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J Biol Chem, Vol. 275, Issue 2, 742-751, January 14, 2000

Solution ¹H NMR Study of the Influence of Distal Hydrogen Bonding and N Terminus Acetylation on the Active Site Electronic and Molecular Structure of Aplysia limacina Cyanomet Myoglobin^*

Bao D. Nguyen, Zhicheng Xia, Francesca Cutruzzolá^§, Carlo Travaglini Allocatelli^§, Maurizio Brunori^§, and Gerd N. La Mar^¶

From the  Department of Chemistry, University of California, Davis, California 95616 and the ^§ Dipartimento di Scienze Biochimiche, University of Rome "La Sapienza," "A. Rossi Fanelli" P. le A. Moro, 5, I-00185 Rome, Italy

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution ¹H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small ~2° rotation of the axial His in rWT relative to true WT and a ~3° rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Myoglobin (Mb)¹ is a member of the globin family of proteins of approximately 140-150 residues that encapsulate heme and exhibit a remarkably strongly conserved fold of seven to eight helices (A-H) despite a high variability in sequence (1-3). The heme is wedged between the E and F helices, and only the axial (proximal) His F8 (eighth residue on helix F) and Phe CD1 (first residue on the loop between the C and D helices) are completely conserved. This conserved globin fold, however, results in a very wide range of functionality, which appears to be controlled primarily by the nature of the "distal" residues at position E7, E11, E10, and B10 that line the ligand binding pocket (4). The major interaction that strongly stabilizes O₂ over CO binding has been shown to involve H-bonding to the bound O₂ by a distal residue, although destabilization of the bound CO by distal steric interaction cannot be completely discounted (4-6). Although the distal H-bond in vertebrate globins is always provided by residue E7 (which is overwhelmingly His, but occasionally Gln (2)), there is much more variation in the nature of the E7 residue and the position of the distal H-bond donor in nonvertebrate globins (3). Such alternate residue H-bond stabilization of the bound O₂ has been established in natural globins from sea hares (Arg E10) (7) and trematodes (Tyr B10) (8-10) and in synthetic globins at position E11 (Asn and Thr) (11).

An effective strategy for determining the role of individual residues is to perform functional and molecular structural studies of site directed mutant globins (4). X-ray crystallography is generally the most effective tool for describing both the global and heme pocket structures of globins (4, 6, 12). The description of the globin active site structure, however, is just as effectively pursued by NMR, particularly in the paramagnetic oxidation/spin states, in which structurally exquisitely sensitive hyperfine shifts impart improved active site resolution over a diamagnetic analog (13-15). Hyperfine shifts reflect electronic and/or magnetic properties of the chromophore, and hence can be extraordinarily sensitive to (and hence render detectable) small structural changes that are unlikely to be detected in either a crystal structure or a solution NMR structure of a diamagnetic analog. Perhaps the best examples of the exquisite sensitivity are the observation of isotope effects on iron-porphyrin covalency due to the distal H-bond to the bound ligand (16-18) and the characterization of small populations of globins with alternate orientation of the heme about the ,-meso axis (19, 20).

The information content is particularly rich in the cyanomet globins, in which the bound cyanide can model both the H-bond acceptor properties of bound O₂ (21-23) and the potential steric tilt/bend of bound CO (17, 24-28). The large dipolar shifts, moreover, guarantee that any heme pocket labile proton can be detected (usually resolved), and its role in H-bonding to the ligand elucidated directly by its placement in the distal pocket (17, 22, 23, 26, 28), and indirectly by the expected influence of such a distal H-bond on the electronic structure of the heme (16-18). The pattern of the dominant heme methyl contact shifts reflect the orientation of the axial His relative to the heme (i.e.  in Fig. 1) (29-33), and the pattern of the dominant meso-H dipolar shift reflects the orientation of the rhombic magnetic axes ( in Fig. 1) (34, 35). The mean of the heme methyl contact shifts has been shown to be sensitive to distal H-bonding to bound cyanide in models (36), but it has not yet been assessed in globins. Lastly, the dominant dipolar shifts for nonligated residues provide information on the orientation of the paramagnetic susceptibility tensor, which can be related to the tilt/bend of the Fe-CN unit and the orientation of the axial His and thereby facilitate the determination of the orientation of mutated residues in the heme cavity (17, 21, 22, 24-26, 28).

