©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alteration of Axial Coordination by Protein Engineering in Myoglobin
BISIMIDAZOLE LIGATION IN THE His Val/Val His DOUBLE MUTANT (*)

Yi Dou , Suzanne J. Admiraal (§) , Masao Ikeda-Saito (¶) , Szymon Krzywda (1), Anthony J. Wilkinson , Tiansheng Li , John S. Olson , Roger C. Prince , Ingrid J. Pickering , Graham N. George

From the (1)Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, From the Department of Chemistry, University of York, York, YO1 5DD, United Kingdom and theDepartment of Crystallography, Faculty of Chemistry, Adam Mickiewicz University, ul. Grunwaldzka 6, 60-720 Poznan, Poland, From the Department of Biochemistry and Cell Biology, and W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77251-1892, From the Exxon Research and Engineering Company, Annandale, New Jersey 08801, From the Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, California 94309-0210

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pig and human myoglobin have been engineered to reverse the positions of the distal histidine and valine (i.e. His(E7) Val and Val(E11) His). Spectroscopic and ligand binding properties have been measured for human and pig H64V/V68H myoglobin, and the structure of the pig H64V/V68H double mutant has been determined to 2.07-Å resolution by x-ray crystallography. The crystal structure shows that the N of His is located 2.3 Å away from the heme iron, resulting in the formation of a hexacoordinate species. The imidazole plane of His is tilted relative to the heme normal; moreover it is not parallel to that of His, in agreement with our previous proposal (Qin, J., La Mar, G. N., Dou, Y., Admiraal, S. J., and Ikeda-Saito, M.(1994) J. Biol. Chem. 269, 1083-1090). At cryogenic temperatures, the heme iron is in a low spin state, which exhibits a highly anisotropic EPR spectrum (g = 3.34, g = 2.0, and g < 1), quite different from that of the imidazole complex of metmyoglobin. The mean iron-nitrogen distance is 2.01 Å for the low spin ferric state as determined by x-ray spectroscopy. The ferrous form of H64V/V68H myoglobin shows an optical spectrum that is similar to that of b-type cytochromes and consistent with the hexacoordinate bisimidazole hemin structure determined by the x-ray crystallography. The double mutation lowers the ferric/ferrous couple midpoint potential from +54 mV of the wild-type protein to -128 mV. Ferrous H64V/V68H myoglobin binds CO and NO to form stable complexes, but its reaction with O results in a rapid autooxidation to the ferric species. All of these results demonstrate that the three-dimensional positions of His and Val in the wild-type myoglobin are as important as the chemical nature of the side chains in facilitating reversible O binding and inhibiting autooxidation.


INTRODUCTION

The nature of the axial heme ligand is an important factor defining the biochemical function of hemoproteins (Dawson, 1988). In myoglobin and hemoglobin, the fifth coordination site of the heme iron is occupied by the imidazole group of the proximal histidine, and exogenous ligands bind to the sixth position (Dickerson and Geis, 1983). There are two highly conserved residues, His(E7) and Val(E11), located in the distal pocket adjacent to the ligand binding site. The distal histidine does not directly interact with the heme iron but forms a hydrogen bond with bound O to stabilize the oxy form of the globins (Phillips and Shoenborn, 1981; Kitagawa et al., 1982; Olson et al., 1988; Springer et al., 1989; Ikeda-Saito et al., 1991). In deoxymyoglobin, N of His(E7) forms a hydrogen bond with a noncoordinated water that must be displacedbefore ligands can bind to the iron atom, thus inhibiting the rate of ligand entry into the heme pocket (Quillin et al.,1993; Springer et al., 1994). The distal histidine also plays apivotal role in regulating ligand reactivity in metmyoglobin (Brancaccio et al., 1994). Mutation of Val(E11) also causes marked changes in the ligand binding properties in myoglobin (Egeberg et al., 1990b; Smerdon et al., 1991; Brancaccio et al., 1994; Quillin et al., 1995). Val interacts with bound ligands, the His side chain, and the noncoordinated water molecule in deoxymyoglobin. Mutations at this position affect ligand binding by a modulation of these interactions (Springer et al., 1994). In addition, His and Val are now known to play key roles in apomyoglobin folding (Hargrove et al., 1994a).

