Showing papers on "Myoglobin published in 1993"
TL;DR: In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms: at high [O2], direct dissociation of the neutral superoxide radical (HO2) from oxymyoglobin dominates, and this process is accelerated by decreasing pH, and at low [ O2], autooxidated occurs by a bimolecular reaction between molecular oxygen and deoxymyoglobin containing a weakly coordinated water molecule.
349 citations
TL;DR: The agreement between calculated and observed titration behavior suggests that this theoretical model captures much of the electrostatic behavior in this system, even though it ignores conformational fluctuations and the differences in mean structures that may exist between crystal and solution.
Abstract: Site-specific titration curves for 12 histidine residues in carbon monoxy sperm whale myoglobin (MbCO) have been determined from two-dimensional (2D) double quantum NMR experiments. Eight of these histidine residues are observed to titrate over the accessible pH range, and pK(a) values have been determined; bounds on the titration midpoints of the remaining four histidines are also reported. Results for residues 48, 81, and 119 differ significantly from those estimated from earlier, one-dimensional studies, but they are in good agreement with values recently determined for metaquomyoglobin. These experimental values (plus those determined earlier for tyrosine titrations) are compared to predictions from crystal structures of myoglobin using a numerical Poisson-Boltzmann model and a Monte Carlo treatment of the multiple-site titration. An extension of existing models is described that accounts for alternate tautomers for histidines. Calculations are reported using several choices for radii and charges, and for five crystal structures, in order to assess the sensitivity of the results to details of the calculations. In general, the agreement between calculated and observed titration behavior suggests that this theoretical model captures much of the electrostatic behavior in this system, even though it ignores conformational fluctuations and the differences in mean structures that may exist between crystal and solution. Interactions among titrating groups are often important; in general, these interactions lead to more gradual individual site titrations (the mean Hill coefficient is about 0.8), and in several cases the interactions are so strong that two side chains need to be considered as a unit and single residues may participate in two-step titrations. It is suggested that histidines involved in such two-step titrations and carboxylic acid residues with abnormally low pK(a) values in the native conformation may be involved in the acid-induced partial unfolding of MbCO.
215 citations
TL;DR: The folding propensities of two peptides (Mb-G and Mb-H), corresponding to the G- and H-helix segments of the myoglobin sequence, are described and a novel method for assessing the distribution of helical populations based on the relative magnitudes of medium-range d alpha beta (i,i+3) NOE connectivities is estimated.
Abstract: Myoglobin has been extensively studied as a model system for protein folding in vitro. As part of an ongoing study of myoglobin folding, we have synthesized a series of peptide fragments corresponding to portions of the sequence of the sperm whale protein. The conformational preferences of these peptides have been investigated by circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution. In this paper we describe the folding propensities of two peptides (Mb-G and Mb-H), corresponding to the G- and H-helix segments of the myoglobin sequence. The Mb-G peptide shows evidence of a very small population of helical conformations in aqueous solution, both by CD and NMR. By contrast, the monomeric Mb-H peptide is found by CD to adopt a significant population (ca. 30%) of ordered helix and by NMR to populate helical conformations in rapid dynamic equilibrium with unfolded states. The Mb-H peptide undergoes a well-characterized, concentration-dependent monomer-tetramer equilibrium. At peptide concentrations greater than 1 mM there is an increase in the population of helix, to approximately 85% according to the CD spectrum, through self-association to form a tetramer. Both medium-range NOE connectivities and a CD spectrum characteristic of ordered helix are observed at low peptide concentrations, establishing that helical conformations are present in the monomeric state of Mb-H. The relative helicity at various sites throughout the Mb-H peptide has been estimated using a novel method for assessing the distribution of helical populations based on the relative magnitudes of medium-range d alpha beta (i,i+3) NOE connectivities. The population of ordered helix is seen to be highest in the center of the peptide sequence; the ends of the peptide show evidence of pronounced fraying.
170 citations
TL;DR: The picosecond evolution of the tertiary conformation of myoglobin after photodissociation of MbCO was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13,110 cm-1 (763 nm) that is sensitive to the out-of-plane displacement of the iron.
Abstract: The picosecond evolution of the tertiary conformation of myoglobin (Mb) after photodissociation of MbCO was investigated at room temperature by probing band III, a weak iron-porphyrin charge-transfer transition near 13,110 cm-1 (763 nm) that is sensitive to the out-of-plane displacement of the iron. Upon photolysis, the iron moves out of the plane of the porphyrin, causing a blue-shift of band III and a concomitant change in the protein conformation. The dynamics for this functionally important motion are highly nonexponential, in agreement with recent molecular dynamics simulations [Kuczera, K., Lambry, J.-C., Martin, J.-L. & Karplus, M. (1993) Proc. Natl. Acad. Sci. USA 90, 5805-5807]. The conformational change likely affects the height of the barrier to ligand rebinding and may explain nonexponential NO rebinding.