The Mbs from the sea hare Aplysia limacina (37), like those from Dolabela auricularia (38), possess a Val E7 but still exhibit high O₂ affinity and reasonably slow O₂ off-rates. Both crystallography (12) and solution ¹H NMR on Aplysia Mb (22, 39) and solution ¹H NMR of Dolabela Mb (40) have demonstrated that Arg E10 can orient into the heme pocket and provide an H-bond to bound ligand. The sharp increase in k_off and decrease in affinity for O₂ in the Aplysia R(E10)T-Mb mutant directly confirms (7) the H-bonding role for this residue. A question that naturally arises is whether the alternate H-bonding residues in sperm whale and Aplysia Mbs can be interchanged solely by interchanging the Val E7/Arg E10 in Aplysia with the His E7/Thr E10 of sperm whale Mb. Similar studies have shown that substitution of the key distal residues can in small or large part transfer an unusual functional property from one globin to another (17, 26, 28). Thus, although the O₂ off-rate increases sharply and its O₂ affinity decreases sharply upon substituting His E7 by Val in sperm whale Mb, a significant portion of the O₂ affinity can be recovered by inserting Arg E10 in the sperm whale H(E7)V/T(E10)R-Mb mutant (41). Solution ¹H NMR of sperm whale H(E7)V/T(E10)R-metMbCN found that Arg E10 side chains oriented to provide an H-bond to the bound cyanide (22). Hence, the distal H-bonding scheme of Aplysia Mb can be transferred, albeit less effectively, to sperm whale Mb. The successful cloning and expression of Aplysia Mb has been reported (7), although the N terminus of the recombinant protein, in contrast to true wild-type (12), is not acetylated (7). Substitution of Arg E10 for the Thr found in mammalian Mb led to very large decrease in O₂ affinity and increase in O₂ off-rate and autoxidation and clearly identified Arg E10 as the source of the H-bond stabilization to the O₂ ligand (7). However, engineering the second residue to convert the Aplysia Mb to mimic the sperm whale Mb pocket, V(E7)H/R(E10)T-Mb, failed to either recover a significant fraction of the O₂ affinity or retard the O₂ off-rates relative to WT Aplysia Mb.

We report herein on the ¹H NMR spectra of the cyanomet complex of the Aplysia mutants V(E7)H-Mb and V(E7)H/R(E10)T-Mb that show that the individual distal His E7 residues are indeed oriented out of the heme pocket and hence cannot participate in any H-bonding interaction with the bound cyanide. On the other hand, comparison of the ¹H NMR spectra of native wild-type (WT) and recombinant wild-type (rWT) Aplysia metMbCN shows that the N-acetylation missing in rWT Mb (7) leads to changes in both the relative stability of the alternate heme orientations in the heme pocket and a small reorientation of the proximal His imidazole plane relative to the F-helix, when compared with true WT.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein Preparation-- A. limacina V(E7)H and V(E7)H/R(E10)T mutant myoglobins were expressed and purified as described previously (7). Cyanide complex of ferric Mb (metMbCN) samples were made by exchanging the protein with a ²H₂O or 90% ¹H₂O and 10% ²H₂O solution containing 50 mM NaCl, 10 mM KCN, 50 mM K₂HPO₄-KH₂PO₄ at pH 8.2 in an Amicon ultracentrifuge cell. The final solutions had a protein concentration of ~1.5 mM.

¹H NMR Spectra-- ¹H NMR spectra were collected on a GE Omega 500 MHz spectrometer. The strongly relaxed signals were optimally detected in WEFT spectra (42). Nonselective T₁ values for the resolved strongly relaxed protons were measured via inversion-recovery experiments. Steady-state NOEs were recorded as described in detail previously (43). The phase-sensitive TOCSY (44), NOESY (45), and conventional magnitude COSY (46) employed the method described by States et al. (47) to provide quadrature detection in the t1 dimension. Solvent suppression, when required, was achieved by direct saturation in the relaxation delay period. 512 blocks were collected with 25.0 kHz spectral width to include all resonances and 10 kHz to improve resolution for the diamagnetic envelope. 128-256 scans were accumulated with a repetition rate of 0.7 or 1.2 s¹ for each block with free induction decays of 2048 complex points. The data were processed as described previously (22); details are given in the figure legends. All two-dimensional data were processed on Silicon Graphics workstation using the software package Felix from Biosym/MSI (San Diego, CA).

Determination of Magnetic Axes-- The magnetic axes were determined as described in detail previously (21, 22, 24-26, 28, 34, 35). Experimental dipolar shifts (n values) for the structurally conserved proximal side of the heme were used as input to search for the Euler rotation angles, (, , ), which transform the molecular pseudosymmetry coordinates (x', y', z' or R, ', ' (Fig. 1)), readily obtained from crystal coordinates (12), into magnetic axes x*, y*, and z*, (where  is diagonal) by minimizing the global error function, F/n,

(Eq. 1)

where _dip(calc) and _dip(obs) are given by

(Eq. 2)

and

(Eq. 3)

respectively. _ax and _rh are axial and rhombic anisotropies, and _DSS(obs) is the observed chemical shift referenced to DSS. _dia is the shift in the isostructural diamagnetic complex that is calculated via _dia = _tetr + _sec + _rc, where _tetr is the shift in an unfolded tetra peptide (48); _sec is the shift of an amino acid proton typical for -helices, -strand, coils, etc. (49); and _rc is the ring current shift (50). Minimizing the error function F/n in Equation 1 was performed over three parameters, , , and , using the _ax and _rh from WT metMbCN (22) or extended to all five parameters to yield both the Euler angles and anisotropies, using the A. limacina MbF crystal coordinates (12), as described in detail previously (22).