Using site-directed mutagenesis, we have prepared myoglobin in which the distal histidine and valine are exchanged, i.e. His(E7) Val and Val(E11) His. Our previous hyperfine-shifted proton resonance studies on this double mutant have shown that the hemin iron is a thermal mixture of low spin and high spin states (Qin et al., 1994). The NMR results also indicated that the imidazole side chains of His(F8) and His(E11) are coordinated to the hemin iron in both the high spin and low spin forms. The axial bond to the distal His appeared to be weakened or strained compared with that for the proximal histidine in both spin states. A tilted His coordination was proposed as the only conformation in which His can be sufficiently close to coordinate to the heme iron in the normal myoglobin tertiary fold. The distorted Fe-His coordination was proposed to produce a weaker ligand field than those in typical bishistidine-ligated hemoproteins, such as b-type cytochromes, and hence, to allow significant population of a high spin state.

In order to evaluate this model for the coordination structure of H64V/V68H myoglobin and to determine the effects of these mutations on the reactivity of the heme, we have measured optical absorption, IR, EPR, and x-ray absorption spectra; CO binding kinetics; and the oxidation/reduction midpoint potential of human H64V/V68H myoglobin and have determined the structure of pig H64V/V68H myoglobin by x-ray crystallography. Both spectroscopic and crystallographic results confirm axial coordination of His as proposed in the previous NMR studies (Qin et al., 1994). The ferrous H64V/V68H double mutant forms stable CO and NO complexes, but its reaction with O results in a rapid formation of ferric species. Thus, proper stereochemical positioning of His and Val is essential for creation of a distal pocket capable of reversible O binding. The altered coordination structure of H64V/V68H myoglobin also serves as a useful model for examining the role of heme pocket amino acids in determining the spectroscopic and redox properties of hemoproteins in general.


EXPERIMENTAL PROCEDURES

Mutagenesis of the wild-type human and pig myoglobin coding sequences was performed as described in the previous papers (Varadarajan et al., 1985; Ikeda-Saito et al., 1991; Smerdon et al., 1991), and the recombinant myoglobins were expressed in Escherichia coli and purified to homogeneity following procedures described in these publications.

Light absorption spectra were recorded using a Hitachi U-3210 spectrophotometer at 20 °C. Extinction coefficients were obtained by the alkaline pyridine hemochromogen method (Antonini and Brunori, 1971). Oxidation-reduction potentials were determined as described previously (Ikeda-Saito and Prince, 1985) using a calomel electrode as the reference electrode. The measurements were carried out in the presence of 10 µM 2-hydroxy-1,4-naphthaquinone, 2-hydroxy-1, 4-anthraquinone, N-methylphenazine methosulfate, and N-ethylphenazine ethosulfate plus 20 µM 2,3,4,5-tetramethylphenylenediamine. Potentials are expressed on the basis of the assumption that the calomel electrode had an E of +247 mV.

X-ray absorption spectra were measured at the Stanford Synchrotron Radiation Laboratory on beam line 7-3 using a Si (220) double crystal monochrometer with a 1-mm vertical aperture and no focussing optics. Twenty 30-min scans were averaged, and the sample was maintained at a temperature close to 10 K in an Oxford Instruments CF1204 flowing liquid helium cryostat. Iron-K edge spectra were measured as fluorescence excitation spectra using a Canberra 13-element detector (Cramer et al., 1988). The extended x-ray absorption fine structure (EXAFS)()oscillations were quantitatively analyzed as described previously (George et al., 1989) using the full multiple scattering code feff (version 6.01) of Rehr and co-workers (Rehr et al., 1991; Mustre de Leon et al., 1991) and included a total of 49 nondegenerate scattering paths. The x-ray energy was calibrated with respect to the first inflection energy of an iron metal foil, which was defined as 7111.3 eV.

EPR spectra were recorded on a Bruker ESP-300 instrument operating at 9.45 GHz as described previously (Ikeda-Saito et al., 1992). Experiments were carried out at an incident microwave power of 1 mW with a field modulation of 0.5 millitesla at 100 kHz. An Oxford flow cryostat (ESR-900) was used for liquid helium temperature measurements. Microwave frequency was monitored by a Hewlett Packard 5350B frequency counter, and a Bruker ER-035M NMR gauss meter was used to determine the magnetic flux density.

IR spectra were measured by a Mattson Galaxy 6020 spectrometer as described previously (Li et al., 1994). CO association rates were measured by a flash photolysis apparatus consisting of two photographic strobe units (Sunpak 544) equipped with thyristor quenching devices, and CO dissociation rates were determined by the ligand replacement method using NO in a Gibson-Dionex stopped flow apparatus (Rohlfs et al., 1990).

Crystals of the H64V/V68H mutant were prepared as described previously for recombinant wild-type pig myoglobin (Dodson et al. 1988) except that 5 mM Tween 60 was included in the hanging drops. A single crystal mounted in a glass capillary in the usual manner was exposed to CuK radiation generated from a rotating copper anode source, and diffraction intensities were recorded on a Mar Research image plate. 110 images were collected in 1° oscillations at 19 °C. Data were processed with the DENZO package of programs (Z. Otwinowski, Yale University). The crystals are in the monoclinic space group, C2, with two molecules in the asymmetric unit. These two molecules are related by a noncrystallographic 2-fold axis of symmetry, which is perpendicular to the crystallographic 2-fold symmetry axis. As a result, the diffraction pattern exhibits pseudo I222 symmetry. As for wild-type and a series of mutant myoglobin crystals analyzed previously, we chose to process the data in the nonstandard space group I2 in which the angle is close to 90 °C rather than in the C2 cell in which = 128 °C and the dimension is 157 Å (see ).

The starting model for the refinement of the H64V/V68H mutant was the wild-type pig metmyoglobin structure (1MYG.PDB) from which water molecules had been removed and the side chains of His and Val truncated in both molecules of the asymmetric unit. Initially, cycles of rigid body refinement were carried out as described by Derewenda(1989). This lowered the R from 35.2 to 30.2%. Cycles of least squares refinement in the program PROLSQ (CCP4, 1979) in which the weighting of the x-ray terms to the geometric terms was 1:2 reduced the R to approximately 22%, at which point 2F - F and F - F electron density maps were calculated and displayed graphically using the program O (Jones et al., 1991). These maps contained electron density peaks for the missing Val and His side chains. There was a noticeable break in the electron density between His and the heme iron in the A molecule in 2F - F maps contoured at 3. The mutated side chains were built into the model, which was subsequently refined further in cycles of the automated refinement procedure (Lamzin and Wilson, 1993). In this procedure, water molecules, which satisfy distance criteria, are introduced automatically in successive cycles into peaks in F- F electron density maps. These water molecules are automatically removed in subsequent cycles if they do not refine satisfactorily. In these intermediate cycles of refinement, the root mean squared deviations of atomic positions from noncrystallographic symmetry were restrained to 0.05 and 2 Å for main and side chain atoms, respectively. The noncrystallographic symmetry restraints were removed in subsequent cycles of least squares minimization refinement procedures in the program PROLSQ (CCP4, 1979). Electron density maps were inspected periodically during refinement, and manual adjustments were made to the model in the program O (Jones et al., 1991).


RESULTS

Spectroscopic and Functional Properties of H64V/V68H Myoglobin

Fig. 1shows the light absorption spectra of ferric and ferrous forms of human and pig H64V/V68H myoglobins (toppanels) and its ferrous CO and NO forms (bottompanels). The ferric form of the human mutant myoglobin has absorption bands at 563 and 525 nm in the visible region and a Soret band at 412 nm, together with a small band around 630 nm. The spectrum of the ferric pig mutant myoglobin is very similar to that of the human mutant, as seen in the rightpanel of Fig. 1. The absorption bands at 563 and 525 nm are attributable to a ferric low spin species, and that at 630 nm is an indication of the presence of a high spin state (Eaton and Hofrichter, 1981). Both human and pig ferric H64V/V68H mutants are mixtures of low spin and high spin species in agreement with the NMR results on the human mutant, which indicated a thermal equilibrium between a low spin (70%) and a high spin state (30%) (Qin et al., 1994). When cyanide, azide, imidazole, and fluoride (up to 50 mM concentration) were added to the ferric H64V/V68H mutants, there were no detectable changes in the light absorption spectrum. Ferric H64V/V68H myoglobin either has very weak ligand affinities or cannot bind these exogenous ligands. Upon reduction by dithionite, both of the mutant myoglobins showed a typical hemochrome-type absorption spectrum with peaks at 563, 529, and 427 nm, indicating that ferrous H64V/V68H myoglobin is a hexacoordinate low spin species. The ferric/ferrous couple of the oxidation-reduction potential of the H64V/V68H mutant is -128 mV at 20 °C in 0.1 M phosphate buffer, pH 7, which is markedly lower than the +54 mV potential of the wild-type protein.


Figure 1: Light absorption spectra of human (left panels) and pig (right panels) H64V/V68H myoglobins in 0.1 M phosphate buffer, pH 7, at 20 °C. Toppanels, spectra for ferric (solidline) and ferrous (brokenline) forms. Bottompanels, spectra for ferrous CO (solidline) and NO (brokenline) forms.



Light absorption spectra of CO and NO forms of ferrous H64V/V68H human and pig myoglobins are similar to those of the corresponding wild-type complexes. The NO and NO complexes of ferrous H64V/V68H human myoglobin exhibit typical six-coordinate nitrosyl EPR spectra, indicating that NO binds to the sixth coordination site of the heme iron with the imidazole of His as a trans axial ligand (data not shown). CO and NO appear to assume the normal His-Fe-L (L = CO or NO) coordination structure.

Reaction of the ferrous H64V/V68H mutants with O resulted in a rapid oxidation of the iron, and the spectrum of the putative oxy form was not detected in a conventional spectrophotometer with a mixing time of 10-30 s. The CO binding parameters for the double mutants are compared in with those for wild-type and H64V human and pig myoglobins. These functional results show that the effects of mutagenesis are virtually identical in the myoglobins from pig and human muscle. In contrast to the single H64V mutant, the double H64V/V68H mutant shows a marked 10-fold reduction in the CO association rate constant and a 4-fold reduction in the dissociation rate constant compared with the wild-type protein. The net result is a 2-3-fold reduction in the CO affinity for the double mutant compared with the 3-fold increase in the CO affinity observed for the single His Val mutant.

The distal pocket structure was further examined by measurements of the IR spectrum of the CO complex. Fig. 2compares the IR spectra of human wild-type and H64V/V68H myoglobin. The major CO stretching band of the wild-type protein at 1944 cm is shifted to 1967 cm in the H64V/V68H double mutant. The CO stretching band of H64V/V68H myoglobin is thus similar to those of the human, pig, and sperm whale H64V single mutants, which have major bands at 1965-1967 cm (Li et al., 1994; Balasubramanian et al., 1993). The order of the CO bond is coupled to the surrounding electric field, and thus the vibrational properties of the bound CO are sensitive to the polarity of the distal pocket (Park et al., 1991; Sakan et al., 1993; Li et al., 1994). Thus, the electrostatic field around the bound CO appears to be the same in both H64V/V68H and H64V myoglobins. This suggests that once the Fe-imidazole bond is broken, the His side chain rotates away from the bound ligand taking up a conformation similar to that of the aromatic side chain of Phe in the crystal structure of V68F myoglobin (Quillin et al., 1995).


Figure 2: IR spectra of recombinant human CO myoglobin in 0.1 M phosphate buffer, pH 7.0, with 1 atmosphere of CO recorded at room temperature (22 °C).



The EPR spectra of human and pig ferric H64V/V68H myoglobins are shown in Fig. 3. A residual high spin signal at g = 6, and a ``large g type'' low spin signal (g = 3.34, and g 2.0) are seen in the human and pig mutant myoglobins. The optical absorption (Fig. 1) and the EPR results show that iron atoms in the human and pig mutants have virtually identical electronic structures. In the low spin signal, the g feature is well defined, the g feature is still visible, but the g feature is not visible under the experimental conditions. The hemin iron is predominantly in a low spin state at cryogenic temperatures, indicating that it is the ground state. The H64V/V68H low spin EPR is somewhat similar to that of b-type cytochromes of mitochondria, neutrophils, and chloroplasts and some model compounds that have a strong g > 3.3 as the major observable spectral feature (Tsai and Palmer, 1983; Salerno, 1984; Salerno and Leigh, 1984; Nitschke and Rutherford, 1989; Walker et al., 1986; Miki et al., 1992). In contrast, cytochrome b and the imidazole complex of metmyoglobin exhibit EPR signals with three well defined components at g 3, g 2.2, and g 1.4 (Blumberg and Peisach, 1971; Hori, 1971; Ikeda-Saito et al., 1974).


Figure 3: EPR spectrum of human (top) and pig (bottom) ferric H64V/V68H myoglobins in 0.1 M phosphate buffer, pH 7, recorded at 6 K.



Fig. 4, A and B present the iron-K edge x-ray absorption edge spectrum and the analyzed EXAFS of ferric human H64V/V68H myoglobin at 10 K. Shiro et al.(1990) have suggested the 1s 3d pre-edge feature at approximately 7112 eV is an indicator of the coordination state of the heme iron. An increase in its intensity indicates a decrease in the molecular symmetry around the iron atom as observed for five coordinate hemoproteins (Shiro et al., 1990). The relatively weak feature at 7112 eV observed for the double mutant is characteristic of a hexacoordinate low spin species and is consistent with the notion that His becomes a heme ligand.


