©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Double Mutant of Sperm Whale Myoglobin Mimics the Structure and Function of Elephant Myoglobin (*)

(Received for publication, April 24, 1995)

Xuefeng Zhao K. Vyas Bao D. Nguyen Krishnakumar Rajarathnam Gerd N. La Mar Tiansheng Li (§) George N. Phillips, , Jr. Raymund F. Eich (¶) John S. Olson (**) Jinshu Ling David F. Bocian

From the  (1)Department of Chemistry, University of California, Davis, California 95616 (2)Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892 (3)Department of Chemistry, University of California, Riverside, California 92521-0403

ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, ^1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K.(1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln(E7) substitution lowers oxygen affinity 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O(2) affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.


INTRODUCTION

Myoglobin is a small, 16-18-kDa, globular hemeprotein which stores molecular oxygen in muscle cells. Because of the similarity of their tertiary structures, myoglobin is frequently used as a model for the alpha and beta subunits of tetrameric hemoglobin. The key physiological properties of myoglobin are its high affinity for oxygen and low rate of spontaneous oxidation to the inactive ferric form (Antonini and Brunori, 1971). Studies of naturally occurring and mutant hemeproteins have shown that ligand affinity and autooxidation are affected markedly by the chemical nature of the amino acids in the heme pocket. In order to understand these functional properties, the molecular structures of a large number of myoglobins and hemoglobins have been determined by x-ray crystallography and solution NMR studies. These studies have been pursued along two lines. Traditionally, naturally occurring genetic or species variants with distinctive functional properties have been investigated (i.e. Bolognesi et al.(1989), Steigmann and Weber(1979), Vainshtein et al.(1978), Yu et al.(1990), Qin and La Mar (1992), and Vyas et al.(1993)). More recently, site-directed mutagenesis has been used either to test structural hypotheses (Olson et al., 1988; Braunstein et al., 1988; Carver et al., 1990; Ikeda-Saito et al., 1991; Balasubramanian et al., 1993; Li et al., 1994; Ling et al., 1994; Huang and Boxer, 1994) or to explore systematically the physiological roles of individual residues (Rohlfs et al., 1990; Egeberg et al., 1990; Smerdon et al., 1991, 1993; Carver et al., 1991, 1992; Quillin et al., 1993, 1995; Lai et al., 1995). A review of the mutagenesis literature is given in Springer et al.(1994).

Dene et al.(1980) sequenced Asian elephant myoglobin and reported that it contains a Gln at position 64 (E7) rather than a His, which is found at this position in all other mammalian myoglobins. This replacement appeared to be neutral since the O(2) affinity and autooxidation rate of elephant myoglobin are very similar to those of pig, human, and, sperm whale Mb (^1)(Table 1, Romero-Herrera et al., 1981; Springer et al., 1994). Romero-Herrera et al.(1981) concluded that in elephant myoglobin, N of Gln(E7) forms a hydrogen bond with bound oxygen which is equivalent in strength to that formed with His(E7) in all other mammalian myoglobins. However, when this idea was tested directly by constructing the His(E7) Gln mutation in recombinant sperm whale myoglobin, the results indicated that this substitution is not neutral. The H64Q mutation causes a 5-fold decrease in K



Studies aimed at understanding why H64Q sperm whale myoglobin does not mimic the functional and spectral properties of elephant myoglobin have been pursued by two approaches. First, La Mar and co-workers (Yu et al., 1990; Vyas et al., 1993) have carried out high resolution ^1H NMR studies of both diamagnetic carbonyl and paramagnetic cyano-met complexes of elephant Mb and compared the results to native sperm whale myoglobin as a standard. The spectra show that, in addition to features for the expected distal Gln(E7), there are also signals which strongly indicate that a phenylalanine side chain is in van der Waals contact with bound ligand in the elephant protein.

Second, Olson, Phillips, and co-workers (Carver et al., 1992; Gibson et al., 1992; Springer et al., 1994; Li et al., 1994) have carried out a systematic study of point mutations at position 29 (B10) in sperm whale myoglobin. Substitution of Phe for Leu(B10) in sperm whale myoglobin leads to a dramatic increase in O(2) affinity and marked decrease in autooxidation rate (Carver et al., 1992). The crystal structure of this mutant shows that C of the Phe side chain makes van der Waals contact with bound ligands. The Fe-O-O complex is stabilized by favorable electrostatic interaction with the positive edge of the phenyl multipole, accounting partly for the 15-fold increase in affinity shown by the L29F mutant. The large Phe side chain also excludes water from the distal pocket of the deoxy form of the mutant. (^2)The favorable multipole and water exclusion effects also inhibit protonation of the Fe-O-O complex, preventing disproportionation into Fe and HO(2) (Brantley et al., 1993).

In an effort to construct a blood substitute prototype, Olson and co-workers (Brantley et al., 1993; Olson, 1994) constructed a double mutant in which Phe was put at the 29 position to inhibit autooxidation by excluding water from the active site and Gln was put at the 64 position to lower O(2) affinity by weakening hydrogen bonding to the bound ligand. Later, it was discovered that this active site configuration, Phe(B10)/Gln(E7), was predicted to occur in elephant Mb by Yu et al.(1990) even though the published sequence for the elephant protein lists a Leu at the B10 position (Dene et al., 1980). To resolve this problem and test the conclusions of Yu et al.(1990), we have compared in detail the structural, spectral, and functional properties of elephant myoglobin and the L29F/H64Q sperm whale mutant. Rate constants for O(2) and CO binding and autooxidation were determined by conventional kinetic techniques. Infrared spectra of the CO complexes of wild-type, L29F, H64Q, and L29F/H64Q sperm whale myoglobins were compared with that of elephant MbCO. Resonance Raman techniques were used to measure the and bands for the same set of mutants and native proteins. The structure of the active site of the cyano-met form of the sperm whale double mutant was determined by solution ^1H NMR and compared with that of the single mutants and elephant myoglobin. Finally, the structure of L29F/H64Q sperm whale MbCO was determined by x-ray crystallography for comparison with the active site parameters determined by NMR and with the crystal structure of native elephant metmyoglobin recently obtained by Bisig et al.(1995).