Dipolar Shift Simulations-- The position of a substituted or perturbed residue can be determined by minimizing a local error function (22-24, 26, 28). This local error function, designated F*(residue)/n' (where n' is the number of protons) to distinguish it from that global error function in Equation 1 is given by the following equation,

(Eq. 4)

where _dip(₁, ₂ . . . ) represents the _dip(calc) as a function of a bond rotations ₁, ₂ . . . using the magnetic axes derived from conserved structural elements. The bond angle that minimizes the residual error function F*(residue)/n' defines structural changes, as described in detail previously (17, 22-24, 26, 28). The molecular modeling was carried out on a Silicon Graphics Indigo work station from available crystal coordinates using the Insight II program (Biosym/MSI).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heme Orientational Heterogeneity-- ¹H NMR spectra for both metMb (51) and metMbCN (19, 20) had shown that in solution, ~20-25% of the globin possesses a heme rotated 180° about the ,-meso axis with respect to the unique orientation reported in the crystal structure (12) (shown in Fig. 1). The metMb spectra at pH 6.0 exhibit low field heme methyl peaks very similar to those of WT (51). However, as shown in the ¹H NMR spectra for the lowest field pair of heme methyls of WT, rWT, V(E7)H/R(E10)T-metMb and V(E7)H-metMb in Fig. 2, the relative intensities of the major component (M) to minor component (m) methyl peaks is similar at a 5.0 ± 0.5:1.0 ratio in WT and rWT, but somewhat lower, with a 3.5 ± 0.3:1.0 ratio, for the two mutants. The upfield portion of equilibrated cyanomet complexes of Aplysia WT, rWT, V(E7)H/R(E10)T-Mb and V(E7)H-Mb ¹H NMR spectra, where the vinyl H peaks (Fig. 3, H₂ for major components and h₂ for minor components) for both isomers resonate (19, 20), are illustrated in Fig. 3, A-D, respectively, and reveal equilibrium major to minor isomer ratios of 3.3:1.0, 2.1:1.0, 3.3:1.0 and 3.3:1.0, respectively; thus, the population of the minor component is clearly greater in rWT than WT protein.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the crystal structure-based reference coordinate system, x', y', z'; the magnetic coordinate system, x*, y*, z*, in which the paramagnetic susceptibility tensor is diagonal; and the electronic coordinate system, x, y, z, in which dxz, dyz are eigenfunctions, as determined by the axial His orientation relative to the x' axis with angle . The reference and magnetic coordinate systems are related by the Euler rotation (, , ) according to (x*, y*, z*) = (x', y', z')(, , ), where  is the tilt of the major axis from the heme normal (not shown),  is the angle between the projection of the tilt on the heme plane and the x' axis (not shown), and  ~  +  corresponds to the location of the rhombic magnetic axes relative to the x' and y' axes (shown). Theoretical considerations demand that  = . Panel A shows the effect of a counterclockwise rotation of the axial His by relative to heme and protein, with the result that the new  =   , and the new  =   . Panel B shows the effect of a counterclockwise rotation of the heme by relative to a stationary axial His and protein matrix. The new  =  + , but the new , still referenced to the original axial x', y', must be represented as  =  + 2.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Low field portion of the 500 MHz 1H NMR spectra showing the low field pair of methyls for both the major (M) and minor (m) heme orientation for the WT (A), rWT (B), V(E7)H/R(E10)T-metMb (C), and V(E7)H-metMb (D) complexes in 1H2O at pH 6.0 and 30 °C.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Upfield portion of the 500 MHz 1H NMR spectra showing the vinyl proton peak for the major (H2c and H2t) and minor (h2c and h2t) isomer of WT (A), rWT (B), V(E7)H/R(E10)T-metMbCN (C), and V(E7)H-metMbCN (D) complexes in 1H2O at pH 8.2 and 25 °C.

