Sperm Whale Myoglobin Mutant Research Articles

Distal pocket polarity in ligand binding to myoglobin: deoxy and carbonmonoxy forms of a threonine68(E11) mutant investigated by X-ray crystallography and infrared spectroscopy.

The crystal structures of the deoxy and carbonmonoxy forms of a distal pocket myoglobin mutant in which valine68(E11) is replaced by threonine have been solved to 2.1- and 2.2-A resolution, respectively. This substitution has been shown previously to cause large decreases in the rate of oxygen binding and to lower the equilibrium association constants for O2 and CO. The synchrotron Laue method was used for the rapid acquisition of X-ray diffraction data to overcome problems caused by the very rapid rate of autooxidation of the mutant protein. The refined deoxy structure shows that the noncoordinated water molecule in the distal pocket is in a position to form strong hydrogen bonds with both the N epsilon-H of the distal histidine64 and O gamma of threonine68 with no other unexpected alterations in the protein structure. In the carbonmonoxy form, the bound ligand is well-defined and inclined away from the two hydrogen-bonding groups, refining to a position in which the Fe-C-O angle is 162 degrees. This value is very close to that previously observed in recombinant wild-type and position-64 (E7) mutants of sperm whale myoglobin (160-170 degrees). The similarity of the CO conformations contrasts with the 150-fold range in equilibrium binding constants (KCO) among the distal pocket myoglobin mutants and indicates that CO affinities cannot be predicted from the coordination geometry of the bound ligand. Furthermore, a comparison of the infrared stretching frequencies of CO in wild-type, valine64 and threonine68 single mutant, and valine64-threonine68 double mutant pig carbonmonoxymyoglobins shows a lack of correlation between KCO and vCO. These effects can be understood in terms of the stability of noncovalently bound water in deoxymyoglobin and electrostatic interactions between bound ligands and the distal pocket residues.

Molecular dynamics simulation of NO recombination to myoglobin mutants.

Molecular dynamics simulations on two coupled electronic surfaces are employed to investigate the geminate recombination of nitric oxide to mutants of sperm whale myoglobin. A model for the ground and the excited states is constructed based on experimental data. The crossing between the surfaces is treated using the Landau-Zener formula. The reaction probability and the recombination curves are calculated directly by histogramming the results of an ensemble of trajectories. The experimental trend is reproduced in which the picosecond recombination rate of different mutants increases in the order Phe29 > Leu29 > Val29 > Ala29. Furthermore, in accord with the experiment on significantly longer time scales an opposite trend is obtained, in which the recombination rate for Ala29 is larger than for Phe29. These results are explained by constrained diffusion of the ligand in the heme pocket. The average and the transient volume of the heme pocket is modified by the 29 mutants.

Kinetics of ligand binding to Pseudoterranova decipiens and Ascaris suum hemoglobins and to Leu-29–>Tyr sperm whale myoglobin mutant

The kinetics of binding of O2, CO, and NO to the octameric, two-domain hemoglobins of the parasitic nematodes Pseudoterranova decipiens and Ascaris suum were determined on nanosecond and picosecond time scales using flash photolysis. The two nematode hemoglobins have very similar kinetic properties. On the picosecond time scale, they exhibit an unusual behavior in showing a geminate reaction with oxygen that is biphasic and dependent on the flash intensity. The geminate reaction with NO is also faster and more complete than for sperm whale myoglobin; however, in contrast to the O2 reaction, it is homogeneous. In addition, the oxygen dissociation rate of P. decipiens hemoglobin, 0.0035 s-1, is as low as that of A. suum hemoglobin, 0.004 s-1 (Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. Lond. B Biol. Sci. 163, 206-214). A mutant of sperm whale myoglobin suggested by sequence alignment of the nematode hemoglobins, Leu-29-->Tyr, did not have kinetic properties similar to them.

Structural and functional characterization of sperm whale myoglobin mutants: role of arginine (E10) in ligand stabilization.