MATERIALS and METHODS

Site-directed Mutagenesis and Protein Purification

A detailed description of the mutagenesis of sperm whale myoglobin was given by Carver et al.(1992). Briefly, the PstI-KpnI fragment of pMb413a (Springer and Sligar, 1987) was subcloned into pEMBL19 plasmids (Boehringer Mannheim) for mutagenesis, sequencing, and expression. The product of these manipulations was designated pEMbS-1. The Kunkel method of oligonucleotide-directed mutagenesis was used to construct the original L29F mutant. The double H64Q/L29F sperm whale mutant was then constructed using the E-helix cassette described by Springer et al.(1989) to make the original H64Q single mutant. The resulting plasmid was sequenced by the dideoxy method as described by Hattori and Sakaki(1986) using a United States Biochemical Corp. sequencing kit. Plasmids containing the mutant gene were transformed into the Escherichia coli TB-1 (Life Technologies, Inc.), expressed constitutively, and purified as described by Springer and Sligar(1987) (see also Lai et al.(1995)). The recombinant myoglobins were concentrated to 1 to 2 mM and stored in liquid nitrogen.

Overall Kinetic Measurements

Association and dissociation rates for O(2) and CO binding were determined using conventional laser flash photolysis and stopped-flow rapid mixing techniques as described in detail by Rohlfs et al.(1990). Equilibrium association constants for O(2) and CO binding were computed as the ratios of the overall rate constants. Autooxidation rates were measured using the techniques described by Brantley et al.(1993). Wild-type sperm whale myoglobin expressed in E. coli has been shown to be identical with native sperm whale myoglobin in terms of tertiary structure and kinetic properties (Springer et al., 1994). Thus, the numerical values presented for wild-type sperm whale myoglobin also apply to the native protein.

Infrared and Resonance Raman Spectroscopy

For the infrared absorption measurements, approximately 30 µl of the MbCO solution (3-5 mM heme) was added slowly to a 1-mm cuvette to obtain a uniform film. The cuvette consists of 2 CaF(2) windows separated by a 56-µm spacer and was purged with nitrogen gas immediately before the sample was added. Spectra were recorded at 2 cm resolution in the region 1800-2100 cm using a Mattson Galaxy 6020 spectrometer interfaced with a Compaq 386 computer. Up to 10,000 interferograms were collected for all samples and the corresponding buffer controls. The final IR spectra were corrected for buffer background by digital subtraction of the sample and control data.

For the resonance Raman measurements, the reduced CO forms of the proteins were prepared by flushing deoxymyoglobin samples with 1 atm of CO under rigorously anaerobic conditions. The cyano-metmyoglobin samples were prepared by oxidation with ferricyanide and addition of potassium cyanide, and then excess KCN and K(3)Fe(CN)(6) were removed by ion exchange chromatography. Approximately 100 µM samples were placed in a quartz spinning cell to prevent photodissociation in the case of MbCO and photoreduction in the case of MbCN (this was judged negligible as measured by the absence of any discernible contribution from the (4) mode of the photoproduct). The resonance Raman spectra were acquired using a triple spectrograph (Spex 1877) equipped with a liquid nitrogen cooled 1152 298 pixel charge-coupled device (Princeton Instruments LN/CCD with an EEV chip) as the detector. Excitation was provided by the 415.4 nm output of a krypton ion laser (Coherent Innova 200-K3). The laser power was typically 5 milliwatts, and the spectral resolution was 4 cm.

^1H NMR Measurements

Cyano-metMb (cyano-metmyoglobin) samples for ^1H NMR were made by adding K(3)Fe(CN)(6) to oxidize the protein, followed by ion exchange chromatography to remove the ions and concentration in an Amicon ultrafiltration cell. The final solution was 4 mM heme in 50 mM NaCl, 10 mM KCN, 50 mM sodium phosphate, pH 8.6; 10% ^2H(2)O was added for the lock.

All the ^1H NMR spectra were collected on the GE Omega 500 MHz spectrometer. To effectively observe rapidly relaxing signals, the slowly relaxing diamagnetic envelope was suppressed by the WEFT pulse sequence (Gupta, 1976). Nonselective T(1)s for the resolved strongly relaxed protons were measured via the inversion-recovery experiment. Steady-state NOEs were recorded as described in detail previously (Emerson and La Mar, 1990a); the ratio of the steady-state NOEs to a common proton upon saturating the 1-CH(3) and 8-CH(3) groups was obtained from the ratio of the amplitudes of the NOEs in the difference traces (Rajarathnam et al., 1993). The phase-sensitive TOCSY (Braunschweiler and Ernst, 1983; Davis and Bax, 1985; Rance 1987), NOESY (Jeener et al., 1979), and magnitude COSY (Bax, 1982) measurements employed the same parameters, and were processed in the same fashion, as described in detail previously (Rajarathnam et al., 1992, 1993, 1994; Qin and La Mar, 1992).

