1H NMR study of the solution molecular and electronic structure of engineered distal myoglobin His64(E7) Val/Val68(E11) His double mutant. Coordination of His64(E11) at the sixth position in both low-spin and high-spin states.

A genetically engineered human myoglobin (Mb) in which the distal His, His64(E7), and the distal Val, Val68(E11), are replaced by Val and His, respectively, has been expressed in Escherichia coli, for the purpose of assessing the potential role of a E11 residue in providing a hydrogen bond donor to the coordinated ligand. Molecular modeling indicates that such an interaction is possible. The 1H NMR spectrum of the ferric form of the double mutant Mb exhibits large hyperfine shifts and strong paramagnetic relaxation for which the temperature dependence of the hyperfine shifts reveals a thermal equilibrium between a low-spin and high-spin state (70, 30% at 25 degrees C, respectively). Standard sequence specific two-dimensional (2D) NMR assignments of the E and F helical backbones allow the identification of the peptide protons for the proximal His93(F8) and substituted distal His68(E11). Steady-state nuclear Overhauser effect from these peptide protons locate strongly hyperfine shifted His93(F8) and His68(E11) side chain protons which dictate that both the imidazole rings are coordinated to the iron. 2D bond correlation and one-dimensional and 2D dipolar correlation experiments locate and assign the resonances for the heme. The pattern of the heme contact shifts in both the low-spin and high-spin state, together with the nature of the temperature dependence of the His93(F8) and His68(E11) resonances, establish that the two His are ligated in the high-spin as well as low-spin forms. The pattern of heme methyl hyperfine shifts in the low-spin state, and the smaller hyperfine shifts for His68(E11) as compared to His93(F8) in the high-spin state, indicate that the axial bond to the distal His68(E11) is weakened or strained as compared with that for the proximal His93(F8) in both spin states. This weak ligation originates from a tilted iron-His68 bond, the only conformation in which His68 can place its imidazole group sufficiently close to bind to the heme iron in the conventional Mb folding. Not only do these results support the belief that distal His is indispensable for the control of the ligand binding in Mb and hemoglobin, but also reveal the significance of the evolution that the stereochemical disposition of both His64 and Val68 are unique and non-exchangeable for interacting with the bound ligand.

High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin.

The highly conserved distal histidine residue (His64) of sperm whale myoglobin modulates the affinity of ligands. In an effort to fully characterize the effects of mutating residue 64, we have determined the high-resolution crystal structures of the Gly64, Val64, Leu64, Thr64 and Gln64 mutants in several liganded forms. Metmyoglobins with hydrophobic substitutions at residue 64 (Val64 and Leu64) lack a water molecule at the sixth coordination position, while those with polar amino acid residues at this position (wild-type and Gln64) retain a covalently bound water molecule. In the Thr64 mutant, the bound water position is only partially occupied. In contrast, mutating the distal histidine residue to glycine does not cause loss of the coordinated water molecule, because the hydrogen bond from the imidazole side-chain is replaced by one from a well-ordered solvent water molecule. Differences in water structure around the distal pocket are apparent also in the structures of deoxymyoglobin mutants. The water molecule that is hydrogen-bonded to the N epsilon atom of histidine 64 in wild-type deoxymyoglobin is not found in any of the position 64 mutant structures that were determined. Comparison of the carbonmonoxy structures of wild-type, Gly64, Leu64 and Gln64 myoglobins in the P6 crystal form shows that the conformation of the Fe-C-O complex is nearly linear and is independent of the identity of the amino acid residue at position 64. However, the effect of CO binding on the conformation of residue 64 is striking. Superposition of deoxy and carbonmonoxy structures reveals significant displacements of the residue 64 side-chain in the wild-type and Gln64 myoglobins, but no displacement in the Leu64 mutant. These detailed structural studies provide key insights into the mechanisms of ligand binding and discrimination in myoglobin.

High-resolution X-ray structures of pig metmyoglobin and two CD3 mutants: Mb(Lys45----Arg) and Mb(Lys45----Ser).

The structure of pig aquometmyoglobin has been refined to a crystallographic R-factor of 19.8% against X-ray diffraction data between 10- and 1.75-A spacing. The final structural model comprises two molecules of pig myoglobin, 233 water molecules, and two sulfate ions. A water molecule is coordinated to each of the heme iron atoms with an average Fe-OH2 bond distance of 2.19 A, and the mean Fe-N epsilon (proximal histidine-93) distance is 2.20 A. In contrast to the structure of sperm whale metmyoglobin, the iron is not significantly displaced from the plane of the heme. At the entrance to the heme pocket, the side-chain amino group of lysine-45 (CD3) is well-defined in the electron density map and forms salt-bridging interactions with the heme 6-propionate and with a sulfate ion. Serine and arginine replacements have been made previously at position 45 to examine the proposal that the CD3 side chain acts as a barrier to ligand entry into the protein. Crystal structures of the arginine-45 and serine-45 mutant metmyoglobins have been solved to 1.9 and 2.0 A resolution, respectively. In both cases the structural changes are confined to the site of mutation. Arginine-45 takes up a conformation closely similar to that observed for this residue in wild-type sperm whale myoglobin, in which it makes more extensive charge-charge and charge-dipole interactions and appears to restrict the movement of the distal histidine away from the ligand. The hydroxyl group of serine-45 is disordered, but it is clear that the effect of the mutation is to open up the solvent-exposed face of the heme pocket.

Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy.

Proton NMR spectroscopy was applied to myoglobin in the ferric, water-liganded form (metMbH2O) and the apo form (apoMb) to probe the structure and stability of the latter. Proteins from sperm whale and horse skeletal muscles were studied to simplify the spectral assignment task. Nuclear Overhauser effects and the response of chemical shifts to variations of pH were used as indicators of residual native holoprotein structure in the apoprotein. The investigation was focused in the histidine side chains and their environment. In metMbH2O, the resonances of all imidazole rings not interacting with the heme were assigned by applying standard two-dimensional methods. These assignments were found to differ from those reported elsewhere [Carver, J. A., & Bradbury, J. H. (1984) Biochemistry 23, 4890-4905] except for His-12, -113, and -116. Only one histidine (His-36) has a pK(a) higher than 7, two (His-48 and His-113) have a pK(a) lower than 5.5, and two (His-24 and His-82) appear not to titrate between pH 5.5 and pH 10. In the apoproteins, the signals of His-113 and His-116, as well as those of His-24, -36, -48, and -119 previously assigned in the horse globin [Cocco, M. J.. & Lecomte, J. T. J. (1990) Biochemistry 29, 11067-11072], could be followed between pH 5 and pH 10. A comparison to the holoprotein data indicated that heme removal has limited effect on the pK(a) and the surroundings of these residues. Five additional histidines which occur in the two helices and connecting loops forming the heme binding site were identified in the horse apoprotein. Four of these were found to have pK(a) values lower than that expected of an exposed residue. The NOE and titration data were proposed to reflect the fact that several holoprotein structural elements, in particular outside the heme binding site, are maintained in the apoprotein. In the heme binding region of the apoprotein structure, the low pK(a)'s suggest local environments which are resistant to protonation.