133 citations
TL;DR: The ion spray mass spectra of three representative heme-containing proteins were studied and the charge state of iron in the expelled heme from myoglobin and hemoglobin appears to be 3+ but 2f for heme expelled from cytochrome c.
108 citations
TL;DR: It is demonstrated that the open conformation plays a significant role in determining the ligand association and dissociation rates and suggest that environmentally induced modulations of the open population could be used as a biomolecular control mechanism for the uptake and delivery of oxygen in muscle cells.
95 citations
TL;DR: Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin and shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process.
88 citations
TL;DR: As part of an extensive dissection of the folding pathway of myoglobin, a series of peptides corresponding to fragments of sperm whale myoglobin have been synthesized and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol.
Abstract: As part of an extensive dissection of the folding pathway of myoglobin, a series of peptides corresponding to fragments of sperm whale myoglobin have been synthesized, and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol. The behavior of short fragments corresponding to the sequences of the G- and H-helices of myoglobin and to the turn region between these helices has been described in accompanying papers. At the next level of complexity, peptide model compounds have been synthesized to explore the longer-range interactions which may take place in protein folding after initial secondary structure formation has occurred. A series of disulfide-bridged dimeric peptides containing the complete sequences of the G- and H-helices of myoglobin were synthesized and their conformational preferences examined. CD spectra indicate that disulfide-bridged peptides consisting of two H-helix sequences (Mb-HssH) and of one G- and one H-helix (Mb-GssH) are highly helical in water solution, as a result of intermolecular association. A 51-residue peptide, Mb-GH51, encompassing the entire G-H helical hairpin of myoglobin, including the turn sequence between the two helices, has been successfully synthesized by standard methods. This peptide was designed to be monomeric in aqueous solution. Mb-GH51 does not appear from CD spectra to contain any additional helix in water solution above what would be expected from an equimolar mixture of the G- and H-helix peptides. NMR spectra indicate that the turn conformation observed in shorter peptide fragments is retained in Mb-GH51 in high population.(ABSTRACT TRUNCATED AT 250 WORDS)
85 citations
TL;DR: It is shown that inclusion of a stabilizing buffer, such as ammonium acetate, in the protein solution is greatly beneficial to the ES/MS detection of intact noncovalent globin/heme complex.
81 citations
TL;DR: A series of peptide fragments of sperm whale myoglobin, corresponding to segments of the region between the G- and H-helices of the protein, have been synthesized and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol.
Abstract: A series of peptide fragments of sperm whale myoglobin, corresponding to segments of the region between the G- and H-helices of the protein, have been synthesized and their conformational preferences investigated using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution and in solvent mixtures containing water and trifluoroethanol The smallest fragment, Mb-GH5, a five-residue peptide with the sequence HPGDF corresponding to the connecting loop between the two helices in the folded protein, adopts highly populated turn conformations in aqueous solution A 25-residue peptide, Mb-GH25, containing the same sequence flanked by contiguous segments of the G- and H-helix sequences, was also found to contain a high proportion of conformers with a turn in this region No helix formation was observed in the flanking sequences in water solution, either in Mb-GH25 or in control 10-residue peptides (Mb-G10 and Mb-H10) with sequences corresponding to the G- and H-helix segments No additional helicity above that of the sum of the components was observed for Mb-GH25, indicating that a helical hairpin structure is not formed in the monomeric peptide in aqueous solution In the presence of TFE, ordered helix is formed in Mb-GH25 according to the CD spectrum, and NMR spectra indicate that this is localized in the N-terminal portion of the peptide NOESY spectra clearly show that the turn conformation is retained under these conditions(ABSTRACT TRUNCATED AT 250 WORDS)
77 citations
TL;DR: The X-ray crystal structure of the ligand-free ferric form of Aplysia limacina myoglobin has been refined at 1.7 A resolution, and its cyanide, thiocyanate and imidazole derivatives studied by difference Fourier techniques at atomic resolution.
TL;DR: The artificial intelligence-superexchange method of estimating the long-range electronic coupling in proteins is used to study electron-transfer reactions in Ru-modified cytochrome c and myoglobin derivatives.
Abstract: The artificial intelligence-superexchange method of estimating the long-range electronic coupling in proteins
that we have developed previously is used to study electron-transfer reactions in Ru-modified cytochrome c and
myoglobin derivatives. Good correlations between theoretical and experimental rate constants are obtained
using the present method. Amino acid paths for electron transfer are analyzed.