The resolved portions of the 500 MHz ¹H NMR spectra of Aplysia native WT, rWT, V(E7)H/R(E10)T-metMbCN, and V(E7)H-metMbCN in ¹H₂O are illustrated in Fig. 4, A-D, respectively, with the previously reported (20) methyl and single proton peaks labeled M_i and H_i for the major component and m_i and h_i for the minor component (where i is either the heme position in Fig. 1 or the position on His⁹⁵(F8)). The spectra are all very similar, as noted previously for mutants and WT sperm whale metMbCN complexes (17, 23-26, 28, 34). However, even cursory examination of Fig. 4, A and B, reveals that the spectra of Aplysia WT and rWT metMbCN differ not only in the ratio of the two isomers noted above, but also in the chemical shifts of heme and axial His resonances, with the heme 1-CH₃ and 5-CH₃ resonating further to low field, and the 3-CH₃ and 8-CH₃ appearing further to high field in rWT than WT metMbCN, clearly showing that the heme electronic structure differs for WT and rWT. This difference in shifts is maintained throughout the alkaline pH range (19). We consider here the ¹H NMR spectral properties and heme pocket structure only for the major isomers in solution for each metMbCN complex; extensive and detailed assignments for the major isomer and some for the minor isomer of WT metMbCN have been reported previously (20, 22).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   20 to 9 and 1.5 to 7 ppm portions of the 500 MHz 1H NMR spectra of WT (A), rWT (B), V(E7)H/R(E10)T-metMbCN (C), and V(E7)H-metMbCN (D) in 1H2O at pH 8.2 and 25 °C. Insets to each trace are for WEFT spectra, which better define the positions of the two axial His ring peaks. Resonances are labeled Mi and Hi for the major and mi and hi for the minor methyl and single proton heme resonances, respectively (where i corresponds to the heme position in Fig. 1). The resolved signal for the axial His95(F8) for the major isomer is also labeled.

metMbCN Assignments-- The procedure for obtaining complete assignment of the heme and the heme cavity residues have been presented in detail for Aplysia WT metMbCN (20) and applied to both WT and numerous mutants of sperm whale Mb (17, 23-26, 34, 35). Hence, NMR data are shown only to substantiate changes in molecular structure from that of WT metMbCN. The complete heme resonances were assigned as reported previously (20) by detecting the characteristic NOESY connections among four low field methyls, two TOCSY-detected three-spin fragments (vinyls), two TOCSY-detected four-spin systems (propionates), and the four strongly relaxed meso-H signals, each with large, temperature-dependent hyperfine shifts. The heme chemical shifts for the four complexes of interest are listed in Table I.

Standard sequential (52) assignments for Aplysia V(E7)H/R(E10)T-metMbCN locate three segments with the inter-residue NOESY contacts characteristic of three -helical fragments, labeled I, AMX_i-Ala_i+1-Z_i+2-Z_i+3-AMX_i+4-Val_i+5; II, AMX_j-Z_j+1-Z_j+2-Thr_j+3-Ile_j+4-AMX_j+5-Z_j+6-Z_j+7; and III, AMX_k-Z_k+1-AMX_k+2-Val_k+3 (Z residue with more than three nonlabile proton signals). Segment I must arise from Phe⁹¹(F4)-Val⁹⁶(F9), with AMX_i+4 exhibiting the large, low field dipolar shifts characteristic of His⁹⁵(F8), as confirmed by the NOE to the peptide NH from a strongly hyperfine-shifted and relaxed labile proton at 14 ppm readily identified as the His⁹⁵(F8) NH (20). The residues exhibit all the NOESY contacts predicted from the WT crystal structure (12), with the exception of small differences in intensity for His F8 protons as considered in detail below.

Fragment II, the TOCSY/NOESY data of which are shown in Fig. 5, is identified by the sequence as due to His⁶³(E7)-Arg⁷⁰(E14), with His⁶³(E7) and Ile⁶⁷(E11) exhibiting significant dipolar shifts, and with residues E10, E11, and E14 exhibiting the NOESY cross peak pattern for rWT connectivities that is identical to that of WT (20) and very similar but not identical (see below) for the mutants relative to rWT. The largest dipolar shifts are displayed by the His⁶³(E7) CHs. Although the NHCHCH₂ fragment of His⁶³(E7) could be unambiguously identified in the two mutants, it was not possible to locate the cross-peak to the ring CH. Lastly, helical segment III arises from Phe¹⁰⁵(G5)-Val¹⁰⁸(G8), with the residues exhibiting NOESY contacts to the heme in all three complexes as observed for WT (20). The complete substituted Thr E10 in the double mutant was readily located by TOCSY; spectral congestion prevented assignment of more than the NHCHCH₂ fragment of Arg⁶⁶(E10) in the single mutant. The dipolar-shifted residue at two interhelical corners, Phe⁴³(CD1), Phe⁹⁸(FG2), and Val¹⁰⁰(FG4), were identified by TOCSY spectra, characteristic paramagnetic relaxation, and the NOESY contacts to the heme predicted by the crystal structure and previously reported for Aplysia WT metMbCN (20). The chemical shifts for nonligated residues are listed in Table II.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Portion of the TOCSY (A) (m = 50 ms) and NOESY (B) (m = 100 ms) spectra of Aplysia V(E7)H/R(E10)T-metMbCN in 1H2O at pH 8.2 and 25 °C showing the sequential assignment for the backbone of helix E residues His63(E7)-Ala70(E14), including the NHCHCH2 portion of His63(E7), F helix residues Gln90(F3)-Val96(F9), and G helix residues Phe105(G5)-Arg108(G8). The NOESY data were processed by applying 30°-shift sine-bell-squared window over 1024 t1 × 256 t2 points prior to zero-filling to 2048 × 1048 data points and Fourier transformation. The TOCSY data were processed by applying an 30°-shifted sine-bell-squared window over 512 t1 × 256 t2 points prior to zero-filling to 2048 × 1048 data points and Fourier transformation.