Magnetic Axes Determination

The magnetic axes were determined as described in detail previously (Emerson and La Mar, 1990b; Rajarathnam et al., 1992, 1993; Qin et al., 1993a). Experimental dipolar shifts for the structurally conserved proximal side of the heme were used as input to search for the Euler rotation angles, R(alpha, beta, ), that transform the molecular pseudosymmetry coordinates (x`, y`, z` or r, `, ` (Fig. 1)) readily obtained from crystal coordinates into magnetic axes, x, y, z, by minimizing the global error function:


Figure 1: Orientation of the coordinate system, x, y, z, for the magnetic susceptibility tensor , relative to the symmetry (x-ray crystallographic) coordinate system x`, y`, z`. A, tilt of the major magnetic axes, z, from the heme normal, z`, given by the angle beta; note that since the z axis is positive on the proximal side, the Fe-CN tilt is defined by the -z axis. B, projection of the tilt of the z axis on the heme plane, defined by the angle alpha from the symmetry coordinate system x` axis, and location of the rhombic magnetic axes, x and y, whose projection on the heme plane is related to the symmetry axes, x`, y`, by the angle kappaalpha + , where alpha, beta, and are the Euler angles that transform the symmetry coordinates into the magnetic coordinates. The angle and modulus r define lateral movement of the E-helix. The arrows labeled a, b, c, d, and e correspond to the Fe-CN orientation (-z direction) in WT- H64V-, H64Q-, L29F-, and L29F/H64Q-cyano-metmyoglobin, respectively.



where

and

Delta, Delta are axial and rhombic anisotropies, and is the observed chemical shift referenced to 2,2-dimethyl-2-silapentane-5-sulfonic acid. is the shift in the isostructural diamagnetic MbCO complex (Dalvit and Wright, 1987; Chiu, 1992) or calculated for protons whose are not available by using the equation (Qin et al., 1993):

where is the shift of an amino acid proton typical for alpha-helices, beta-strand, coils, etc. (Wishart et al., 1991), and is the heme-induced ring current shift of the proton based on the WT coordinates by using the eight-loop model (Cross and Wright, 1985). Minimizing the error function F/n in was performed over three parameters, alpha, beta, , using available Delta and Delta, or extended to all five parameters to yield both the Euler angles and anisotropies as described in detail previously (Rajarathnam et al., 1992).

Distal Pocket Structure by NMR

The position of the E-helix was assessed by the relative intensities of the steady-state NOEs from the heme 1-CH(3) and 8-CH(3) to Val(E11) CH and Ala(E14) CH(3), as described in detail previously (Rajarathnam et al., 1993). The orientation of Gln(E7) was obtained by determining the sequential bond angles, starting with alpha-beta, that optimally reproduced via and the R(alpha, beta, ), Delta, Delta, (obs) () for the residue, as described elsewhere in detail (Qin et al., 1993a). For Phe(B10), the side chain orientation was determined separately by finding the sequential bond angles that correctly reproduce both the relative iron-induced relaxation (T(1)) and (obs), as described previously (Qin et al., 1993a; Rajarathnam et al., 1993). Distances to the iron for proton a, R(a), was obtained from:

where a known proton, i.e. His(F8) NH with R(a) = 5.0 Å and T = 31 ± 3 ms, yields R(b) when T is measured. The molecular modeling was carried out on a Silicon Graphics personal IRIS for the MbCO structure using the MIDAS program.

X-ray Crystallography

Crystallization was carried out in concentrated ammonium sulfate solution using the batch method described by Phillips et al.(1990). The CO form of the double mutant crystallized in the hexagonal P6 space group with one molecule per asymmetric unit. Diffraction data were collected with a Rigaku R-AXIS IIC imaging plate system as described elsewhere (Quillin et al., 1993). The unit cell dimensions were a = b = 91.39 Å, c = 45.87 Å. The data set was 97% complete to a resolution of 1.7 Å, comprising 151,315 total measurements with 24,699 unique reflections. R was 10.9%.

The coordinates of wild-type MbCO were used as the starting model in the refinement of the double mutant structure (Protein Data Bank entry 1MGK, Brookhaven National Laboratory; Quillin et al. (1993)). Initial difference maps were calculated using the program X-PLOR (Brunger et al., 1989). The Gln(E7) and Phe(B10) side chains were then introduced into the difference map using the molecular modeling program CHAIN (Sack, 1988). Constrained least-square refinements and map calculations were performed by X-PLOR using the Engh and Huber parameter set for the ferrous myoglobins (Engh and Huber, 1991). After several cycles of refinement, manual refitting, and solvent placement, the crystallographic R-factor converged to 17.3% with root mean square bond deviations of 0.02 Å. Coordinates for the L29F/H64Q MbCO structure have been deposited in the Brookhaven Protein Data Bank (1MCY).


RESULTS

O(2)and CO Binding and Autooxidation

The rate and equilibrium constants for ligand binding to wild-type, H64Q, L29F, and L29F/H64Q sperm whale and elephant myoglobins are listed in Table 1. he last column in this table lists the corresponding autooxidation rate constants. The H64Q substitution causes a marked increase in the rate of O(2) dissociation due to weakening of hydrogen bonding between residue 64 (E7) and the bound ligand (Springer et al., 1994; Quillin et al., 1993). The net result is a 5-fold decrease in K

IR and Resonance Raman Spectra

Infrared spectra of CO bound to recombinant sperm whale and native elephant myoglobins are shown in Fig. 2. Wild-type sperm whale myoglobin shows two components in the IR spectrum of its CO complex, a major peak at 1944 cm (A substrate) and a smaller one at 1932 cm (A(3) substrate, for a review see Li et al.(1994)). The L29F mutation causes a shift to a single component at = 1932 cm. The peak position for the H64Q mutant is the same as that of wild-type sperm whale myoglobin, but there is a loss of absorbance in the 1930 cm region and an increase above 1950 cm. Both L29F/H64Q sperm whale and native elephant myoglobin show intermediate, more symmetrical peaks centered at 1938 cm.