1H NMR and ligand-binding data were used to assess the role of residue Arg(E10) in ligand stabilization of several site-directed mutants, all carrying the His(E7) to Val substitution, obtained using a synthetic sperm whale myoglobin gene. Arg(E10) was previously found to form a hydrogen bond with the ligand in fluoro-, azido- and cyanomet derivatives of Aplysia limacina myoglobin, which lacks the distal His(E7) [Qin, J., La Mar, G. N., Ascoli, F., Bolognesi, M., & Brunori, M. (1992) J. Mol. Biol. 224, 891-897]. NMR analysis of the paramagnetically induced relaxation, hyperfine shift patterns, and dipolar connectivities shows that Arg(E10) also falls into the distal pocket in the engineered sperm whale myoglobin mutants and resides at an H-bonding distance from the Fe(3+)-bound cyanide. The rate constant for cyanide dissociation from the ferrous derivative was determined by stopped-flow experiments; the ligand stabilization achieved by Arg(E10) is similar to that exerted by His(E7) in wild-type sperm whale myoglobin, and both are very different from the His(E7)Val single mutant. Contrary to that for the wild-type, the cyanide dissociation rate constant for the mutant containing Arg(E10) is essentially independent of pH (from 6 to 9), as expected on the basis of the guanidinium group of Arg having a pK > 10. This finding is consistent with the NMR data in which the chemical shift of the Arg(E10) N epsilon H is insensitive to pH (6-9), as is also observed in Aphysia limacina cyanometmyoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of a distal site double mutant of sperm whale myoglobin at 1.6 Å resolution

The three-dimensional structure of sperm whale myoglobin His 64(E7)→Val,Thr 64(E10)→Arg double mutant has been studied by X-ray crystallography at 1.6 Å resolution, and refined to a crystallographic R-factor of 0.197. The Arg 67(E10) side chain is extended in the direction of the ligand binding site, and its NH1 atom is at a distance of 3.11 Å from the NH1 atom of Arg 45(CD3), which is also pointing towards the distal site. Both are kept in this position by hydrogen bonding and electrostatic interactions with a solvent sulfate ion, located amongst the two, on the protein surface. No liganded water molecule is present at the sixth coordination position of the Fe(III) heme.

Heterolytic versus homolytic peroxide bond cleavage by sperm whale myoglobin and myoglobin mutants

Despite extensive work on homolytic versus heterolytic peroxide bond cleavage by iron porphyrins, the nature of the corresponding reactions catalyzed by myoglobin (Mb) and hemoglobin (Hb) remains unclear. These hemoproteins react with peroxides to give a ferryl (Fe IV =O) complex and a protein radical, but the coupling of dioxygen bond cleavage to protein radical formation is obscure. This process is examined here with the help of 4-hydroperoxy-4-methyl-2,6-di-tert-butyl-cyclohexa-2,5-dien-1-one (BHTOOH), a compound that is converted heterolytically to an alcohol and homolytically to several rearranged products

Determination of the orientation of the magnetic axes of the cyano-MetMb complexes of point mutants of myoglobin by solution 1H NMR: influence of his E7 .fwdarw. Gly and Arg CD3 .fwdarw. Gly substitutions

A method is described for obtaining the axes of the diagonal paramagnetic susceptibility tensor for the low-spin cyanide complexes of distal point mutants of ferric sperm whale myoglobin (metMbCN). It relies on using the crystal coordinates of the wild-type (WT) protein for that portion of the molecule unperturbed by the point mutation, together with the experimental dipolar shifts, to search for the Euler rotation that correctly converts the crystal coordinates to the magnetic axes

Selective examination of heme protein azide ligand-distal globin interactions by vibrational circular dichroism