Structural Differences between WT and rWT-- The difference in heme methyl contact shifts between WT and rWT dictates (see under "Discussion") a difference in the orientation of the axial His⁹⁵(F8) imidazole plane relative to the heme (29, 30, 32, 33), with  in Fig. 1 predicted to decrease (rotate counter-clockwise in Fig. 1A) in rWT relative to WT. However, the altered heme methyl contact shift pattern does not distinguish between rotation of the heme relative to a stationary His⁹⁵(F8) (Fig. 1B) and rotation of the His⁹⁵(F8) imidazole ring relative to a stationary heme and protein (Fig. 1A). These alternate movements, however, can be distinguished by changes either in distance between heme methyls (i.e. 1-CH₃) and the E helix backbone (i.e. Ile⁶⁷(E11) CH) to detect heme rotation (24, 34) or between the His⁹⁵(F8) NH and the F helix to detect rotation of His⁹⁵(F8), as described in detail previously (34).

Saturation of the heme 1-CH₃ peak in WT and rWT metMbCN leads to identical NOEs to Ile⁶⁷(E11) CH, as shown in Fig. 6, A and B, dictating that the heme orientation relative to the protein matrix is the same in WT and rWT. On the other hand, saturating the His⁹⁵(F8) NH signal in WT and rWT reveals numerous differences in the pattern of NOEs, as shown in Fig. 7. Quantitative comparison of the NOEs is complicated by overlap of the His⁹⁵(F8) NH for both the major and minor isomers in each globin and the fact that the His⁹⁵(F8) CH shifts differ, but also overlap for the two isomers, rendering the interpretation of the NOEs to the CH peaks ambiguous. Analysis of the WT crystal structure reveals that a small 2-3° rotation of His⁹⁵(F8) ring does not significantly alter the distance between His⁹⁵(F8) NH and Phe⁹¹(F4) CH, but increases the His⁹⁵(F8) NH distance to His⁹⁵(F8) CH and decreases the His⁹⁵(F8) NH distance to the Phe⁹¹(F4) ring protons for counter-clockwise change in , shown in Fig. 1A. Comparison of panels A and B of Fig. 7 shows that the NOE to His⁹⁵(F8) CH is significantly larger and to the Phe⁹¹(F4) ring protons is significantly smaller, in rWT compared with WT, establishing that His⁹⁵(F8) rotates counter-clockwise in rWT relative to WT. The magnitude of the NOEs intensity differences are consistent with a His ring rotation of ~2°.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Portion of the 500 MHz 1H NMR NOE difference spectra in 1H2O at pH 8.2 and 25 °C upon saturating the resolved 1-CH3 signal in WT (A), rWT (B), and V(E7)H/R(E10)T-metMbCN (C). The NOE difference spectra reflect identical 1-CH3 intensity, allowing comparison of NOE intensity among the three complexes. Note significantly enhanced intensity to Ile67(E11) in the double mutant relative to either WT or rWT metMbCN.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   The 5.5-11 ppm spectral window of 500 MHz 1H NMR NOE difference spectra in 1H2O at pH 8.2 and 25 °C upon saturating the axial His95(F8) NH signals in WT (A) and rWT (B) Aplysia metMbCN. The spectra reflect the same intensity of the NH resonance allowing comparison of the NOEs to axial His95(F8) CH and Phe91(F4) ring CHs. Note larger NOE to His95(F8) CH and smaller NOEs to the Phe91(F4) ring CHs in rWT relative to WT metMbCN.

Heme Orientation in the Mutants-- Differences in the meso-H hyperfine shift pattern (34) between WT and H(E7)V/R(E10)T-metMbCN indicate (see under "Discussion") that the relative rotational position of the heme and axial His differ in rWT and the double mutant metMbCN. Saturation of the His⁹⁵(F8) NH signal does not shed light on whether the axial His ring has rotated because the key Phe⁹¹(F4), Ala⁹²(F5), and His⁹⁵(F8) CH signals are degenerate (not shown). However, saturation of the heme 1-CH₃ signal in the double mutant leads to a ~30% larger NOE to Ile⁶⁷(E11) CH than in rWT (Fig. 6C), and the implied decrease in the distance of 5% requires a 3° counter-clockwise rotation of the heme in the mutant relative to conserved His⁹⁵(F8) and protein matrix, as illustrated in Fig. 1B. The heme rotational position of the single mutant was found unchanged from that of rWT on the basis of the magnitude of the 1-CH₃ NOE to Ile⁶⁷(E11) CH (not shown).