TL;DR: It is shown that the extent of the iron displacement out of the plane of the porphyrin nitrogens is the main structural parameter controlling the Fe-NHis band features, such as the dependence of its frequency and intensity on the protein conformation and number of the axial ligands.
TL;DR: The effects of mutation of the conserved serine92 residue to alanine, valine, and leucine in pig myoglobin have been determined and the overall affinities are increased regardless of the chemical nature of the liganded species, indicating that the reactivity of the heme iron itself has been raised.
Abstract: The effects of mutation of the conserved serine92 residue to alanine, valine, and leucine in pig myoglobin have been determined. In myoglobin crystal structures, the hydroxyl group of serine92 is within hydrogen-bonding distance of the N delta-H of histidine93, whose N epsilon coordinates the iron atom of the heme prosthetic group. The association equilibrium constants of the ferrous forms of the mutant myoglobins for O2, CO, and methyl and ethyl isocyanide are increased 1.3-13-fold relative to the wild-type protein. The rates of azide association with the mutant ferric proteins at neutral pH are decreased by factors of 2-5 consistent with an increased affinity for the iron-bound water molecule which must be displaced. The dissociation rates for azide appear to be decreased 4-10-fold, suggesting that the affinity of the mutant proteins for this ligand is also higher. Thus, the overall affinities are increased regardless of the chemical nature of the liganded species, indicating that the reactivity of the heme iron itself has been raised. Time courses for association of methyl and ethyl isocyanide at high concentrations show fast and slow phases in which the absorbance at 445 nm drops and then rises, respectively. Comparison of these traces with spectra following the reaction of isocyanide ligands with chelated proton heme in soap micelles indicates that the slow phase is associated with the breaking of the iron-proximal histidine bond and the binding of a second isocyanide species in the proximal heme pocket.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: If that the barrier to rebinding depends on the out-of-plane iron position, the time dependence obtained from the simulation can explain the nonexponential room-temperature geminate recombination of NO.
Abstract: Molecular dynamics simulations of myoglobin after ligand photodissociation show that the out-of-plane motion of the heme iron has a rapid subpicosecond phase followed by a slower nonexponential process involving more global protein relaxation. Individual trajectories show rather different behavior, suggesting there is an inhomogeneous component to the relaxation. The calculated time dependence of the iron motion over 100 ps is in excellent agreement with the frequency shift of band III of the heme group [see Lim, M., Jackson, T. A. & Anfinrud, P. A. (1993) Proc. Natl. Acad. Sci. USA 90, 5801-5804]. If that the barrier to rebinding depends on the out-of-plane iron position, the time dependence obtained from the simulation can explain the nonexponential room-temperature geminate recombination of NO.
TL;DR: Examination of the catalytic activities of the possible Tyr-->Phe mutants, the His-64-->Val mutant, and a protein combining all the tyrosine and histidine mutations indicates that tyrosines may participate in, but are not essential for, protein-mediated epoxidation.
TL;DR: The ratio NADH/NAD is decreased in well-oxygenated cells with increased work; steady-state NAD reduction is increased with increasedWork when oxygen delivery is limited; and functional myoglobin ensures an oxygen supply to the mitochondria of working cells.
TL;DR: A method for the measurement of discrimination between 16 O and 18 O in the reversible interaction of dioxygen with proteins is described and sees a trend in equilibrium isotope effects in proceeding from ancestral oxygen carriers, hemocyanin and hemerythrin, 18 (K) obs =1.0184 and 1.0113, respectively.
Abstract: A method for the measurement of discrimination between 16 O and 18 O in the reversible interaction of dioxygen with proteins is described. According to this method, total dioxygen (±protein) in reaction vessels is converted to carbon dioxide and analyzed by isotope ratio mass spectrometry. We see a trend in equilibrium isotope effects in proceeding from ancestral oxygen carriers, hemocyanin and hemerythrin, 18 (K) obs =1.0184 and 1.0113, respectively, to myoglobin and hemoglobin, 18 (K) obs =1.0054 and 1.0039, respectively
TL;DR: A transition-state analysis suggests that protein conformational relaxation coupled to the displacement of the iron from the heme plane is an unlikely cause for the nonexponential rebinding of nitric oxide.