Figure 2: Fourier transform infrared spectra of CO-myoglobin complexes at pH 7, 25 °C.



Resonance Raman spectra of the CO-myoglobin and cyano-metmyoglobin complexes in the regions of the Fe-ligand stretching vibrations ( and ) are shown in Fig. 3and 4, respectively (for a review of these modes in hemoglobins and myoglobins see Yu and Kerr(1988)). The assignments for the and modes were confirmed via experiments with CO and CN (viz. Ling et al. (1994)). The single L29F mutation causes an increase in from 509 for wild-type MbCO to 525 cm, and the single H64Q replacement causes a small decrease to 507 cm. When both substitutions are present, as in the double mutant and elephant myoglobin, is 513 cm. Similar results are observed for the Fe-CN complexes (Fig. 4). The L29F mutation causes a 7 cm increase in ; the H64Q replacement causes a 7 cm decrease; whereas the values for the double mutant and elephant myoglobin are roughly equal to that of wild-type sperm whale myoglobin. Taken together, the spectral results in Fig. 2-4 argue strongly that elephant myoglobin contains a Phe(B10) residue which interacts with bound ligands in a manner identical with that observed in the L29F/H64Q double mutant of sperm whale myoglobin. These data also emphasize the sensitivity of the Fe-CO and Fe-CN bond orders to amino acid substitutions in the distal pocket.


Figure 3: Soret-excitation resonance Raman spectra of CO-myoglobin complexes at pH 7, 25 °C.




Figure 4: Soret-excitation resonance Raman spectra of cyano-metmyoglobin complexes at pH 7, 25 °C.



NMR Structural Studies: Assignment of Conserved Residues

The 500-MHz ^1H NMR spectra for H64Q, L29F, (^3)and L29F/H64Q cyano-metmyoglobin in ^2H(2)O are shown in Fig. 5. The nonlabile proton signals for these three proteins reflect a pattern similar to, but not identical with, that of wild-type sperm whale myoglobin and other point mutants (Rajarathnam et al., 1992, 1993; Qin et al., 1993a, 1993b). The significantly altered hyperfine shift pattern, as demonstrated earlier for other point mutants, must reflect changes in the magnetic axes of the ferric ion. The strategy for assigning the heme and conserved residues has been presented and discussed in detail previously for both sperm whale and Aplysia limacina cyano-metmyoglobin (Emerson and La Mar, 1990a; Qin and La Mar, 1992; Rajarathnam et al., 1992, 1993; Qin et al., 1993a, 1993b). Representative two-dimensional NMR data for L29F/H64Q cyano-metmyoglobin are shown to assign and characterize structurally the two mutated residues Gln(E7) and Phe(B10).


Figure 5: Hyperfine-shifted portions of the 500-MHz ^1H NMR reference spectra. A, H64Q-cyano-metmyoglobin at 35 °C at pH 8.6 in ^2H(2)O; B, L29F-cyano-metmyoglobin at 35 °C at pH 8.6 in ^2H(2)O; C, L29/H64Q-cyano-metmyoglobin at 35 °C at pH 8.6 in ^2H(2)O; D, in ^1H(2)O; E, fast inversion recovery spectrum of L29F/H64Q-cyano-metmyoglobin in ^1H(2)O collected at 12 s to emphasize broad, efficiently relaxed proton signals. F, WEFT-NOE difference spectrum of L29F/H64Q-cyano-metmyoglobin upon irradiating 1-CH(3) in ^1H(2)O at 20 °C. G, super WEFT-NOE spectrum of L29F/H64Q-cyano-metmyoglobin in ^2H(2)O at 35 °C at a repetition rate of 29 s.



The ^1H NMR spectrum of L29F/H64Q-cyano-metmyoglobin in ^1H(2)O is shown in Fig. 5D. Two hyperfine shifted and strongly relaxed labile proton resonances are found in the low field window by comparison to the trace in ^2H(2)O shown in Fig. 5C. One of these signals is apparent only in a partially relaxed spectrum in ^1H(2)O which reveals a strongly relaxed NH proton overlapping the 1-CH(3) signal that is absent in the same spectra in ^2H(2)O (Fig. 5E). The super-WEFT trace of L29F/H64Q-cyano-metmyoglobin in ^2H(2)O shown in Fig. 5G suppresses the diamagnetic envelope, retains the resolved strongly relaxed proton signals for CH of Phe(B10), CH, CHs of Phe(CD1) and CH of Ile(FG5), and reveals strongly relaxed protons under the aromatic window, i.e. CH of His(FG3) and the ring CHs of His(F8).

Complete sets of resonances for the heme groups in all three mutants were assigned as described by Qin and La Mar(1992). Two series of sequential backbone NOESY cross-peaks uniquely locate the segments Leu-Thr(F4-FG1) and Lys-Ala(E6-E17) and the hyperfine shifted side chain signals (except for Gln) are located by TOCSY and COSY (Qin and La Mar, 1992; Qin et al., 1993a, 1993b). Ile(FG5) is identified by COSY and its NOESY contacts to the 4-vinyl and 5-CH(3). His(FG3) ring protons are identified by the characteristic short T(1) (20 ms) for CH and the dipolar contact of CH to 6H. A strongly relaxed (T(1) 20 ms for CH) AMM`XX` spin system with NOESY cross-peaks to 5-CH(3) locates the hyperfine shifted Phe(CD1). TOCSY and MCOSY locate three weakly hyperfine shifted Phe side chains which characteristic NOESY cross-peaks to Phe(CD1), Gln(E7) CH, and 2-vinyl/1-CH(3) identify Phe(B14), Phe(CD4), and Phe(H15), respectively (Emerson and La Mar 1990a; Rajarathnam et al., 1993). NOESY cross-peaks for 2-H and 3-CH(3) to an upfield methyl peak at -0.44 ppm with apparent COSY cross-peaks to 1.0 and 0.6 ppm locate the CH(2)-CHs of Ile(G8). Parallel studies on the two single chemical shifts for the heme model of L29F/H64Q cyano-metmyoglobin, as well as more limited data on the two single mutants, are listed in Table 2. (^4)