Determination of Magnetic Axes-- The magnetic axes for rWT and WT Aplysia metMbCN are essentially identical, as expected from the fact that the dipolar shifts for nonligated residues are largely indistinguishable (Table II), with  = 65°,  = 7.0°, and  = 25° (and with uncertainties of ± 10°, ±1°, and ± 10°, respectively) with _ax = 2.38 × 10⁸ m³/mol and _rh = 0.55 × 10⁸ m³/mol, as reported previously² (22). Both five-parameter and three-parameter searches based on the WT (35) _ax and _rh using a variety of input data sets yielded highly clustered angles for each mutant with that using the same 21 proximal side protons yields  = 75°,  = 11°, and  = 35° for V(E7)H/R(E10)T-metMbCN and  = 95°,  = 8.5°, and  = 25° for V(E7)H-metMbCN, with the optimized anisotropies in the five-parameter search inconsequentially altered from those of WT. In each case, the correlation between _dip(obs) and _dip(calc) was excellent not only for the input data protons but also for the majority of the distal side, excluding the mutated residues (not shown).

Orientation of Distal His⁶³(E7) and Thr⁶⁶(E10) in Mutants-- Introduction of Thr⁶⁶(E10) into Aplysia metMbF crystal structure (12) with the orientation as found in sperm whale carbonmonoxymyoglobin (6) resulted in an excellent correlation between _dip(obs) and _dip(calc) (not shown). In contrast, the _dip(obs) for one His⁶³(E7) CH in both mutants is much larger than predicted by the His⁶³(E7) orientation of sperm whale carbonmonoxymyoglobin. Plots of the residue error function (Equation 4) for the CHs of His⁶³(E7) as a function of ₁ are shown in Fig. 8 for V(E7)H/R(E10)T-metMbCN (line A) and V(E7)H-metMbCN (line B). A His⁶³(E7) orientation in the heme pocket such as in sperm whale Mb corresponds to ₁ = 163°, as shown by the vertical arrow on the left, which predicts shifts clearly inconsistent with those observed in either mutant. However, the plots in Fig. 8 reveal a clear minimum, where ₁ ~ 40° for each mutant.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Plot of the residue error function, F*/n, as a function of 1, for the distal His63(E7) CH2 of V(E7)H/R(E10)T-metMbCN (line A) and V(E7)H-metMbCN (line B) using the magnetic axes determined for each mutant. The 1 value for the distal His orientated into the distal pocket as in sperm whale MbCN is shown by a vertical arrow at 1 = 163°.

	DISCUSSION

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Heme Cavity Structure of the Sperm Whale Mb Mimic-- The dipolar shifts for the His⁶³(E7) in the Aplysia Mb mutants show that ₁ is rotated by ~120° from that found in sperm whale Mb (6). The magnitude and direction of the change in ₁ orients the imidazole ring out of the heme pocket and in the direction of the protein surface. The temperature dependence of the _dip(obs) for the His⁶³(E7) CHs correlates well with that for other dipolar shifted protons (21), indicating that the His⁶³(E7) orientation is relatively well defined by ₁ ~ 40° and does not represent any equilibrium between an "in" and "out" orientation, as observed in crystals of Chironomus HbIII (53). The His⁶³(E7) orientation deduced herein for the mutant metMbCN complexes is completely consistent with the observation (7) of low O₂ affinity and rapid O₂ off-rate indicative of the absence of significant H-bond stabilization of the bound O₂. Superposition of the crystallographically defined heme cavities of sperm whale (6) and Aplysia (12) Mb indicates that the His E7 CH is ~1.1 Å closer to the iron in Aplysia than sperm whale Mb, such that the His E7 "in" orientation in Aplysia Mb, like that of sperm whale, could not be accommodated in the ligated state.

Effect of N-Acetylation on WT Mb Structure-- A logical link between the orientation of the His⁹⁵(F8) ring and N-acetylation in Aplysia Mb is found in the unique, direct interaction between the N terminus and the beginning of the F helix in the form of an H-bond between Leu² NH to the Ala⁷⁸(EF) carbonyl at the end of the E-helix, as depicted (12) in Fig. 9. Deletion of the acetyl group (7) can be expected to seriously impact this interaction. The absence of N-acetyl group in rWT metMbCN leads to ~0.3 Kcal/mol decrease in the relative stabilization of the major versus minor heme orientation and a ~2° counterclockwise rotation for the axial His plane relative to WT metMbCN. The change in the ratio of the heme orientations demands a small difference in the contacts between the protein and pyrroles A and B of the heme, and the rotation of the His suggests a small translation of the F-helix relative to the heme (rather than heme relative to the F-helix) (see below).