Abstract: We have begun to use molecular dynamics to simulate the kinetics of nitric oxide rebinding to myoglobin after photodissociation. Rebinding was simulated using a potential function that switches smoothly between a nonbinding potential and a binding potential as a function of the position and orientation of the ligand, with no barrier arising from the crossing of potential surfaces of different electron spin. In 96 of 100 trajectories, the ligand rebound in < 15 ps. The kinetic progress curve was obtained by determining the time in each trajectory at which the ligand rebound and then calculating the fraction of unbound ligands as a function of time. The curve can be well reproduced by a simple model based on the dynamics of a Langevin particle moving on a one-dimensional potential of mean force calculated from nonreactive protein trajectories. The rate of escape from the energy well adjacent to the heme is in good agreement with the value calculated from experimental data, suggesting that a multiple-well model provides a plausible explanation for the nonexponential rebinding kinetics. A transition-state analysis suggests that protein conformational relaxation coupled to the displacement of the iron from the heme plane is an unlikely cause for the nonexponential rebinding of nitric oxide.
TL;DR: A strong correlation is found between the final barrier height to CO recombination and theCO stretch frequency: as compared to wild type, the barrier is smaller in those mutants that have a higher CO stretch frequency (vCO) and vice versa.
Abstract: The infrared spectra of CO bound to human myoglobin and myoglobin mutants at positions His-64, Val-68, Asp-60, and Lys-45 on the distal side have been measured between 100 and 300 K. Large differences are observed with mutations at His-64 and Val-68 as well as with temperature and pH. Although distal His-64 is found to affect CO bonding, Val-68 also plays a major role. The variations are analyzed qualitatively in terms of a simple model involving steric interaction between the bound CO and the distal residues. A strong correlation is found between the final barrier height to CO recombination and the CO stretch frequency: as compared to wild type, the barrier is smaller in those mutants that have a higher CO stretch frequency (vCO) and vice versa. Possible reasons for this correlation are discussed. It is emphasized that the temperature and pH dependence of both the kinetics and the infrared spectra must be measured to obtain a consistent picture.
TL;DR: Results are consistent with a primary role for the structure of the distal pocket in determining diffusion of ligands away from the metal atom and as a result the time course of picosecond ligand rebinding.
TL;DR: The correlation of electronic coupling with tunneling length for myoglobin is improved significantly by the inclusion of multiple pathways, and single dominant pathways adequately describe the electronic couplings in cytochrome c but do not satisfactorily account for the myoglobin couplings.
Abstract: Site-directed mutants of human myoglobin have been prepared and characterized; each protein has a single
surface-modifiable histidine (at position 48, 70, or 83). The proteins were modified by covalent attachment of pentaammineruthenium (a_5Ru) to the surface histidine and substitution of zinc mesoporphyrin IX diacid (ZnP) for the heme. Donor-acceptor separations (edge-edge distances d) in the modified proteins are 9.5 A, His70; 12.7 A, His48; and 15.5 A, His83. Rates of photoinduced electron transfer in these ruthenium-modified myoglobins were measured by transient absorption spectroscopy. The ^(3)ZnP* → Ru^3+ rate constants are 1.6 × 10^7 (His70), 7.2 × 10^4 (His48), and 4.0 × 10^2 s^-1 (His83) (-ΔG°= 0.82 eV); charge-recombination (Ru^2+ → ZnP^+) rates are 1.1 × 10^5 (His48) and 7.3 × 10^2 s^-1 (His83) (-ΔG° = 0.96 eV). Activationless (maximum) rates assuming h = 1.3 eV are 7.2 X lo7 (His70), 3.3 × 10^5 (His48) and 1.8 × 10^3 s^-1 (His83). Distant electronic couplings, which limit the maximum rates in the modified myoglobins, have been analyzed along with data from Ru-modified cytochromes c in terms of a tunneling pathway model. Single dominant pathways adequately describe the electronic couplings in cytochrome c but do not satisfactorily account for the myoglobin couplings. The correlation of electronic coupling with tunneling length for myoglobin is improved significantly by the inclusion of multiple pathways.
TL;DR: It is found that ligand recombination is nonexponential for all the mutants and that both the geminate amplitude and rate show large variations, and a molecular mechanism for ligand binding that is consistent with the results is discussed, and supporting evidence for this mechanism is examined.
