Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

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 approximately 50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were approximately 5-15-fold higher than those for the native protein. All mutants studied (except His-64----Tyr) showed approximately 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.     

The effects of amino acid substitution at position E7 (residue 64) on the kinetics of ligand binding to sperm whale myoglobin

Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64----Gly substitution produced a sterically unhindered myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Gly64 (approximately 10(7) M-1 s-1) much greater than Val64 approximately Leu64 (approximately 10(6) M-1 s-1) greater than Met64 greater than Phe64 approximately His64 approximately Gln64 (10(5)-10(3) M-1 s-1) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimolecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the His64----Arg and His64----Asp mutants were abnormally high (1-5 x 10(6) M-1 s-1), suggesting that Arg64 and Asp64 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the association rate constants for O2 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except His64----Gln approached or were larger than those for chelated model heme (i.e. approximately 1 x 10(8) M-1 s-1 for O2 and approximately 1 x 10(7) M-1 s-1 for CO), whereas the corresponding rate parameters for myoglobin containing either Gln64 or His64 were 5- to 10-fold smaller. This result suggests that a major kinetic barrier for O2 and CO binding to native myoglobin may involve disruption of polar interactions between His64 and water molecules found in the distal pocket of deoxymyoglobin. Finally, the rate and equilibrium parameters for O2 and CO binding to the His64----Gln, His64----Val, and His64----Leu mutants were compared to those reported previously for Asian elephant myoglobin (Gln-E7), Aplysia limacina myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).     

Controlling ligand binding in myoglobin by mutagenesis.

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.     

EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants.

Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli. The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy. We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule. Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket. To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val. Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue.     

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution 1H nuclear magnetic resonance determination of hydrogen bonding of the E10 (66) Arg side-chain to the bound ligand in Aplysia cyano-met myoglobin.

A combined one-dimensional nuclear Overhauser effect, paramagnetic-induced relaxation and two-dimensional sequence-specific 1H n.m.r. assignment of the spectrum of portions of the distal pocket of Aplysia cyano metMyoglobin (metMbCN) has been carried out in order to establish the presence and identity of distal residues in the heme pocket. In the absence of the usual distal E7 His in Aplysia Mb (E7 Val), the sequence-specific assignment of the E7 and E10 residues, together with their hyperfine shift patterns, relaxivities and dipolar connectivities to each other and the remainder of the E helix, reveal that the E10 Arg is turned into the pocket and hydrogen bonds to the bound cyanide group. We have previously found a similar rearrangement of the E10 Arg in Aplysia fluoro metMyoglobin, and the stabilizing effect of this residue was proposed to be responsible for the slow rate of cyanide dissociation from rapidly reduced ferrous Aplysia myoglobin. Based on the similar distal E7 His hydrogen-bonding interaction to the bound ligand in the crystal of sperm whale MbO2 and in solution of its cyano met complex, we propose that the E10 Arg similarly hydrogen bonds to the bound O2 in Aplysia MbO2 and accounts for its strong ligand binding and slow dissociation rate.     

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.     

Myoglobin mutants giving the largest geminate yield in CO rebinding in the nanosecond time domain.

We have measured the rebinding of carbon monoxide (CO) to some distal mutants of myoglobin (Mb) in the time range from 10(-8) to 10(-1) s by flash photolysis, in which the photodissociated CO rebinds to the heme iron without escaping to the solvent water from the protein matrix. We have found that the double mutants [His64-->Val/Val68-->Thr (H64V/V68T) and His64-->Val/Val68-->Ser (H64V/V68S)] have an extremely large geminate yield (70-80%) in water at 5 degreesC, in contrast to the 7% of the geminate yield of wild-type Mb. The CO geminate yields for these two mutants are the largest in those of Mb mutants reported so far, showing that the two mutants have a unique heme environment that favors CO geminate rebinding. Comparing the crystal structures and 1H-NMR and vibrational spectral data of H64V/V68T and H64V/V68S with those of other mutants, we discuss factors that may control the nanosecond geminate CO rebinding and CO migration in the protein matrix.     

Tyrosine B10 inhibits stabilization of bound carbon monoxide and oxygen in soybean leghemoglobin

Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of a library of E7, E11, and B10 mutants indicate significant differences in the role of electrostatic interactions in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin, strong hydrogen bonds from several closely related conformations of the distal histidine (His(E7)) side chain preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of His(E7) is confined to a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of Tyr(B10) appears to "fix" the position of His(E7), probably by donating a hydrogen bond to the Ndelta atom of the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established by a favorable staggered geometry of the proximal histidine. The interaction between His(E7) and Tyr(B10) prevents overstabilization of bound oxygen. If hydrogen bonding from His(E7) were as strong as it is in mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport in root nodules.     

Solution (1)H NMR study of the influence of distal hydrogen bonding and N terminus acetylation on the active site electronic and molecular structure of Aplysia limacina cyanomet myoglobin

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

Ligand binding to a hemoprotein lacking the distal histidine. The myoglobin from aplysia limacina (Val(E7))

The time course of ligand recombination to the myoglobin from Aplysia limacina, which has Val(E7), was measured following photolysis by flashes of 35 ps to 300 ns with a time resolution of 10 ps or 1 ns. CO shows only biomolecular recombination. O2 has a small geminate reaction with a half-time of tens of picoseconds, but no nanosecond geminate reaction. NO has two picosecond relaxations with half-times of 70 ps (15%) and 1 ns (80%) and one nanosecond relaxation with a half-time of 4.6 ns. The biomolecular rates for O2 and NO are the same: 2 x 10(7) M-1 s-1. Methyl and ethyl isonitriles have a geminate reaction with a half-time of 35 ps. Ethyl isonitrile has, in addition, a nanosecond relaxation (25%) with a half-time of 100 ns. t-Butyl isonitrile has four geminate relaxations (10 ps, 35 ps, 1 ns, and 1 microseconds). Analysis of the results suggests much easier movement of ligand between the heme pocket and the exterior than in sperm whale myoglobin (His(E7]. The reactivity of the heme is little different, placing the effect of the differences from sperm whale myoglobin on the distal side of the heme.     

Application of molecular dynamics and free energy perturbation methods to metalloporphyrin-ligand systems II: CO and dioxygen binding to myoglobin.

The protein contribution to the relative binding affinity of the ligands CO and O2 toward myoglobin (Mb) has been simulated using free energy perturbation calculations. The tautomers of the His E7 residue are different for the oxymyoglobin (MbO2) and carboxymyoglobin (MbCO) systems. This was modeled by performing two-step calculations that mutate the ligand and mutate the His E7 tautomers in separate steps. Differences in hydrogen bonding to the O2 and CO ligands were incorporated into the model. The O2 complex was calculated to be 2-3 kcal/mol more stable than the corresponding CO complex when compared to the same difference in an isolated heme control. This value agrees well with the experimental value of 2.0 kcal/mol. In qualitative agreement with experiments, the Fe-C-O bond is found to be bent (theta = 159.8 degrees) with a small tilt (theta = 6.2 degrees). The contributions made by each of the 29 residues--within the 9.0-A radius of the iron atom--to the free energy difference are separated into van der Waals and electrostatic contributions; the latter contributions are dominant. Aside from the proximal histidine and the heme group, the residues having the largest difference in free energy in mutating MbO2-->MbCO are His E7, Phe CD1, Phe CD4, Val E11, and Thr E10.     

Human carbonic anhydrase I: Effect of specific site mutations on its function

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.     

X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin at 2.0 A resolution. Stabilization of the fluoride ion by hydrogen bonding to Arg66 (E10)

The X-ray crystal structure of the fluoride derivative of Aplysia limacina ferric myoglobin has been solved and refined at 2.0 A resolution; the crystallographic R-factor is 13.6%. The fluoride ion binds to the sixth co-ordination position of the heme iron, 2.2 A from the metal. Binding of the negatively charged ligand on the distal side of the heme pocket of this myoglobin, which lacks the distal His, is associated with a network of hydrogen bonds that includes the fluoride ion, the residue Arg66 (E10), the heme propionate III, three ordered water molecules and backbone or side-chain atoms from the CD region. A comparison of fluoride and oxygen dissociation rate constants of A. limacina myoglobin, sperm whale (Physeter catodon) myoglobin and Glycera dibranchiata monomeric hemoglobin, suggests that the conformational readjustment of Arg66 (E10) in A. limacina myoglobin may represent the molecular basis for ligand stabilization, in the absence of a hydrogen-bond donor residue at the distal E7 position.     

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.