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Schematic representation of the position of the F-helix relative to the heme face and the position of the H-bond between the N terminal Leu2 NH and the carbonyl of Ala78 at the EF corner. The arrow over the heme indicates the direction of movement of the F-helix (by 0.07 Å) relative to the heme face that would result in a counterclockwise rotation (of ~2°) of the axial His ring in order to maintain an unstrained Fe-His bond.

Rotation of the His⁹⁵(F8) about ₂ in the Aplysia Mb crystal structure shows that the ring N ligated to the iron translates laterally by ~0.03 Å per 1° counterclockwise rotation of the axial His, in the direction indicated by the arrow in Fig. 9. The conserved contact shifts for the axial His argue against such a distortion for the iron-His bond. The ~2° axial His imidazole rotation, however, would leave the axial His bond intact if the F-helix translated relative to the heme by ~0.07 Å in the direction of the -meso position as shown by the arrow in Fig. 9. Such a movement of the central portion of the F-helix is consistent with a ~0.15 Å movement of the N terminus of the F-helix in the same direction. The direction of the movement of the F-helix terminus would suggest that the deletion of the N-acetyl group either strongly destabilizes or abolishes the H-bond between the N terminus and the EF corner in rWT. It is noted that such a small translation of the F-helix could account for the altered stabilities for the two heme orientations, inasmuch as Phe⁹¹(F4) represents an important contact with pyrrole A.

Heme Methyl Shifts as Indicator of Distal H-Bonding-- It has been observed that the heme mean methyl hyperfine shift in low spin hemin models with ligated cyanide are systematically dependent on the H-bond strength of the solvent, with decreasing H-bond donation leading to increasingly upfield shifted heme methyl shifts (36). Comprehensive published assignments of sperm whale WT (43) and mutants (17, 23, 24, 26), in fact, support a correlation between heme mean methyl shift and presence of labile protons in contact with the bound ligand, as shown in Table III. Sperm whale H(E7)V-metMbCN exhibits a ~1.1 ppm upfield bias relative to WT for the mean methyl shift, and the shift of H(E7)Q mutant moved slightly to the high field of WT, confirming a likely weaker interaction for Gln than His. Similarly, the addition of second H-bond (albeit weaker) via Tyr²⁹(B10) results in a ~0.8 ppm low field bias to the shift.

The _DSS(CH₃) in Aplysia V(E7)H/R(E10)T-metMbCN is found 0.8 ppm to upfield of WT Aplysia metMbCN, confirming the absence of a distal H-bond. Interestingly, Aplysia V(E7)H-metMbCN exhibits a _DSS(CH₃) intermediate between WT and the double mutant, suggesting the possibility that the Arg⁶⁶(E10) may have a weak interaction with the distal ligand. The labile proton for Arg⁶⁶(E10), observed clearly in both WT (22) and rWT Aplysia metMbCN, was not detected in the V(E7)H-metMbCN NMR spectrum. However, even a weak H-bond or only a fractional populated, dynamic H-bond would lead to rapid exchange with solvent. Indeed the O₂ off-rates were found to be very similar for both the single and double mutants (7).

The present data confirm a valuable role of the variable heme mean methyl shift as an indicator of differentiation distal H-bonding among a series of mutant metMbCN complexes. However, because it has been shown that the heme mean methyl shift varies with the axial His orientation (33), as defined by  in Fig. 1, it may serve as an indicator of distal H-bonding among a series of point mutants, but not among a series of natural genetic variants that exhibit significantly different , such as Aplysia ( = 22°) and sperm whale ( = 5°) metMbCN.

Effect of His/Heme Rotation on ¹H NMR Spectral Parameters-- The perturbed NOE pattern between heme and the protein matrix or axial His and protein matrix indicate that  differs slightly between WT and rWT metMbCN and between rWT and V(E7)H/R(E10)T-metMbCN. Such changes in  are expected (32, 54) to lead to changes in  (Fig. 1). The values of  obtained from the magnetic axes, however, have ±10° uncertainties (35) and indicate that  for the four complexes of interest is the same within the uncertainties. Nevertheless, changes in  and  manifest themselves in extraordinarily sensitive manners in the heme hyperfine shift patterns (30-34). It is recognized that the asymmetry in the heme methyl hyperfine shift pattern is dominated by the contact interaction that imposes strong heme methyl shifts dependence of the on the His/heme orientation defined by  in Fig. 1. In contrast, it has been shown (34, 55) that the asymmetry of the heme meso-H shifts is dominated by the rhombic term of the dipolar shift, which reflects  in Fig. 1. Theoretical considerations (54) lead to the expectation that  = . The changes in heme methyl shifts going from WT  rWT metMbCN, based on published modeling of the contact shift (33), estimate a ~2° counterclockwise rotation of .