Vibrational circular dichroism (VCD) spectra of the antisymmetric stretch of azide ligated to the heme of a series of evolutionarily diverse and site-directed mutant hemoglobins and myoglobins are anomalously intense and demonstrate an intriguing sensitivity to subtle protein-ligand interactions. The antisymmetric stretch of the azide ligand covalently bound to the low-spin iron shows an anisotropy ratio of -9.5 X 104 for sperm whale and horse myoglobin which decreases to -8.0 X 104 for human and carp hemoglobin and Chironimus thummi thummi III monomeric hemoglobin. The VCD spectra of these heme-azide complexes depend upon the interactions of the azide ligand with distal heme pocket residues such as the E7 distal histidine and El 1 valine. The site-directed mutants of sperm whale (distal histidine substituted GIy E7) and human (distal valine substituted Asn El 1) myoglobin have vanishingly small anisotropy ratios (<-0.5 X 1(T 4), while elephant myoglobin (distal histidine substituted GIn E7) shows an anisotropy ratio of-6.4 X ICT 4. The azide ligand ionically bound to a high-spin iron shows a vanishingly small VCD intensity.

A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate.

Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix notation) to examine the effects of distal pocket size on the rates of ligand binding and autooxidation. Leu29 was replaced with Ala, Val, and Phe using the synthetic gene and Escherichia coli expression system of Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8961-8965). Structures of the ferric forms of Val29 and Phe29, and the oxy form of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography. The ferric mutant proteins are remarkably isomorphous with the wild type protein except in the immediate vicinity of residue 29. Thus, the protein structure in the distal pocket of myoglobin can accommodate either a large "hole" (i.e. Ala or Val) or a large side chain (i.e. Phe) at position 29 without perturbation of tertiary structure. Phe29 oxymyoglobin is also identical to the native oxy protein in terms of overall structure and interactions between the bound O2 and His64, Val68, Phe43, and Ile107. The distance between the nearest side chain atom of residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the Phe29 protein and 4.9 A in native myoglobin. The equilibrium constants for O2 binding to Ala29, Val29, and Leu29 (native) myoglobin are the same, approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29 protein is markedly greater, 15 x 10(6) M-1. This increase in affinity is due primarily to a 10-fold decrease in the O2 dissociation rate constant for the Phe29 mutant and appears to be the result of stabilizing interactions between the negative portion of the bound O2 dipole and the partially positive edge of the phenyl ring. Increasing the size of residue 29 causes large decreases in the rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37 degrees C. Thus, the Leu29----Phe mutation produces a reduced protein that is remarkably stable and is expressed in E. coli as 100% MbO2. The selective pressure to conserve Leu29 at the B10 position probably represents a compromise between reducing the rate of autooxidation and maintaining a large enough O2 dissociation rate constant to allow rapid oxygen release during respiration.

Control and recognition of anionic ligands in myoglobin

Equilibrium and kinetic experiments on site-directed mutants of a synthetic sperm whale myoglobin (Mb) gene have been performed. Results on the reactivity on both ferric and ferrous wild type and mutants Mb's are presented. Analysis of ligand binding to His(E7) Val and His(E7) Val-Thr(E10) Arg mutants compared to wild-type sperm whale, horse and Aplysia limaelna Mb's, shows that the introduction of an arginyl residue at the topological position E10 greatly enhances the stability of the various Mb:heme ligand adducts. Alternative mechanisms of ligand stabilization may therefore be operative in Mb's lacking the distal histidine.

Distal and proximal ligand interactions in heme proteins: correlations between carbon-oxygen and iron-carbon vibrational frequencies, oxygen-17 and carbon-13 nuclear magnetic resonance chemical shifts, and oxygen-17 nuclear quadrupole coupling constants in [C17O-] and [13CO]-labeled species

We have obtained the oxygen-17 nuclear magnetic resonance (NMR) spectra of a variety of C17O-labeled heme proteins, including sperm whale (Physeter catodon) myoglobin, two synthetic sperm whale myoglobin mutants (His E7----Val E7; His E7----Phe E7), adult human hemoglobin, rabbit (Oryctolagus cuniculus) hemoglobin, horseradish (Cochlearia armoracia) peroxidase (E.C. 1.11.1.7) isoenzymes A and C, and Caldariomyces fumago chloroperoxidase (E.C. 1.11.1.10), in some cases as a function of pH, and have determined their isotropic 17O NMR chemical shifts, delta i, and spin-lattice relaxation times, T1. We have also obtained similar results on a picket fence prophyrin, [5,10,15,20-tetrakis(alpha, alpha, alpha, alpha, alpha-pivalamidophenyl)porphyrinato]iron(II) (1-MeIm)CO, both in solution and in the solid state. Our results show an excellent correlation between the infrared C-O vibrational frequencies, v(C-O), and delta i, between v(C-O) and the 17O nuclear quadrupole coupling constant (e2qQ/h, derived from T1), and as expected between e2qQ/h and delta i. Taken together with the work of others on the 13C NMR of 13CO-labeled proteins, where we find an excellent correlation between delta i(13C) and v(Fe-C), our results suggest that IR and NMR measurements reflect the same interaction, which is thought to be primarily the degree of pi-back-bonding from Fe d to CO pi* orbitals, as outlined previously [Li, X.-Y., & Spiro, T.G. (1988) J. Am. Chem. Soc. 110, 6024]. The modulation of this interaction by the local charge field of the distal heme residue (histidine, glutamine, arginine, and possibly lysine) in a variety of species and mutants, as reflected in the NMR and IR measurements, is discussed, as is the effect of cysteine as the proximal heme ligand.

The role of Val68(E11) in ligand binding to sperm whale myoglobin. Site-directed mutagenesis of a synthetic gene.

Site-directed mutants of sperm whale myoglobin were prepared to probe the functional role of the highly conserved distal pocket valine residue, Val68(E11). This amino acid was replaced with Ala, Ile, and Phe to examine the effects of the side chain volume at position 68 on ligand binding. Three double mutants were also constructed in which the distal His64(E7) was replaced with Gly and Val68 was replaced with Ala, Ile, and Phe to determine the effects of size at position 68 in the absence of the distal histidine. Association and dissociation rate constants for O2, CO, and alkyl isocyanide binding were measured by stopped-flow rapid mixing, conventional flash, and laser photolysis techniques at pH 7, 20 degrees C. The association rate constants for the binding of all eight ligands to the single mutants decreased in the order Ala68 greater than Val68 (native) greater than Ile68 myoglobin, indicating that the 68(E11) residue is part of the overall kinetic barrier. A similar pattern was observed for the association constants of the double mutants: Gly64/Ala68 greater than Gly64/Val68 greater than Gly64/Ile68. Thus, increasing size of the E11 side chain inhibits the rate of ligand binding even in the absence of histidine at position 64. Substitution of Ala for Val68 had little effect on O2 affinity but did increase the affinities for CO and isocyanide binding. The affinities for all of the ligands were decreased for the Ile68 mutant. The ligand binding affinities for the Gly64/Ala68, Gly64/Val68, and Gly64/Ile68 myoglobins displayed an analogous trend to that of the single mutants, indicating that the equilibrium interactions between the position 64 and 68 side chains and the bound ligand are roughly additive. Both the association rate constants and dissociation rate constants for O2 and isocyanide binding were decreased for the Phe68 mutant myoglobin. These kinetic parameters result in little change in O2 affinity and an increase in isocyanide affinity, relative to the native protein. Thus, the large benzyl side chain of phenylalanine at position 68 inhibits the rate of ligand movement up to and away from the iron atom but not the final bound state.

Discrimination between Oxygen and Carbon Monoxide and Inhibition of Autooxidation by Myoglobin: Site-directed mutagenesis of the distal histidine

Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64→Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40–350-fold. The His-64→Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were ∼50–1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were ∼5–15-fold higher than those for the native protein. All mutants studied (except His-64→Tyr) showed ∼10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O2 and CO association and dissociation rate constants resulted in 3–14-fold increased CO affinities, 10–200-fold decreased O2 affinities, and 50–380-fold greater M (KCO/KO2) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.