The proximal residues, Leu(F4)-Ala(F9), His(FG3), Ile(FG5), and His(H15), of the double mutant exhibit heme-residue and inter-residue NOESY cross-peak patterns and paramagnetic induced relaxativities that are unchanged from those observed for wild-type cyano-metmyoglobin (Emerson and La Mar, 1990a; Rajarathnam et al., 1992). Thus, the structure on the proximal side of the heme group is highly conserved in all three mutants. For the most part, the unchanged distal residues also display intra- and inter-residue NOESY cross-peak patterns and paramagnetic relaxativity very similar to those observed for wild-type sperm whale myoglobin. The exceptions represent small structural changes required to accommodate the mutated E7 and B10 residues.

Assignment of Gln(E7) and Phe(B10) Resonances

Gln(E7) in L29F/H64Q-cyano-metmyoglobin is detected in COSY spectra (Fig. 6A) as two spin fragments, NH-CH CH and CH`-CH(2), which are connected by a steady-state NOE between CH and CH (the CH-C`H COSY cross-peak is too close to the diagonal to detect). The strongly relaxed (T(1) = 13 ± 4 ms) labile proton detected under 1-CH(3) in Fig. 5E yields NOEs to Gln(E7) CHs and Thr(E10) CH(3) (Fig. 5F) These data place NH of Gln(E7) at a distance of 4.4 ± 0.2 Å away from the iron (). The geminal partner NH` is too close to the water signal to detect. The complete Gln(E7) assignment in the single H64Q mutant was obtained in a similar fashion.


Figure 6: NMR spectra of of L29F/H64Q-cyano-metmyoglobin. A, the portions of the MCOSY spectrum in ^1H(2)O, at 35 °C showing spin connectivities for the Phe(B10) and Gln(E7). The MCOSY data are are processed by applying an unshifted sine-bell-squared window over 512 T(1) 512 T(2) points prior to zero-filling to 2048 2048 data points and Fourier transformation. B, the portions of the NOESY spectrum ( = 50 ms) of L29F/H64Q-cyano-metmyoglobin ^1H(2)O, pH 8.6 at 35 °C, illustrating the cross-peaks of Phe(B10) and Gln(E7). The data were processed by applying a 30°-shifted sine-bell-squared window over 1024 T(1) 256 T(2) points prior to zero-filling to 2048 2048 data points and Fourier transformation. The box indicates that the cross-peak is observed in the other dimension while the triangles stand for the cross-peaks observed at a lower contour level.



The spectra of both L29F and L29F/H64Q cyano-metmyoglobins exhibit two additional relaxed and low-field hyperfine shifted signals with an AMM`XX` spin system for a rapidly reorienting Phe ring that must arise from Phe(B10) (Fig. 6A). The relaxation properties yield R of 4.9 ± 0.2 (T(1) = 27 ± 3 ms) for CH of Phe(B10) in both mutants, with the mean R for CHs 5.8 ± 0.2 Å (T(1) = 72 ± 7 ms) in the L29F mutant and 6.2 ± 0.2Å (T(1) = 115 ± 15 ms) in the L29F/H64Q double mutant. Phe(B10) in L29F/H64Q cyano-metmyoglobin does not exhibit NOE connectivities to heme resonances but does exhibit NOESY cross-peaks to Gly(E8), Ile(G8), Phe(B14), and Val(E11) (Fig. 6B), as well as a steady-state NOE to Gln(E7) (not shown). These NOE connections are expected from the crystal structures of both the single L29F (Carver et al., 1992) and double L29F/H64Q MbCO mutants (Table 4) and are similar to those displayed by the Leu(B10) residue in native and wild-type MbCO (Dalvit and Wright, 1987; Quillin et al., 1993). These contacts among the distal pocket residues are described schematically in Fig. 7.




Figure 7: The distal heme pocket structure of L29F/H64Q-cyano-metmyoglobin and NOE connectivities. Dashed and solid lines indicate heme-residue and inter-residue NOE/NOESY contacts, respectively.



Determination of the Magnetic Axes

Fitted Euler angles for the magnetic axes, alpha, beta, , and Delta, Delta for the three mutants and wild-type sperm whale myoglobin are listed in Table 3. The correlation between (obs) and (calc) are excellent and the residual error functions low. The results from a previous determination for H64V cyano-metmyoglobin are included for comparison. On the basis of systematic studies of variable input data sets, Rajarathnam et al.(1993) showed that the angles alpha, beta, kappa(alpha + ) vary by leq10, leq1, and leq10°, respectively, and that these values qualitatively represent the uncertainties in the angle determinations. The inclusion of anisotropies in the least square search leads to insignificant changes in the Euler angles and the anisotropies. This is consistent with the conserved g values observed in the low temperature EPR spectra of various cyano-metmyoglobin mutants (Chiu, 1992).



Solution Structure of the Distal Pocket

The NOEs from 8-CH(3) to CH of Val(E11) and CH(3) of Ala(E14) are larger in L29F/H64Q cyano-metmyoglobin than in the wild-type protein and very similar to those found in H64V cyano-metmyoglobin. In the latter mutant, these changes have been shown to arise from an 0.8-Å lateral move of the E-helix toward the iron (Rajarathnam et al., 1993). Movement of the E-helix by 0.8 Å yields an improved fit between (obs) and (calc) for the E-helix backbone protons (not shown).

The orientation of Gln(E7) in L29F/H64Q cyano-metmyoglobin was determined by searching for rotation angles, (1), (2), (3), which most accurately reproduced the magnetic axes of the single and double mutant and the observed dipolar shifts for the proton j on the fragment CH(j) as the i-j bond was rotated (). Starting with the alpha-beta bond, each subsequent search for C(j)Hs (j = beta,, etc) was carried out at the optimized angle for the previous search as described in detail by Qin et al. (1993a). The orientation of Gln(E7) in the double mutant is nearly identical with that determined for the single mutant and places NH 4.2 Å from the iron atom, a distance which is consistent with that obtained independently by relaxation data. Since this orientation determined by ^1H NMR differs inconsequentially from that found in the two crystal structures, a more global search to optimize the conformation was not pursued.

Both dipolar shifts () and relaxation data () were used as independent constraints to estimate the orientation of Phe(B10) in L29F/H64Q cyano-metmyoglobin. As shown in Fig. 8(solid squares), T(1) (CH) = 25 ± 3 ms and R(CH) = 4.9 ± 0.2 Å indicate a (1) value of -98°, while the dipolar shifts suggest a value of -95° (not shown). The optimized value for (2) when (1) is fixed at -95° is 0° (open circles, Fig. 8). The dipolar shift was found to be much less sensitive to (2), but also yielded values consistent with those obtained from relaxation data.


Figure 8: Distance versus (1) and (2) plots. The distance of the affected proton(s) from iron,^5 <R> for the averaged Phe(B10) CHs as a function of (1) (solid squares, lower scale) and (2) (open circles, upper scale) for Leu(B10) Phe substituted in the sperm whale WT MbCO crystal structure, with the E-helix movement laterally over the heme by 0.8 Å toward the iron. The values of (1) and (2) are consistent with the <R> obtained from T(1) data, as shown by dashed boxes.



High Resolution Crystal Structure of L29F/H64Q Sperm Whale MbCO

A stereo drawing of the active site of L29F/H64Q myoglobin is shown in Fig. 9A. A comparison of the exact geometries of the side chains adjacent to the bound ligand with those observed in the structures of the corresponding single mutants is presented in Table 4. The mutated side chains do not interact sterically and occupy positions in the double mutant which are nearly identical with those observed in the crystal structures of the single mutants. The bound ligand is in van der Waals contact with both N of Gln(E7) and the C atom of Phe(B10), explaining the additivity of the functional and spectral effects shown in Table 1and Fig. 2-6. There is also a close correspondence between the interatomic distances computed from the NOE intensities and the hyperfine shifts observed in the solution NMR experiments with cyano-met complexes. These results support the NMR geometry determinations and indicate that the structures of the distal pockets in solution are similar to, if not identical with, those in solution.


Figure 9: Crystal structures of L29F/H64Q sperm whale and elephant myoglobins. A, electron density map for the distal pocket of L29F/H64Q sperm whale CO myoglobin. The benzyl ring of Phe is in a position to interact directly with the bound ligand. The N atom of Gln is in a position to hydrogen-bond, albeit weakly, with the bound ligand and occupies a position similar to that of a corresponding atom in His which is found in almost all mammalian myoglobins. B, stereoplot comparison of the structures of the distal pockets of L29F/H64Q sperm whale myoglobin (black) and asian elephant myoglobin (white, Bisig et al.(1995)). The arrangements of the side chains are very similar in both proteins which serves to explain their similar functional properties. The drawings were made with MOLSCRIPT (Kraulis, 1991).



Finally, Bisig et al.(1995) have just determined the high resolution crystal structure of native Asian elephant metmyoglobin. As shown in Fig. 9B, a Phe residue is present at the 29 position in the elephant protein, and the conformations of the E7 and B10 side chains are almost identical with those in the sperm whale double mutant.


DISCUSSION

Resolution of the Elephant Mb Sequence Problem

Elephant myoglobin has represented an enigma in attempting to correlate structure with function in hemeproteins. This protein exhibits almost the same O(2) affinity and resistance to autooxidation as the more typical mammalian myoglobins from pig, man, and sperm whale even though a Gln is present at the E7 position (Table 1; Springer et al., 1994). Until the mutagenesis work of Springer et al.(1989), it was thought that this H64Q substitution was neutral and that the amide N stabilized bound O(2) to the same extent as the corresponding atom in His(E7). However, as shown in Table 1, the single H64Q mutation causes a substantial decrease in oxygen affinity and an increase in autooxidation rate in sperm whale myoglobin. This result led to a search for other amino acid replacements that might account for the differences between the properties of the single H64Q sperm whale mutant and native elephant protein.

Solution ^1H NMR studies of elephant myoglobin indicate the presence of an additional phenylalanine side chain in the distal portion of the heme pocket, designated Phe^A, which is not seen in other mammalian myoglobins. The signals associated with Phe^A indicate that C makes van der Waals contact with the bound ligands (Yu et al., 1990; Vyas et al., 1993). The decrease in size of the distal cavity and its enhanced apolar character was proposed to be the origin of the normal, low autooxidation rate of elephant myoglobin. Detailed NMR characterization of Phe^A indicated that it is located at the B10 position (Vyas et al., 1993). The difficulty with this interpretation is that the published sequence of elephant Mb lists Leu at this position, as is the case in all other known mammalian myoglobin sequences (Dene et al., 1980).

The ^1H NMR spectrum of L29F/H64Q cyano-metmyoglobin is essentially identical with that of elephant cyano-metmyoglobin (Table 2). This correspondence between elephant myoglobin and the L29F/H64Q sperm whale mutant has been examined quantitatively. The NOE contacts for Phe(B10) in L29F/H64Q cyano-metmyoglobin are identical with those previously reported for Phe^A in elephant cyano-metmyoglobin (see Fig. 7; Vyas et al. (1993)). In addition, the magnetic axes for elephant cyano-metmyoglobin are indistinguishable from those of L29F/H64Q cyano-metmyoglobin and correctly predict the dipolar shifts for both Gln(E7) and Phe^A when they are placed at position B10 in elephant myoglobin. The orientations predicted for the elephant protein are also identical with those determined experimentally in the crystal structure of the L29F/H64Q mutant of sperm whale MbCO (Table 4). Finally, the presence of Phe(B10) in elephant myoglobin has now been confirmed directly by Bisig et al.(1995) who have determined the structure of Asian elephant aquo-metmyoglobin. Thus, there is no longer a controversy; a phenylalanine is clearly at position 29 (B10) in Asian elephant myoglobin.

Interpretation of the Functional Properties of Elephant Myoglobin

The chemical mechanisms involved in O(2) and CO binding to myoglobin are presented in Fig. 10. As shown, the binding of ligand requires displacement of the distal pocket water molecule hydrogen bonded to His(E7) and in-plane movement of the iron atom to form a hexacoordinate complex. The requirement for water displacement inhibits the binding of both ligands. In the case of oxygen, this inhibition is overcome by formation of a strong hydrogen bond between N of His(E7) and the second bound oxygen atom (Fig. 10, top right). As a result, O(2) is bound much more tightly in myoglobin than in simple model heme compounds. The polar interaction between His(E7) and bound carbon monoxide is much weaker due to the apolar nature of the Fe-C-O complex and cannot compensate for the inhibitory effect of water displacement. Thus, it is the polarity of the distal histidine, and not steric hindrance, as previously believed, which discriminates in favor of O(2) and against CO binding (Springer et al., 1994).


Figure 10: Mechanism for O(2) and CO binding. The scheme was taken from Springer et al. (1994).



The effects of mutagenesis are readily explained in terms of this electrostatic mechanism. In Gln(E7) sperm whale myoglobin, the N atom of the amide side chain is farther from bound ligands and shows greater disorder in its position than the corresponding atom in the His side chain of the native protein (Quillin et al., 1993). As a result, O(2) affinity is 5-fold smaller due to weaker hydrogen bonding. The large phenyl side chain in Phe(B10) sperm whale myoglobin displaces bound water in the deoxy structure and places another partial positive charge near the bound ligand (Carver et al., 1992). Both of these effects cause a marked enhancement of O(2) binding. In both the L29F/H64Q double mutant and elephant Mb, the weakened hydrogen bonding due to the presence of Gln is almost compensated by the presence of the Phe side chain. A possible explanation for the reduced k

The currently accepted mechanism of autooxidation is shown in Fig. 11(Brantley et al., 1993). Under aerobic conditions, the dominant pathway for autooxidation is protonation of the Fe(II)-O(2) complex and dismutation into Fe(III) and the neutral superoxide radical HO(2). In native oxymyoglobin, the hydrogen bond between the neutral side chain of His(E7) and the bound ligand decreases the rate of autooxidation by preventing net protonation of the Fe-O(2) complex since the pK for forming the imidazolate anion is geq12. This same interaction also enhances O(2) affinity by roughly the same amount, accounting for the tight inverse coupling between K


Figure 11: Mechanism for autooxidation. The scheme was taken from Brantley et al.(1993).



Vibrational Properties of Bound CO

Although weak in terms of free energy, electrostatic interactions in the distal pocket are also a major determinant of the stretching frequencies observed for the Fe-C-O complex in myoglobin (Park et al., 1991; Ray et al., 1994; Li et al., 1994; and Ling et al., 1994). Positive fields enhance back bonding causing an increase in the order of the Fe-CO bond and a decrease in the order of the C-O bond as electrons are pulled into the nonbonding orbitals of the terminal oxygen atom (see Fig. 10, bottom right, and Li et al.(1994)). The increase in positive field due to the phenyl multipole in the single Phe(B10) mutant causes a decrease in and an increase in (Fig. 2Fig. 3Fig. 4). Weakening of the positive field due to the H64Q substitution causes an increase in absorbance at higher values and a shift of to lower values. Making both substitutions results in little net change in electrostatic field near the bound ligand compared to that in the wild-type protein as judged by the peak positions for and . However, the exact features of the IR and resonance Raman spectra of elephant myoglobin and the L29F/H64Q mutant are distinct from those of native sperm whale myoglobin (Fig. 2-4).

Structure of the Heme Cavity

The ^1H NMR data for the proximal side of the heme groups in all three mutants and the two native proteins indicate a highly conserved molecular structure. As found by both x-ray crystallography and solution ^1H NMR, the introduction of point mutations in the distal pocket yields an unperturbed proximal side and only local structural changes in the vicinity of the distal mutation (Carver et al., 1992; Quillin et al., 1993; Rajarathnam et al., 1992, 1993; Qin et al., 1993b). The NMR data for L29F/H64Q cyano-metmyoglobin indicate a small lateral transition of the E-helix, on the order of 0.8 Å and in the direction of the iron, as was observed previously for the H64V mutant (Rajarathnam et al., 1993). The remainder of the conserved distal pocket, including the CD corner, appears relatively unperturbed. The NOE contacts among distal residues and between each of the residues and the heme are essentially the same as those observed for wild-type sperm whale myoglobin (Fig. 7).

Gln(E7) and Phe(B10) in L29F/H64Q cyano-metmyoglobin occupy positions similar to those of His(E7) and Leu(B10) in the wild-type protein. The smaller bulk of Gln relative to His may be responsible for the small lateral movement of the E-helix. However, a comparable shift of the E-helix is observed in the crystal structure of the single L29F mutant (Carver et al., 1992). The orientation of the Gln(E7) side chain is indistinguishable when comparing both the solution and crystal structures of single and double mutants. In each case, the NH is in a position to provide a hydrogen bond to either bound cyanide in the cyano-metmyoglobin complex or O(2) in the oxy complex.

The side chain of Phe(B10) has a similar orientation in the single and double mutants of sperm whale myoglobin and in native elephant myoglobin. In all cases, CH is close enough to the iron (5 Å) to be in van der Waals contact with the bound ligand. The aromatic ring in the NMR solution structure of the double mutant appears to be rotated when compared with that in the single mutant in solution. This structural difference is suggested by the weaker paramagnetic relaxation of the Phe(B10) CHs signal (relative to CH) in L29F/H64Q cyano-metmyoglobin (T(1)(CHs) 115 ms, R = 6.2 Å) than that observed in L29F cyano-metmyoglobin (T(1)(CH) = 72 ms, R = 5.8 Å). This difference in (2) between the double and single mutants is not observed in the crystal structure (Table 4). However, more ^1H NMR data are needed to substantiate this small difference between the solution and crystal structures.

Protein Engineering and the Evolution of Elephant Mb

The L29F/H64Q mutant was originally constructed to be a blood substitute prototype with moderately low O(2) affinity, large rate constants, and increased resistance to autooxidation (Olson, 1994). The substitutions were chosen on the basis of the mechanisms shown in Fig. 10Fig. 11. The large Phe group was put at the 29 position to exclude solvent from the active site and increase the resistance to autooxidation. By itself, this single substitution is not useful because it markedly increases oxygen affinity and would cause myoglobin to inhibit cytochrome oxidase activity (Carver et al., 1992). The H64Q mutation was introduced to lower K


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants GM36243 (to D. F. B.), HL16087 (to G. N. L.), AR40252 (to G. N. P.), and GM35649 and HL47020 (to J. S. O.); Robert A. Welch Foundation Grants C-1142 (to G. N. P.) and C-612 (to J. S. O.), and grants from the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Protein Chemistry Dept., Amgen Inc., 1840 DeHavilland Drive, Thousands Oaks, CA 91320.

Recipient of a predoctoral fellowship from National Institutes of Health Training Grant GM08280.

**
To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry & Cell Biology, Rice University, MS# 140, 6100 Main St., Houston, TX 77005-1892. Tel.: 713-527-4762 or 713-527-4861; Fax: 713-285-5154; olson{at}rice.edu.

(^1)
The abbreviations used are: Mb, deoxymyoglobin; MbCO, carbonmonoxy myoglobin; NOESY, two-dimensional nuclear Overhauser effect spectroscopy; COSY, two-dimensional bond correlation spectroscopy; MCOSY, magnitude COSY; TOCSY, two-dimensional total correlation spectroscopy; WEFT, water-eliminated Fourier transform.

(^2)
The structure of L29F deoxymyoglobin has been determined to 1.7 Å by M. L. Quillin as a part of his Ph.D. thesis work. The orientations of the His(E7) and Phe(B10) side chains are very similar to those reported for the met, CO, and oxy structures of this mutant. A novel feature of the L29F deoxymyoglobin structure is the absence of an internal, distal pocket water molecule due to the large size of the Phe residue. In native and wild-type sperm whale deoxymyoglobin, a non-coordinated water molecule is found hydrogen-bonded to N of His(E7) and restricts access to the heme iron atom (Quillin et al., 1993).

(^3)
The freshly prepared sample of L29F-cyano-metmyoglobin, initially prepared by adding CN-metMb, exhibited a single set of peaks indicative of a unique molecular structure. However, within a short time (1 day), a second set of peaks arise which finally account for 30% of the protein. These two sets of peaks likely arise from heme orientational disorder which is a characteristic equilibrium property of L29F-cyano-metmyoglobin, but not metMb. The presence of the second set of peaks leads to severe spatial overlap which, with the limited sample size available, restrict the extension of the two-dimensional NMR assignments to only those necessary to define the magnetic axes.

(^4)
The 9-proton input set I (references to set E in Rajarathnam et al.(1992)) includes CH of Leu(F4); CH, CH of His(FG3); CH, CH, CH, CH, CH(3), CH(3) of Ile(FG5). The 14-proton input data set II (referenced to as set D` in Rajarathnam et al.(1992)) has, in addition to the 9 protons identified above, CH, CH(3) of Ala(F5) and CH, CHs, CHs of Phe(H15). The 18-proton input data set III (Qin et al., 1993a) has, in addition to the signals of data set II, CHs of Gln(F6), Ser(F7), Ala(F9) and CH(3) of Ala(F9). For L29F-cyano-metmyoglobin, CH of Ile(FG5) and CH of His(FG3) have not been assigned; instead of these protons, the CHs, and CHs of Tyr(G4) have been used.

(^5)
The aromatic ring of Phe(B10) is assumed to be fixed at (2) in the pocket, but undergo a rapid 180° flip to average the CHs and CHs. The average distance to the iron, r, for the CHs is given by r(i) = 0.5(r(1) + r(2))(Eq. 6)where r(1) and r(2) are the distances of the CH(1) and CH(2) from iron. The T(1) of the averaged signal can be calculated using .


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