For the meso-H, the hyperfine shift asymmetry can be cast in the form,

(Eq. 5)

as discussed previously (34). The observed values for the four metMbCN complexes are included in Table I. The calculated value, (meso-H)_calc, is obtained using the dipolar shifts predicted by the magnetic axes (34), as follows.

(Eq. 6)

For WT metMbCN, (meso)_calc = 5.26 ppm, which is in excellent agreement with the observed value of 5.21 ppm in Table I. It is noted, however, that in rWT metMbCN, (meso)_obs becomes more positive to 4.94, which corresponds to a calculated (meso-H) with  decreased by ~2° ( in Fig. 1A). Hence, the change in pattern of methyl contact shifts and meso-H dipolar shifts independently confirm both the 2° decrease in  detected by altered NOEs of His⁹⁵(F8) NH to the F helix and the counter-rotation rule (54) that demands that  = . With the exception of the heme and axial His, only very minor shift differences are observed between WT and rWT metMbCN (see Table II). The observed shift changes, moreover, correlate well in the direction and, at least qualitatively, in magnitude with changes in predicted dipolar shifts due to a change in  by 2°, as shown in Fig. 10.

View larger version (14K): [in this window] [in a new window]   Fig. 10.   Comparison of the difference in observed shifts, (DSS(obs)rWT-DSS(obs)WT), and the difference in predicted dipolar shifts, (dip(calc)rWT-dip(calc)WT), between WT and rWT A. limacina metMbCN due only to the ~2° difference in rhombic magnetic axes that result from the rotation of the axial His plane in rWT relative to WT Mb. The solid symbols represent the His95(F8), and the open markers are for nonligated residues.

Evidence for a change in  in comparing rWT and V(E7)H/R(E10)T-metMbCN is difficult to detect in the heme methyl contact shift pattern, because the mean shift decreases by ~0.8 ppm on abolishing the distal H-bond (see above). The (meso-H)_obs, nonetheless, is much more negative in the double mutant (6.16 ppm) than in rWT (4.94 ppm), and this difference is accounted for in Equation 6 by a ~4-5° increase in . Again, the changes in  are in opposite direction to the changes in , and both confirm the direction of the rotation of the heme. It is noted that  ~ 4-5° is larger than the  ~ 3° deduced from the NOE data. However, as detailed consideration shows in Fig. 1B, if the heme rather than the axial His rotates, the experimental , defined with respect to the original reference coordinates x',y', must change by 2 for a net heme rotation by .

Conclusions-- Solution ¹H NMR spectra of Aplysia metMbCN show that the insertion by mutagenesis of His E7 in both V(E7)H-Mb and V(E7)H/R(E10)T-Mb mutants is oriented out of the heme pocket and not able to provide a stabilizing H-bond to bound ligands. Comparison of WT and rWT metMbCN, moreover, shows that the abolished N-acetylation in rWT leads to both a change in the relative stabilities of the alternate heme orientations and a small change in the orientation of the axial His ring in each isomer. It is shown that the rotation of axial His in rWT and of the heme in the double mutant relative to WT lead to a series of changes in the asymmetry of the heme methyl dominant contact shifts and heme meso-H dominant dipolar shifts that are completely consistent with the currently accepted relationship between the active site molecular and electronic structures. Lastly, analysis of the heme mean methyl shift among sperm whale and the present Aplysia metMbCN mutants reveals that it serves as a valuable empirical indicator of distal H-bonding to cyanide. The facility with which the electronic structure changes can be related to small, but possibly functionally relevant, changes in molecular structure, is a testament to the exquisite sensitivity of hyperfine shifts to molecular structure.

	FOOTNOTES

^* This research was supported by National Institutes of Health Grant HL 16087 (to G. N. L.) and by Grant 97.04083.CT04 from the CNR of Italy (to M. B.).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. Tel.: 530-752-0958; Fax: 530-752-8995; E-mail: lamar@indigo.ucdavis.edu.

² It is noted that the convention for x', y', z' differs from that used previously for A. limacina metMbCN (22) by a 45° rotation in the heme plane and referencing of the  to the +x' rather than the x' axis (35), so that (new) = (old), (new) = (old) + 135°, and (new) = (old) 45°.

	ABBREVIATIONS

The abbreviations used are: Mb, myoglobin; DSS, 2,2-dimethyl-2-silapentane-5-sulfonate; metMbCN, cyanide complex of ferric myoglobin; NOE, nuclear Overhauser effect; NOESY, two-dimensional nuclear Overhauser spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; WT, wild-type; rWT, recombinant WT; V(E7)H/R(E10)T-Mb, Val⁶³(E7)  His/Arg⁶⁶(E10)  Thr-Mb; V(E7)H-Mb, Val⁶³(E7)  His-Mb.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES