¹H NMR characterization of the cyanomet complex of the D1 domain of A. suum metHbCN, which demonstrates that the distal H bonding network is essentially identical to that in HbO₂, that the Fe-CN unit appears tilted strongly away from the heme normal in the direction of the observed terminal oxygen in HbO₂, and that the distal pocket is sufficiently crowded to strongly tilt the Fe-CN vector and to impede the reorientation of the Tyr³⁰(B10) ring.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Preparation-- Native protein samples were prepared as described previously (15, 43). The cyanide complexes were prepared by adding KCN to the protein solution approximately in a molar ratio of 10:1 buffered with 50 mM phosphate, 200 mM NaCl at pH 7.2. ²H₂O sample was prepared by repeatedly washing protein with ²H₂O in the same buffer with a Centricon (Amicon Inc.), and pH was read directly from the pH meter without the isotope effect correction; the final protein concentration was ~2 mM.

NMR Spectra-- All ¹H NMR spectra were recorded on a GE  500 spectrometer operating at 500 MHz. Chemical shift values were referenced to 2,2-dimethyl-2-pentane-5-sulfonate (DSS) through the residual water signal. Reference spectra were collected with ¹H₂O saturation. Steady-state NOE and inversion-recovery spectra were collected at a repetition rate of 3 s¹ (34). The residual water signals were removed from the free induction decay by convolution difference. The nonselective spin lattice paramagnetic relaxation times for the resolved peaks were derived from two-parameter exponential least square fits using only short (50 ms) delays. Estimates for distance to the iron for proton i, R_Fe-i were obtained from the nonselective paramagnetically dominated T₁ values using the following relation.

(Eq. 1)

where the reference T_1j = 150 ms (R_Fe-j = 6.1 Å) for a heme methyl, or T_1j = 30 ms (R_Fe = 5.1 Å) for the His(F8) NH, provided upper and lower limits, respectively (44). Interproton distances, r_ij, were estimated from steady-state NOEs, _i-j to protons with paramagnetically dominated (nonselective) T₁ values, via the following two equations.

(Eq. 2)

where

(Eq. 3)

NOESY (45) and TOCSY (46-48) spectra were collected over a temperature range of 20-35 °C in ¹H₂O. Two different spectral windows and mixing times were used for NOESY 25.0 KHz using 2048 complex points at 3 scans/s with a mixing time of 35 ms to optimize the observation of the hyperfine shifted signals and 10.0 KHz at 1 scan/s with a mixing time of 100 ms to cover the diamagnetic window at optimal digital resolution. The clean TOCSY spectra were collected over 12.0 KHz at 2 scans/s with a spin locking time of 35 ms using the MLEV-17 mixing scheme (47). All the two-dimensional data sets were processed on a Silicon Graphics workstation either using the software package FELIX from Biosym/MSI (San Diego, CA) or AZARA generously provided by Wayne Boucher (Department of Biochemistry, University of Cambridge). To increase the resolution, data sets were apodized with a sine-bell-squared function shifted by 20-40 ° in both dimensions and zero-filled once in t₁ dimension. The spectral assignments were largely facilitated with the aid of ANSIG package (49).

Magnetic Axes-- The magnetic axes² were determined as described in detail previously (34, 50, 51). Experimental dipolar shifts, _DSS(obs), for protons on nonligated residues for structurally conserved portions (relative to A. suum HbO₂) of the heme environment were used as input to search for the Euler rotation angles,  (, , ), that transforms the iron-centered pseudo-symmetry coordinates (x', y', z', or R, ', ') (Fig. 1C)), readily obtained from the Ascaris HbO₂ (domain I) crystal coordinates (8), into magnetic axes, x, y, z, i.e. (x, y, z) = (x', y', z')(, , ), by minimizing the error function using the Levenberg-Marguardt method (52, 53).

(Eq. 4)

with _dip(calc) given by the following equation.

(Eq. 5)

where _ax = _zz  1/2(_xx+_yy) and _rh = _xx  yy are the axial and rhombic anisotropies of the diagonal paramagnetic susceptibility tensor, . The tilt of the major magnetic axis, z, from the heme normal is given by , the projection of this tilt on the heme plane relative to the x' axis is given by , and the location of the rhombic axes projected on the heme plane is approximated by  =  + , as labeled in Fig. 1C. _dip(obs) is given by the following equation.

(Eq. 6)

_DSS(obs) is the observed chemical shift referenced to DSS. _DSS(dia) is the isostructural diamagnetic shift, which, in this case, was calculated by using: _DSS(dia) = _tetr + _sec + _rc, where _tetr is the shift in an unfolded tetra peptide (54), _sec is the shift of an amino acid proton resulted from the protein secondary structure (55), and _rc is the heme-induced ring current shift (56). Minimizing the error function, F/n, in Equation 4 was performed over five parameters, _ax, _rh, , , and , using the HbO₂ crystal coordinates. For the iron ligated porphyrin and axial His, the hyperfine shift is obtained via the following equation.

(Eq. 7)

which yields the contact shift via the following equation.

(Eq. 8)

Molecular Modeling-- Protons were added to the crystal coordinates of A. suum HbO₂ using the program INSIGHT II (MSI). This provided unique coordinates for all protons of interest except the Tyr³⁰(B10) hydroxyl proton; hence its position was determined from the ¹H NMR spectral parameters.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A schematic representation for selected heme cavity proximal (squares) and distal (circles) residues and their disposition relative to the heme is shown in Fig. 1. The heme is shown in Fig. 1A seated as reported in the crystal structure of HbO₂ (8), and in Fig. 1B as found in the solution structure of metHbCN. The resolved portions of the ¹H NMR spectra of A. suum D1 metHbCN in ¹H₂O and ²H₂O are shown in Fig. 2 (A and B, respectively). The region downfield of ~9 ppm resolves four three-proton (methyls) and four single-proton signals as well as one two-proton peak at 11 ppm that overlaps a methyl peak. Ten single-proton peaks and four methyl peaks can be resolved at some temperature in the upfield portion of the spectra. The comparison between ¹H₂O and ²H₂O reveals that three of the most strongly relaxed resolved protons are labile.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Schematic representation of select heme cavity residues on the proximal (square) and distal (circles) sides of the heme and the heme orientation as reported in the crystal structure of A. suum Hb O2 (A) and as determined herein by NMR (B). The heme is labeled with the Fisher notation, and the substituents are labeled M (methyl), V (vinyl), and P (propionate). The expected (on the basis of the crystal structure with the rotated heme) and observed inter-residue and residue-heme dipolar contacts are shown in B by double-sided arrows. The iron-centered, crystal structure-based coordinate system (x', y', z') is shown in C, as is the magnetic coordinate system (x, y, z), where  is diagonal. The two systems are related by the Euler rotation,  (, , ), [x, y, z] = [x', y', z']  (, , ), where  is the tilt of the major magnetic axes from the heme normal,  is the angle between the projection of the tilt on the heme plane and the x' axis, and  = ~ +  define the projection of the rhombic axes on the x', y' plane.  is the orientation of the proximal His imidazole ring plane relative to the NA-Fe-NC vector (x' axis). It is noted that the convention for x', y', z' differs from that used previously by a 45 ° (34, 39-41, 44, 50) rotation in the heme plane and referencing  to the +x' rather than x' axis, so that (new) = (old), (new) = (old) + 135 °, and (new) = (old)  45 °.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   . Resolved portions of the 500 MHz 1H NMR reference spectra of ~2 mM A. suum D1 metHbCN, pH 7.0, at 30 °C in 1H2O (A) and in 2H2O (B). The assigned resolved signals are labeled by the Fisher notation for the heme and by the one-letter code for the residue and sequence position. Also shown are steady-state NOE difference spectra upon saturating the low field Tyr30(B10) OH signal (C) and upon saturating the upfield Gln(E7) NH2 (D). The intensity of the saturated signals in traces C and D are identical. The detected NOEs are assigned as presented in the text. An asterisk indicates off-resonance saturation.

Although the present ¹H NMR data on metHbCN confirm a highly conserved arrangement relative to each other, of both proximal and distal residues (see below), the data also demonstrate that the heme is oriented differently in the cavity from that originally reported in the crystal structure (8). Thus assignments for the heme and axial His are pursued first, followed by the key residues (i.e. Phe⁴⁴(CD1) and Met¹⁰³(FG5)) that invariably place strongly relaxed protons in resolved spectral windows and hence can be unambiguously assigned based solely on the conserved globin fold. These two residues are then used to uniquely orient the heme in the cavity. The remainder of the assignments are then presented on the basis of characteristic TOCSY-detected spin systems, with heme contacts expected on the basis of the heme orientation deduced above. Because the protocol for heme and resolved residue assignments in similar cyanomet globins has been described in detail (13), NMR data are illustrated only for the key heme-Phe⁴⁴(CD1) contact that determines the heme orientation and for the distal H bond donors, Tyr³⁰(B10) and Gln⁶⁴(E7).

Heme Assignments-- The heme substituents could be unambiguously assigned as described in detail elsewhere (13). Two TOCSY-detected vinyl and one propionate groups exhibit NOESY cross-peaks to low field resolved methyls that pair 1-CH₃, 2 vinyl and 3-CH₃, 4 vinyl; NOESY cross-peaks between two of the heme methyls (Fig. 3D) assign the 1-CH₃ and 8-CH₃, and a NOESY cross-peak between the remaining methyls and the one detected propionate uniquely assigns the pyrrole substituents. Common NOESY cross-peaks for the two substituents flanking a meso position, together with large low field intercepts in a Curie plot (not shown), identify the meso-Hs (34). The heme assignments, chemical shifts, T₁ values, and slopes in a Curie plot, are listed in Table I.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   A and B, portions of the TOCSY (15-ms mixing time) spectra showing the scalar correlation for the Phe44(CD1) and Tyr30(B10) rings. C and D, portions of the NOESY spectrum that illustrates the dipolar contact between the two heme methyls that uniquely assigns 1-CH3 and 8-CH3 and the dipolar contact of the Phe44(CD1) ring with both the 1-CH3 and 8-CH3 of the heme, which uniquely characterize the orientation of the heme in the pocket as rotated by 180 ° about the ,-meso axis relative to that found in the HbO2 crystal structure.

Assignment of Key Resolved Resonances-- The 19.0 ppm labile proton, when saturated (not shown), exhibits NOEs to a labile proton at 12 ppm, which is part of a nonlabile proton NMR spin system diagnostic of the axial His⁹⁷(F8) CH₂CH-NH fragment. A very broad and strongly relaxed (T₁ = ~3 ms) upfield, nonlabile proton peak must arise from the axial His ring (34), and a NOE to the CH upon saturating this peak (not shown) establishes that it is the His⁹⁷(F8) CH. The TOCSY-detected CHN_pH backbone and several CHs of the members of the F-helix, Asp⁹⁴(F6) through Arg⁹⁸(F10), could be located via the standard expected helical N_i-N_i+1, _i-N_i+1 contacts (not shown) (54). Although these backbone assignments could not be extended to F4 because of spectral congestion, the expected strong NOE between His⁹⁷(F8) NH and Leu⁹²(F4) locates the latter residue CH.

A resolved (15 ppm), strongly relaxed, nonlabile single proton peak exhibits the TOCSY connectivity (Fig. 3, A and B) and variable temperature slope and intercepts of a rapidly reorienting aromatic ring. The relaxation properties (T₁ = ~20 ms) alone dictate that it must arise from the completely conserved Phe⁴⁴(CD1) (42). TOCSY connections (not shown), moreover, involving two sets of upfield hyperfine shifted residues identify AMXPT and AM(X₃)(Y)₃ spin systems, which, moreover, exhibit several NOESY cross-peaks to each other (shown schematically in Fig. 1). The spin topology, their significant dipolar shifts, and their inter-residue contacts uniquely identify these two residues as Met¹⁰³(FG5) and Val¹⁰¹(FG3). An upfield, resolved, strongly relaxed methyl peak with no TOCSY connectivities exhibits strong NOESY cross-peaks to the terminus of the AMXPT spin system, locating the CH₃, which, together with the NOESY cross-peak to His⁹⁷(F8) (shown schematically in Fig. 1), confirms the assignment of Met¹⁰³(FG5). A Tyr ring with contacts to Val¹⁰¹(FG3) identifies Tyr⁴³(C7).

Orientation of the Heme-- The Phe⁴⁴(CD1) ring exhibits NOESY cross-peaks to both the 1-CH₃ and 8-CH₃ of the heme (Fig. 3, C and D) and not to the 5-CH₃ and 4-vinyl, as predicted by the crystal structure (Fig. 1A), clearly establishing that the heme is oriented differently from that in the crystal by a 180 ° rotation about the ,-meso-axis (Fig. 1B). This conclusion is further confirmed by detecting the characteristic dipolar contact between Met¹⁰³(FG5) and the 1-CH₃ (rather than the 4-vinyl predicted by the crystal structure), as shown schematically in Fig. 1, and between Val¹⁰¹(FG3) and 8-CH₃ (rather than the 5-CH₃, predicted by the crystal structure). Hence all subsequent assignments are determined by using the crystal structure as a guide but with the heme rotated by 180 ° about the ,-axis, as shown in Fig. 1B.

Distal Pocket Residues-- The extreme low field, strongly relaxed (T₁ = ~11 ms) labile proton peak at 22 ppm does not participate in a NOESY map, but when it is saturated (Fig. 2C), it exhibits a strong NOESY cross-peak to a two-proton signal under the 8-CH₃. This signal under the 8-CH₃ in turn exhibits a TOCSY cross-peak to 9.0 ppm. The strong relaxation of the labile proton and the intercept in Curie plots for the TOCSY detected fragment uniquely identify the complete ring of Tyr³⁰(B10). A strong NOE to the Phe⁴⁴(CD1) CH, together with the latter T₁ pf ~20 ms, yields, with Equation 2, a 2.8 ± 0.3 Å estimate for the Tyr³⁰(B10) OH to Phe⁴⁴(CD1) CH distance. Similarly, the relaxation properties (T₁ = ~24 ms) of an upfield shifted labile proton dictate that it must arise from the only other residue that can place labile protons so close to the iron, the terminal N₂H of Gln⁶⁴(E7).³ Saturation of this peak results in a very strong (~15%) NOE to a proton near 4 ppm. The large NOE from this upfield labile proton to 4 ppm identifies the latter as the geminal partner of the saturated peak. The strong NOE from Tyr³⁰(B10) OH to the 4 ppm Gln N₁H confirms the assignment and argues for the assignment of the 4 and 6 ppm peaks to the N₁H and N₂H (see below). The ratio of the steady-state NOEs to the 4 ppm Gln⁶⁴(E7) NH₁ peak upon saturating the Gln⁶⁴(E7) NH₂ and Tyr³⁰(B10) OH (~0.5), together with the fixed ~1.9 Å distance between N₁H and N₂H, leads to a 2.0 ± 0.2 Å estimate for the Tyr³⁰(B10) OH to Gln⁶⁴(E7) N₁H distance.

Additional assignments (data not shown), include two upfield resolved methyls (one strongly relaxed) that are part of a five-spin system diagnostic of a Ile, with a strongly relaxed CH that exhibits the NOESY cross-peaks to 5-CH₃ and 4-vinyl (as predicted by the 180 ° rotated heme) for Ile⁶⁸(E11); this residue exhibits the expected NOESY cross-peaks to the Tyr³⁰(B10) ring (shown schematically in Fig. 1). Common NOESY contacts to 4-vinyl and 5-CH₃ for a TOCSY-detected Ala uniquely identify Ala⁷¹(E14). TOCSY spectra detect three Phe rings with weak hyperfine shifts. They are assigned to Phe³⁴(B14), Phe⁶⁰(E3), and Phe¹⁴⁰(H15) based on their predicted dipolar contacts to Phe⁴⁴(CD1) and Tyr³⁰(B10), only to Phe⁴⁴(CD1), and to 3-CH₃, respectively. Two low field shifted TOCSY-detected fragments with slopes and intercepts indicative of aromatic protons, together with NOESY cross-peaks to Met¹⁰³(FG5) identify the C₁H-C₁H and C₂-C₂H portions of Trp¹⁰⁸(G5); the remainder of the ring protons could not be located because of likely strong relaxation and near degeneracy with other protons and in position under the residual solvent peak. The observed inter-residue and residue-heme dipolar contacts are summarized in Fig. 1B. Spectral congestion precluded further assignments. The assignments, chemical shifts, and T₁ values for the residues described in Fig. 1 are listed in Table II.

Magnetic Axes-- The orientation of the magnetic axes² was found to be essentially independent of the selection of input data or whether the anisotropies were also determined or held constant at the values determined for sperm whale metMbCN (50, 51). The resulting orientation of  is defined by  = 29.5° ± 1.0 ° (tilt from the heme normal),  = 159 ± 10 ° (direction of tilt projected on the heme plane), and  =  +  = 59 ± 10 ° (rhombic axes projected on the heme plane). The residual error function, F/n, is small in all cases (~ 0.05 ppm²), and the resulting correlation between observed and calculated dipolar shifts is very good, as illustrated in Fig. 4. The magnitude of the tilt of the major magnetic axis, z, from the heme normal (z' axis),  = ~30 °, is nearly twice as much as that observed previously in cyanomet globins (34, 50, 51). The large magnitude of the tilt of the major magnetic axis from the heme normal indicated by the complete magnetic axes determination also reveals itself clearly in the analysis of the dipolar shift pattern for individual residues. Thus the nodal surface for the axial dipolar shift can be mapped by considering the magnitude and direction of dipolar shifts of residues near the nodal surface. The plots in Fig. 5 for the protons whose shift direction/magnitude reflect primarily the axial geometric factor node are shown as a function of tilt angle . The agreement with the experimental shifts is acceptable within 30 ± 10 °.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Plot of dip(obs) (via Equation 6) versus the dip(calc) (via Equation 5) for A. suum metHbCN at 30 °C obtained from the optimized magnetic axes with ax = 2.3 × 108 m3/mol, rh = 0.46 × 108 m3/mol,  = 159 °,  = 29.1 °, and  = 59 °. The open circles represent the data used as input to determine the magnetic axes. The points for the Gln NH2 and Tyr(B10) ring protons, which were not used as input, are shown by closed squares and closed circles, respectively. The solid line represents unit slope for a perfect fit.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Plot of predicted dipolar shifts for three protons whose shift directions depend critically on the magnitude of the tilt, , of the major magnetic axis, z, from the heme normal, z'. , Phe44(CD1) CHs; , Val101(FG3) CH; , Val101(FG3) CH. The predicted values at the optimized  ~30° compare very well with the observed dipolar shifts at ~30 ° for each proton.

The Orientation of Tyr³⁰(B10) and Gln⁶⁴(E7)-- Predicted _dip values for the Tyr³⁰(B10) ring and Gln⁶⁴(E7) NHs, based on the HbO₂ crystal coordinates (8), are included in Fig. 4 as filled circles and filled squares, respectively. It is observed that the uniquely placed protons on the Gln(E7) N₂H result in shifts that are well predicted, indicating that this residue in met HbCN maintains the same orientation relative to the iron as in HbO₂. The R_Fe = 4.5 ± 0.4 Å estimated from the T₁ = 25 ms for the Gln⁶⁴(E7) N₂H is consistent with the crystallographic R_Fe = 4.1Å. Moreover, the predicted dipolar shifts for the Gln⁶⁴(E7) nonlabile side chain protons are small and are consistent with the likely appearance of protons in the poorly resolved and very crowded aliphatic envelope. In the case of Tyr³⁰(B10), the dipolar shifts are very well predicted for the ring, which is consistent with conserved ₁, ₂ angles with respect to HbO₂. The placement of the proton on the hydroxyl oxygen crystal coordinates, however, unlike the NHs of Gln⁶⁴(E7), is not unique. Hence the _dip(calc) (Fig. 6A), distance to Phe⁴⁴(CD1) CH (Fig. 6B)_, distance to Gln⁶⁴(E7) N₁H (Fig. 6C), and distance to the iron, R_Fe (T₁ = 10 ms, R_Fe = 4.0 ± 0.4 Å) (Fig. 6D) for the Tyr³⁰(B10) OH are calculated as a function of the dihedral angle between the H-O-C and ring planes, ₃, as illustrated in Fig. 6; the observed values are shown by shaded regions. It is clear that each of the four observable values are optimally predicted for the angle ~20 ° in Fig. 6, which leads us to conclude that we have uniquely spatially located the labile proton for the distal Tyr³⁰(B10).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Plot of predicted parameters for the Tyr30(B10) hydroxyl proton as a function of rotation, 3, of the dihedral angle between the H-O-C and Tyr ring planes. A, dip(calc) as obtained from the optimized magnetic axes. BD, distances from Tyr30(B10) hydroxyl proton to the Phe44(CD1) CH proton (r(Y30 OH-CH F44)) (B); the Gln64(E7) N1H (r(Y30 OH-N1H Q64)) (C); and the iron (RFe) (D). The experimental estimates for each of these parameters are shown by shaded ovals.

Mobility of the Tyr³⁰(B10) Ring-- The Tyr³⁰(B10) CH resonance overlaps, at least in part, the 8-CH₃ peak (Fig. 2A) over the accessible temperature range but appears to broaden selectively as the temperature is lowered. The line broadening, however, can be quantitated by observing only the steady-state NOE for the averaged CH peak upon saturating the Tyr³⁰(B10) OH, as shown in Fig. 7. Plotting the ln (linewidth) versus reciprocal temperature shows a plot with selective increase in slope at low temperature for the CHs peak, which yields an estimated exchange contribution of 20 Hz at 30 °C. This value, together with the _dip(calc) for the individual Tyr³⁰(B10) CHs, results in an estimated shift difference of 4.4 ppm, which at 500 MHz, results in a reorientation rate of 5 × 10⁶ s¹ using the standard equation for chemical exchange in the first exchange limit (57).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   The steady-state NOE difference traces that show the NOE to the Tyr30(B10) CH peak upon saturating the Tyr30(B10) OH proton at 15 (A), 20 (B), and 35 °C (C). A plot of ln (linewidth) versus T1 for the CH () and 5-CH3 () plot is shown in (D).

	DISCUSSION

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Heme Pocket Molecular and Electronic Structure-- The pattern of the heme methyl contact shifts has been proposed to largely reflect the orientation of the axial His imidazole ring relative to a heme pyrrole-Fe-pyrrole axis (12, 58-61). For an axial His oriented along such an N-Fe-N axis, large contact shifts are predicted and observed primarily for the pyrroles normal to the His plane. Thus the contact shift patterns among different globins are modulated separately by the orientation of the His relative to the heme and the orientation of the heme about the ,-meso axis. In cases where the axial His is oriented close to meso-Fe-meso vectors (11, 62), the four pyrroles exhibit comparable contact shifts (13, 63). A. suum metHbCN, like mammalian globins, exhibits large contact shifts for 1-CH₃ and 5-CH₃, arguing for orientation of axial His along the N_B-Fe-N_D vector of the heme if the heme and the axial His⁹⁷(F8) were orientated similarly. However, the heme methyl contact shift pattern in A. suum metHbCN is achieved by completely different means than in sperm whale metMbCN. Thus, as shown in Fig. 1, the axial His ring (as viewed from the proximal side) is rotated by ~60 ° ( = 65 °) relative to that in sperm whale Mb ( = 6 °), which should result in larger 3-CH₃, 8-CH₃ than 1-CH₃, 5-CH₃ contact shifts, if the heme were seated in the pocket the same as in sperm whale Mb. However, the rotation of the heme by 180 ° about the ,-meso axis, when compared with sperm whale Mb, reverses this pattern and leads to larger 1-CH₃, 5-CH₃ contact shifts than 3-CH₃, 8-CH₃ contact shifts. Thus the fortuitous similarity in the heme contact shift pattern in A. suum metHbCN and mammalian globins is due to off-setting influences of the differences in the orientation of both the axial His and the heme.

The determined heme methyl contact shifts, together with the x-ray determined axial His orientation, thus independently confirm that the heme in A. suum metHbCN in solution is rotated by 180 ° about the ,-meso axis relative to that reported in the HbO₂ crystal structure. These results also suggest that caution should be exercised in assigning a heme orientation based on heme methyl contact shifts in a cyanomet globin unless the orientation of the axial His is known. The re-evaluation of the x-ray diffraction data to reconcile the alternate heme orientation in the crystal and solution has shown that the heme in the crystal is, in fact, rotated by 180 ° about the ,-meso axis from that originally reported (8)⁴ and the same as found by ¹H NMR in solution.

Theoretical considerations (61, 64) confirmed in model compounds (65, 66) dictate that if the orbital ground state is determined by the axial His(F8) bonding, the rhombic axes, , and the angle between the heme N-Fe-N and imidazole plane,  (Fig. 1C), obey the counter-rotation rule where  = . The present results conform quite well to these predictions, as shown in Fig. 8. The temperature dependence of the heme methyl shifts reveals that the 1-CH₃, 5-CH₃ exhibit positive slopes that are steeper than Curie (T¹) behavior, whereas the 3-CH₃, 5-CH₃ exhibit slopes that are negative or exhibits anti-Curie behavior. This effect is expected on the basis of thermal population of the excited orbital state, where the lone spin on the iron becomes delocalized into pyrroles B and D (60, 67-69). Lastly, the magnetic axes reported above allow the determination of _dip for the axial His, which, in turn, provides _con for each of the positions, as shown in Table I. Thus only the CH exhibits large contact shifts that are very similar to those reported for sperm whale metMbCN (70) and confirms an essentially conserved axial His-Fe bond in A. suum relative to sperm whale Mb.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Plot of  (orientation of axial His relative to the heme plane) against  ( +   180 °) for sperm whale myoglobin (51) (), Aplysia limacina myoglobin (40) (), Scapharca inequivalvis hemoglobin (63) (), and A. suum hemoglobin (). The line represents a perfect counter-rotation model.

Distal Hydrogen Bonding Network-- The excellent correlation between the observed and crystal-structure predicted values for _dip and T₁ for the Tyr³⁰(B10) ring and Gln⁶⁴(E7) N₂H side chain shows that their dispositions in metHbCN are essentially quantitatively conserved relative to those in the HbO₂ crystal structure (8). The position of the Tyr³⁰(B10) hydroxyl proton, deduced from its relaxation, NOESY, and dipolar shift constraints, with an ₃ value of ~20 °, is precisely in the position to make an ideal H bond to the strongly tilted cyanide ligand. The short interproton distance between the Tyr³⁰(B10) OH and Gln⁶⁴(E7) NH₁, moreover, is consistent with an H bond between the latter proton and the Tyr hydroxyl O. Hence the present ¹H NMR data provide strong support that both Tyr³⁰(B10) and Gln⁶⁴(E7) serve as a H bond donors to bound cyanide ligand to in a manner that is essentially the same as for the bound O₂ in HbO₂. It is concluded that cyanide serves as a valid model for the H bonding experienced by a ligated O₂ molecule.

Distal Pocket Crowding-- Flash photolysis experiments have suggested a compact and constrained heme pocket for A. suum Hb (19). Significant crowding in the distal pocket is evident in two ¹H NMR spectral parameters. The major magnetic axis (Fe-CN tilt) is tilted from the heme normal by ~30 °, nearly twice as much as in other globins (34, 39-41, 51, 63). This can be rationalized by the disposition of the Tyr³⁰(B10) ring, which provides a steric barrier to ligation along the heme normal. The orientations of the Tyr³⁰(B10) ring and the Fe-CN tilt (if only tilted and not primarily bent) determined herein place the two residues in van der Waals' contact between the Tyr O and the N of the bound cyanide. However, the tilt of the major magnetic axis (~30 °) is in the same direction (toward pyrrole C), as is the tilt of the proximal His⁹⁷(F8) imidazole plane (by ~10 °) observed for HbO₂ (8), so that the large tilt in the major magnetic axis, and hence the Fe-CN tilt, could have a significant contribution (to ~10 °) from the proximal His tilt (32). The present results suggest that the crystal structure of A. suum HbCO would find the CO off axis to a degree that is much larger than found in other carbonyl globins. The role of the tilt for the axial His(F8) in contributing to either Fe-CO (32) or Fe-CN tilt could be addressed by either the crystal structure of the carbonyl complex or the solution ¹H NMR determination of the magnetic axes of the cyanomet complex, for the A. suum Hb mutant where the covalent connection between the axial imidazole and the F-helix backbone is severed in the His(F8)  Gly mutant (71, 72), allowing an exogenous imidazole to bind in the preferred normal to the heme.