Figure 4: A, iron-K edge x-ray absorption spectrum of human ferric H64V/V68H myoglobin recorded near 10 K. B, EXAFS Fourier transform of human ferric H64V/V68H myoglobin. The transform was phase-corrected for the first-shell Fe-N scattering. The inset shows a curve-fitting analysis (brokenline) of the EXAFS data (solidline).



The EXAFS analysis indicates that the first shell Fe-N distance is 2.02 Å (with the usual EXAFS accuracy of ± 0.02 Å). This is typical of ferric low spin bisimidazole complexes regardless of low spin EPR type (Walker et al., 1986; Inniss et al., 1988). The EXAFS resolution, as determined by the k range of the data, is approximately 0.11 Å, and we conclude that the Fe-N distances of the histidine ligands differ by less than 0.11 Å from the mean distance of 2.02 Å in the ferric H64V/V68H double mutant. Outer shell Debye-Waller factors were semiempirically approximated using a correlated Debye model assuming a sample temperature of 10 K and a Debye temperature of 1200 K, the latter being determined from the first shell Fe-N Debye-Waller factor of 0.0025 Å. A planar heme ring was assumed with final (refined) distances of 3.05, 3.40, and 4.24 Å for the -, -, and -carbons, respectively. No improvements in the fit were obtained by allowing the histidine rings to move from a position normal to the heme, and we conclude that in this case EXAFS lacks the sensitivity to detect such displacements in the presence of the intense backscattering from the porphyrin carbons.

X-ray Structure of Pig H64V/V68H Myoglobin

The proposed bisimidazole coordination in H64V/V68H myoglobin has also been examined by x-ray crystallography. Initial attempts to crystallize the human H64V/V68H mutant resulted in small needle shaped crystals that did not provide useful x-ray diffraction. Fortunately, pig H64V/V68H myoglobin formed single crystals suitable for x-ray analysis, and the structure of the ferric form has been determined and refined at 2.07-Å resolution. The data collection and refinement statistics are listed in . The refined model, which includes all protein atoms except Gln and Gly in both molecules of the asymmetric unit, comprises 2,653 atoms including 200 water molecules. The final R is 17.5% (free R = 22.7%) for 24,848 reflections in the resolution range of 10.0-2.07 Å. The overall structure of pig H64V/V68H myoglobin is closely similar to that of wild-type pig myoglobin. Following least squares overlap of the A and B molecules of the mutant myoglobin with the corresponding molecules of wild-type pig myoglobin, the root mean square deviation in main chain atomic positions is 0.24 and 0.25 Å, respectively. The root mean square deviation in the positions of main chain atoms following overlap of the A and the B molecules of the H64V/V68H asymmetric unit is 0.16 Å.

Details of the iron ligation and heme and histidine stereochemistry are given in I. The electron density for His is very clear in both molecules of the asymmetric unit and provides unambiguous evidence of ligation of the imidazole side chain to the iron (Fig. 5). However, the orientation of the imidazole side chain with respect to the heme plane is far from optimal for overlap of the bonding orbitals of the imidazole's N and the heme iron. As a result, the Fe-N distance in this bond is rather long (2.3 Å). The N-H group of His is ideally placed to make a strong hydrogen bonding interaction (2.7 Å) with the carbonyl oxygen of Val. Superposition of the double mutant and wild-type myoglobin structures by least squares minimization of the root mean square differences in the main chain atomic positions shows a significant change in the position of the heme group relative to the protein backbone (Fig. 6). In both A and B molecules of the mutant, the movement can be described by a lateral translation of the heme group of 0.5 Å toward the EF corner and about an 8° rotation of the heme about an axis through the - and -meso carbon atoms. The effect of this change, coupled with a small movement of the protein backbone, is to bring the C atom of residue 68 0.6 Å closer to the iron in the H64V/V68H double mutant than in the wild-type myoglobin.


Figure 5: Stereoviews of the heme pocket of pig H64V/V68H myoglobin. 2F - F electron density contoured at 1.8 is displayed on the heme and surrounding residues. These are shown for the A and B molecules of the asymmetric unit in the top and bottompanels, respectively.




Figure 6: Stereo view of the superposition of the heme environments of pig wild-type myoglobin (thin lines) and H64V/V68H myoglobin (thick lines). The overlap minimizes the root mean squared deviation in the positions of main chain atoms of residues 1-151 in the chains of the respective molecules. These are shown for the A and B molecules of the asymmetric unit in the top and bottompanels, respectively. The coordinated water molecule in the wild-type structure and solvent molecules in both structures have been omitted for clarity.



A curious feature of the maps is a break in the 2F- F electron density between the iron atom and the proximal histidine in the A molecule. This was observed in the initial maps and persists in F maps and in F - F maps calculated after the heme and His and His had been omitted from the coordinate set for several cycles of refinement and omitted from the subsequent phase calculation. In the refined model, the imidazole side chain of the proximal histidine (His) in the A molecule has an altered conformation with respect to the plane of the pyrrole nitrogens, and the Fe-N His distance is unusually long (2.3 Å). The principal structural change is the increase in the His angle, from 60° in the B molecule to 78° in the A molecule.

The unusual conformation of the proximal histidine was insensitive to manual modelling and refinement steps taken to impose a more standard geometry. Similar imidazole stereochemistry is not seen in the B molecule even though the main chain atoms of the F helices in the A and B molecules of the asymmetric unit are closely superimposable. Neither the unusual imidazole conformation nor the break in the electron density are observed in wild-type pig myoglobin or in a dozen or so mutant pig myoglobins that form crystals isomorphous with those of the H64V/V68H double mutant. One possible explanation of the differences observed in the Fe-His stereochemistry between the A and B molecules is the different crystal environments inhabited by the two molecules. As mentioned earlier, however, the crystal form is pseudo-I222, and the intermolecular contacts made by the A and B molecules of the asymmetric unit are closely similar. One difference of note may be the side chain amide of Gln, which in the B molecule is involved in crystal contacts with main chain groups of residues 149 and 150 in an adjacent A molecule; these interactions are not matched in detail by Gln in the A molecule. Whether and how this influences the structure of the side chain at position 93 is difficult to evaluate with data that extend to 2.1-Å spacing because the magnitudes of the main chain atomic positional changes are so small (0.1-0.2 Å). It is noticeable, however, that there is greater symmetry in the iron coordination in the A molecule than in the B molecule with the N-H of His making stronger interactions with Ser -OH and Leu >C=O (I). These interactions may make favorable energetic contributions that compensate for what appears to be a weaker Fe-His coordinate bond in the A molecule.


DISCUSSION

An important goal of the present study is to correlate the spectroscopic properties of the H64V/V68H myoglobin mutant with its structure as determined by x-ray crystallography. Ideally both sets of data would be collected on protein from one species. However, human H64V/V68H myoglobin did not yield crystals suitable for x-ray structural determination. The crystal structure of pig H64V/V68H myoglobin was therefore used for interpretation of the spectroscopic and functional properties of both pig and human H64V/V68H myoglobins. Optical absorption spectra of the human double mutant are very similar to those of the corresponding pig protein. The hemochrome type spectrum is seen in both of the proteins, indicating a hexacoordinate low spin state in the ferrous forms. The CO association and dissociation rates of human H64V/V68H are similar to those of the pig mutant. In the ferric state, the pig and human mutants show virtually identical spin state compositions, indicating similar ligand field. The g value of low spin complexes is a function of the bound axial ligand geometry (Walker et al., 1986; Inniss et al., 1988), and the EPR results show the virtually identical axial ligand geometry in human and pig H64V/V68H myoglobins. Thus, the heme pocket structure of human H64V/V68H myoglobin appears to be spectroscopically identical to that of pig H64V/V68H myoglobin, and comparisons with the crystal structure of the latter protein should be valid.

Axial Coordination and Electronic Structure in the H64V/V68H Mutant

The crystal structure of pig H64V/V68H myoglobin demonstrates coordination of the imidazole of His to the sixth position of the ferric iron to form a bisimidazole heme complex. A salient feature is that the imidazole plane of His(E11) is not perpendicular to the heme ring. This is because the conventional myoglobin fold does not allow the E11 imidazole side chain to approach the iron in a conformation perpendicular to the heme. The x-ray crystallographic result verifies the heme ligand geometry predicted by NMR (Qin et al., 1994). On the basis of the heme methyl contact shift patterns and the significantly smaller contact shift observed for His NH than for the His NH, Qin et al.(1994) concluded that the Fe-His bond is weaker than the Fe-His bond in both low spin and high spin complexes. The tilted His and the longer Fe-imidazole N bond observed in the x-ray structure of the pig double mutant also indicate a weaker ligand field when compared with more conventional bis-histidine ligated proteins such as ferricytochrome b (Ikeda-Saito et al., 1974; Lee et al., 1990). This weakening accounts for 30% population of the ferric high spin state at ambient temperatures (Qin et al., 1994).

The x-ray structure shows the constraints placed upon the possible orientations of the position 68 imidazole by the surrounding globin. In x-ray studies with V68F sperm whale myoglobin, Quillin et al.(1995) have shown that the extra bulk of the aromatic substituent can be accommodated in the back of the distal pocket without significant alteration in the tertiary structure. In H64V/V68H myoglobin, the favorable coordination and hydrogen bonding of His to the iron and Val >C=O, respectively, appear to drive heme reorientation and small adjustments of the protein main chain. As a result, the iron atom is 0.6 Å closer to the C atom of residue 68 in the double mutant protein than it is in the wild-type myoglobin. This reorientation is facilitated by the absence of the large polar side chain of His, which is replaced by valine.

Another key feature of the H64V/V68H structure is that the coordinated His imidazole plane is significantly displaced from being parallel to the imidazole plane of the proximal His. This is quite different from the geometries found in cytochrome b or the imidazole complex of metmyoglobin, where the planes of the axial ligands assume nearly parallel orientations (Durley and Mathews, 1994; Lionetti et al., 1991). The nearly perpendicular orientation of His and His imidazoles appears to be the primary cause of the large g low spin EPR signal in the H64V/V68H double mutant. Walker, Huynh, Scheidt, Strouse, and their co-workers (Walker et al., 1986; Inniss et al., 1988) demonstrated that large g low spin EPR signals are associated with a perpendicular orientation of the imidazole ligands. The tilted Fe-His bond, which reduces the ligand field, thereby decreasing the tetragonal splitting, also favors a large g low spin EPR signal.

Sligar and his co-workers (Sligar et al., 1987) have demonstrated that the replacement of one of the axial histidine ligands by methionine in cytochrome b modulates the spin and coordination states of the ferric cytochrome and that properties of the abnormal subunits of M-type hemoglobins can be modeled to some extent in engineered myoglobin mutants with altered axial ligation (Egeberg et al., 1990a). However, designing and engineering a hemoprotein with a highly anisotropic low spin EPR signal has not been done before. Choma et al.(1994) and Robertson et al.(1994) reported the design and synthesis of heme-peptide complexes. Although the redox potentials of the heme-helix complexes were between -200 and -100 mV and similar to that of H64V/V68H myoglobin, these model compounds exhibited low spin EPR spectra typical of ordinary bisimidazole heme complexes (g 3.0). To our knowledge, H64V/V68H myoglobin is the first mutant with a large g low spin EPR signal and will serve as a good model for understanding the origin of this type of EPR signal in naturally occurring hemoproteins.

Iron Reactivity

The His Val/Val His double mutation causes marked decreases in the reactivity of the iron atom with exogenous ligands in both the ferric and ferrous oxidation states. This is clearly due to direct chelation of His with the iron atom. The single His Val mutation increases markedly the association rate constants for azide, CO, NO, and O binding by creating a larger pathway into the heme pocket, by preventing direct water coordination in the ferric state, and by maintaining a completely apolar, anhydrous binding site in the ferrous state (Rohlfs et al., 1990; Quillin et al., 1993; Brancaccio et al., 1994). In contrast, the distal imidazole-iron bond in the H64V/V68H double mutant must be broken before ligand can bind to either oxidation state, causing marked decreases in the rate constants for association.

The Val His substitution also causes a decrease in the rate of CO dissociation relative to either wild-type protein or H64V myoglobin. The decreased size of the distal pocket due to His is expected to inhibit movement of dissociated ligands away from the iron atom, as is observed for Phe sperm whale myoglobin (Egeberg et al. 1990a; Carver et al., 1990). The latter mutant shows lowered dissociation rate constants, increased extents of geminate recombination, and markedly decreased quantum yields for complete photodissociation into the solvent phase (Carver et al., 1990; Quillin et al., 1995). Presumably similar phenomena occur in the H64V/V68H double mutant accounting for the lower CO dissociation rate constant and extremely low quantum yield for NO photodissociation.

The net decrease in CO affinity observed for the double mutant is due to the requirement for disruption of the Fe-His bond before CO can bind. This effect is substantial, 10 fold, when K for the double mutant is compared with that of H64V myoglobin, but it is more moderate, 2-3-fold, when compared with that of the wild-type protein. In the native protein, a distal pocket water molecule has to be displaced before ligands can bind, whereas in the H64V protein no water is present near the iron atom in the deoxy structure (Quillin et al., 1993; Cameron et al., 1993; La Mar et al., 1994).

H64Y myoglobin is another well characterized mutant in which a distal residue coordinates to the ferric heme iron. Spectroscopy and x-ray crystallography have shown direct binding of the Tyr phenol group at the sixth position of the ferric heme iron (Egeberg et al., 1990a; Hargrove et al., 1994b; Maurus et al., 1994). In the H64Y mutant, azide and cyanide can still bind to the iron by displacing the coordinated Tyr (Brancaccio et al. 1994). Ferric H64Y myoglobin is in a high spin state, whereas H64V/V68H myoglobin is largely in the low spin state (Egeberg et al., 1990a; Qin et al., 1994). The ligand field in the H64V/V68H mutant is stronger than that in the H64Y mutant. A stronger Fe-His bond in the double mutant effectively prevents azide or cyanide binding to the ferric iron atom. H64Y deoxymyoglobin is pentacoordinate with His as a proximal ligand. In contrast, H64V/V68H deoxymyoglobin is hexacoordinate with His bound to the iron atom. However, once CO is bound, the imidazole side chain probably adopts a conformation pointing away from the iron atom since of the bound ligand in the H64V/V68H mutant indicates an apolar environment (Fig. 2).

Hargrove et al. (1994a) have shown that the H64V/V68H double mutation markedly inhibits the rate of hemin loss from myoglobin preventing measurement of this process at room temperature, pH 7.0. This is another manifestation of direct coordination between His and the iron atom since the single H64V mutation increases the rate of hemin loss 10-20-fold. However, once hemin is removed, the H64V/V68H apomyoglobin unfolds into a random coil due to the presence of the polar His residue in the normally apolar region between the B, E, and G helices (Hargrove et al., 1994a).

Conclusion

Our present work demonstrates unequivocally that the imidazole group of histidine at position 68 forms an axial bond with the heme iron. Unlike His, His cannot form a hydrogen bond with bound ligands. As a result the double mutant has lowered reactivity toward both ferric and ferrous ligands in comparison with wild-type myoglobin. Rapid autooxidation of the ferrous form and the unusual instability of H64V/V68H apomyoglobin (Hargrove et al., 1994a) further demonstrate that the stereochemical positioning of histidine at position 64 is essential for myoglobin function. Reversal of the distal valine and histidine produces a protein incapable of O transport.

  
Table: Rate and equilibrium constants for CO binding for human and pig myoglobin recombinants

All of the data were determined in 0.1 M phosphate buffer, pH 7.0, at 20 °C. Equilibrium constants, K, were calculated as k / k.


  
Table: Data collection and refinement statistics for pig H64V/V68H myoglobin


  
Table: 1953066862p4in Angles between the normals to the specified pair of planes.


FOOTNOTES

*
Portions of the work that were carried out at Case Western Reserve University, Rice University, and the University of York, were supported by National Institutes of Health (NIH) Grants GM 51588 (to M. I.-S.) and GM 35649 and HL 47020 (to J. S. O.), Grant GR/H 68864 from the Science and Engineering Research Council, United Kingdom (to A. J. W.), a grant-in-aid from the American Heart Association (to M. I.-S.), Grant C-612 from the Robert Welch Foundation (to J. S. O.), and British Council Travel Grant WAR/922/015 (to S. K.). The purchase of the Bruker EPR instrument was in part supported by NIH Grant RR05659 (to M. I.-S.). Part of the work done at Stanford Synchrotron Radiation Laboratory (SSRL), which is funded by the Department of Energy (DOE), Office of Basic Energy Sciences. The SSRL Biotechnology Program is supported by the Biomedical Resource Technology Program, National Center for Research Resources, NIH, and by the DOE, Office of Health and Environmental Research. 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.

The crystallographic coordinates and structure factors (code 1MNI) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
Present address: Dept. of Biochemistry, Stanford University, Stanford, CA 94309.

To whom correspondence should be addressed. Tel.: 216-368-3178; Fax: 216-368-3952; E-mail: mis2@po.cwru.edu.

The abbreviation used is: EXAFS, extended x-ray absorption fine structure.


ACKNOWLEDGEMENTS

We thank Drs. M. Jaskólski and G. Murshudov for expert assistance in x-ray data collection and structure refinement, respectively.


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