Abstract: The kinetics of CO recombination to site-specific mutants of human myoglobin have been studied by flash photolysis in the temperature range 250-320 K on the nanosecond to second time scale in 75% glycerol at pH 7. The mutants were constructed to examine specific proposals concerning the roles of Lys 45, Asp 60, and Val 68 in the ligand binding process. It is found that ligand recombination is nonexponential for all the mutants and that both the geminate amplitude and rate show large variations. The results are interpreted in terms of specific models connecting the dynamics and structure. It is shown that removal of the charged group at position 45 does not substantially affect the barrier height for escape or entry of the ligand; therefore the breakage of the salt bridge linking Lys 45, Asp 60, and a heme propionate is ruled out as the rate-determining barrier for this process. On the other hand, it is found that the escape barrier decreases roughly as size of the residue at position 68 increases, in the order Ala > Val > Asn > Leu. The residue at position 68 is also a major contributor to the final barrier to rebinding, but the barrier height shows no correlation with residue size and is more dependent on the stereochemistry of the residue. A molecular mechanism for ligand binding that is consistent with the results is discussed, and supporting evidence for this mechanism is examined
TL;DR: Haem proteins had a greater prooxidant effect than inorganic iron in raw and heated pork muscle residue when these pro oxidants were present at levels approaching those in red meats.
01 Jan 1993
TL;DR: Hemes and heme proteins are vital components of essentially every cell of every living organism and the transport of electrons in the respiratory chains of organisms as diverse as bacteria, yeasts, algae, plants, and animals.
Abstract: Hemes and heme proteins are vital components of essentially every cell of every living organism. Their roles in cells include
1
the transport of dioxygen in the red blood cells of higher animals (hemoglobin);
2
the storage of dioxygen in the muscles of higher animals (myoglobin);
3
the transport of electrons in the respiratory chains of organisms as diverse as bacteria, yeasts, algae, plants, and animals, and in photosynthetic cells from those of the simplest photosynthetic bacteria to those of higher plants (cytochromes a, b, c, d, f);
4
synthesis, modification and degradation of fatty acids, steroid and adrenal hormones, anesthetics and xenobiotics (cytochromes P-450);
5
activation and metabolism of hydrogen peroxide (peroxidases, myeloperoxidase, haloperoxidases, catalases, etc.); and
6
metabolism of the oxides of nitrogen and sulfur (nitrite reductase, sulfite oxidase, etc.).
TL;DR: Linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix.
Abstract: The peptide backbones in folded native proteins contain distinctive secondary structures, alpha-helices, beta-sheets, and turns, with significant frequency. One question that arises in folding is how the stability of this secondary structure relates to that of the protein as a whole. To address this question, we substituted the alpha-helix-stabilizing alanine side chain at 16 selected sites in the sequence of sperm whale myoglobin, 12 at helical sites on the surface of the protein, and 4 at obviously internal sites. Substitution of alanine for bulky side chains at internal sites destabilizes the protein, as expected if packing interactions are disrupted. Alanine substitutions do not uniformly stabilize the protein, either in capping positions near the ends of helices or at mid-helical sites near the surface of myoglobin. When corrected for the extent of exposure of each side chain replaced by alanine at a mid-helix position, alanine replacement still has no clear effect in stabilizing the native structure. Thus linkage between the stabilization of secondary structure and tertiary structure in myoglobin cannot be demonstrated, probably because of the relatively small free energy differences between side chains in stabilizing isolated helix. By contrast, about 80% of the variance in free energy observed can be accounted for by the loss in buried surface area of the native residue substituted by alanine. The differential free energy of helix stabilization does not account for any additional variation.
TL;DR: The reactivity of Trolox C, a water-soluble analog of vitamin E, toward protein radicals formed during the oxidation of metmyoglobin and the therefrom derived molecular products was examined in terms of the ability of the phenolic antioxidant to prevent specific oxidative reactions involving tyrosyl radicals and to reduce the molecular products to functional hemoproteins.
TL;DR: 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.
TL;DR: Measurements for a series of single amino acid mutants of human myoglobin on the distal side of the heme pocket are reported to examine specific structural determinants involved in this conformational relaxation and to determine the nature of the coupling between relaxation and the functional process of ligand binding.
Abstract: We have recently reported spectroscopic evidence for structural relaxation of myoglobin (Mb) following photodissociation of MbCO (Lambright, D. G., Balasubramanian, S., & Boxer, S. G. (1991) Chem. Phys. 158,249-2601, In this paper we report measurements for a series of single amino acid mutants of human myoglobin on the distal side of the heme pocket (positions 45, 64, and 68) in order to examine specific structural determinants involved in this conformational relaxation and to determine the nature of the coupling between relaxation and the functional process of ligand binding. The kinetics of ligand binding and conformational relaxation were monitored by transient absorption spectroscopy in the Soret spectral region, and the results are analyzed using a four-state ligand binding model. Two principal results emerge: (1) amino acid substitutions in the distal heme pocket affect the kinetics of the nonequilibrium conformational relaxation and (2) the rate of ligand escape from the protein matrix is not significantly perturbed by the distal heme pocket mutations.