Aromatic rings in the heme pocket of globins are generally found with sufficient local flexibility to yield only rotationally averaged ¹H NMR signals (37, 73), despite the apparent close packing suggested by the crystal structures. Thus, Phe(CD1) is generally found packed tightly against the heme surface but nevertheless exhibits an ¹H NMR spectrum that is rapidly averaged by the 180 ° ring flips. The Tyr³⁰(B10) ring exhibits an averaged NMR spectrum, but the rotation contributes significantly to the linewidth, and standard analysis in the fast exchange limit (57) using the _dip(calc) for the individual CHs results in a rotation rate of ~1 MHz. A comparison can be made to globins with Phe rather than Tyr(B10) and with a Gln(E7), i.e. elephant Mb and the sperm whale Leu²⁹(B10)  Phe/His⁶⁴(E7)  Gln and Leu²⁹(B10)  Phe/His⁶⁴(E7)  Gln/Val⁶⁸(E11)  Phe Mb mutants, for which the B10 ring exhibited "normal" linewidth indicative of much faster reorientation (39, 51). Whether the constraints on the Tyr³⁰(B10) ring in A. suum Hb result from "pinning down" the extremity via the H bond to the ligand or from the tight van der Waals' contacts with the aromatic ring is not known but could be elucidated in a comparison of the solution ¹H NMR spectra of WT and Tyr(B10)  Phe A. suum mutant Hb.

Conclusions-- The present NMR data provide support that the heme pocket of A. suum Hb is highly constrained, as evidenced by larger tilts from the heme normal for Fe-CN than previously observed and slow reorientation of the Tyr³³(B10) ring. The heme is shown to be rotated by 180 ° about the ,-meso axis relative to that originally reported in the crystal (8), and the pattern of heme methyl contact shifts is shown to be consistent with the deduced heme orientation. The Tyr³³(B10) and Gln⁶⁴(E7) side chain labile protons in met HbCN are located at essentially the same positions as found in the HbO₂ crystal and hence provide H bonds to the bound cyanide and establish that the metHbCN is a valuable structural model for aspects of both HbO₂ and HbCO. However, although the Fe⁺³-CN unit can serve as limited structural models for the Fe⁺²-CO and Fe⁺²-O₂ units in globins, cyanide ligation rates unfortunately are not functionally relevant to O₂ or CO binding. This is due to the fact that free cyanide at physiologic pH range is protonated, so that both the on- and off-rates involve protonation/deprotonation steps that are strongly influenced by local pocket polarity that modulates the cyanide pK. Thus the cyanide on- and off-rates directly relate to neither distal steric nor H bonding effects (28, 29).

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL16087 (to G. N. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry, University of California, 1 Shields Ave., Davis, CA 95616-5295. Tel.: 530-752-0958; Fax: 530-752-8995; E-mail: lamar@indigo.ucdavis.edu.

^§ Recipient of a Burroughs Wellcome Fund Scholar Award in Molecular Biology.

² It is noted that the conventions for x', y', and z' differ from that used previously (33, 34, 40, 50) by a 45 ° rotation (see text) in the heme plane and referencing  to the +x', rather than x' axis, so that (new) = (old), (new) = (old) + 135 °, and (new) =  (old)  45° (51).

³ Denotation of NHs of Gln⁶⁴ was based on the x-ray structure (8).

⁴ F. S. Mathews, personal communication.

	ABBREVIATIONS

The abbreviations used are: Mb, myoglobin; metMbCN, cyanide complex of ferric myoglobin; metHbCN, cyanide complex of ferric hemoglobin; Hb, hemoglobin; NOESY, two-dimensional nuclear Overhauser spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; DSS, 2,2-dimethyl- 2-silapentane-5-sulfonate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES