Showing papers on "Myoglobin published in 2003"
TL;DR: The heart and those striated muscles that contract for long periods, having available almost limitless oxygen, operate in sustained steady states of low sarcoplasmic oxygen pressure that resist change in response to changing muscle work or oxygen supply.
Abstract: The heart and those striated muscles that contract for long periods, having available almost limitless oxygen, operate in sustained steady states of low sarcoplasmic oxygen pressure that resist change in response to changing muscle work or oxygen supply. Most of the oxygen pressure drop from the erythrocyte to the mitochondrion occurs across the capillary wall. Within the sarcoplasm, myoglobin, a mobile carrier of oxygen, is developed in response to mitochondrial demand and augments the flow of oxygen to the mitochondria. Myoglobin-facilitated oxygen diffusion, perhaps by virtue of reduction of dimensionality of diffusion from three dimensions towards two dimensions in the narrow spaces available between mitochondria, is rapid relative to other parameters of cell respiration. Consequently, intracellular gradients of oxygen pressure are shallow, and sarcoplasmic oxygen pressure is nearly the same everywhere. Sarcoplasmic oxygen pressure, buffered near 0.33 kPa (2.5 torr; equivalent to approximately 4 micro mol l(-1) oxygen) by equilibrium with myoglobin, falls close to the operational K(m) of cytochrome oxidase for oxygen, and any small increment in sarcoplasmic oxygen pressure will be countered by increased oxygen utilization. The concentration of nitric oxide within the myocyte results from a balance of endogenous synthesis and removal by oxymyoglobin-catalyzed dioxygenation to the innocuous nitrate. Oxymyoglobin, by controlling sarcoplasmic nitric oxide concentration, helps assure the steady state in which inflow of oxygen into the myocyte equals the rate of oxygen consumption.
470 citations
TL;DR: In this paper, the normal modes of myoglobin, their lifetimes, the speed of sound, and mean free path were calculated to determine the coefficient of thermal conductivity and thermal diffusivity for the protein.
Abstract: The normal modes of myoglobin, their lifetimes, the speed of sound, and mean free path are calculated to determine the coefficient of thermal conductivity and thermal diffusivity for the protein. A propensity is found for frequency differences of pairs of normal modes localized to nearby regions of the protein to be several hundred cm-1. As a result, the anharmonic decay rate of higher frequency, localized normal modes, calculated by perturbation theory, is typically nearly independent of temperature, consistent with results of pump−probe studies on myoglobin. The thermal diffusivity of myoglobin at 300 K is calculated to be 14 A2 ps-1, the same as the value for water. The thermal conductivity at 300 K is found to be 2.0 mW cm-1 K-1, about one-third the value for water.
162 citations
TL;DR: It is demonstrated that surface proteins contribute to the binding of hemoproteins to S. pyogenes, and an ABC transporter from the iron complex family named sia for streptococcal iron acquisition is identified, which is part of a highly conserved, iron regulated, 10-gene operon.
Abstract: The hemolytic Streptococcus pyogenes can use a variety of heme compounds as an iron source. In this study, we investigate hemoprotein utilization by S. pyogenes. We demonstrate that surface proteins contribute to the binding of hemoproteins to S. pyogenes. We identify an ABC transporter from the iron complex family named sia for streptococcal iron acquisition, which consists of a lipoprotein (siaA), membrane permease (siaB), and ATPase (siaC). The sia transporter is part of a highly conserved, iron regulated, 10-gene operon. SiaA, which was localized to the cell membrane, could specifically bind hemoglobin. The operon's first gene encodes a novel bacterial protein that bound hemoglobin, myoglobin, heme-albumin, and hemoglobin-haptoglobin (but not apo-haptoglobin) and therefore was named Shr, for streptococcal hemoprotein receptor. PhoZ fusion and Western blot analysis showed that Shr has a leader peptide and is found in both membrane-bound and soluble forms. An M1 SF370 strain with a polar mutation in shr was more resistant to streptonigrin and hydrogen peroxide, suggesting decreased iron uptake. The addition of hemoglobin to the culture medium increased cell resistance to hydrogen peroxide in SF370 but not in the mutant, implying the sia operon may be involved in hemoglobin-dependent resistance to oxidative stress. The shr mutant demonstrated reduced hemoglobin binding, though cell growth in iron-depleted medium supplemented with hemoglobin, whole blood, or ferric citrate was not affected, suggesting additional systems are involved in hemoglobin utilization. SiaA and Shr are the first hemoprotein receptors identified in S. pyogenes; their possible role in iron capture is discussed.
154 citations
TL;DR: A method that is capable of rapid on-line digestion and lending itself to high-throughput identification of proteins is demonstrated, applied to the digestion of human transferrin, a proteolytically resistant protein.
Abstract: Proteolytic digestion is an important step in protein identification by peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing. Traditional methods of protein digestion require extended incubation times and have difficulty with proteolytically resistant proteins. Here, we describe a method in which a protein solution was combined with a mixed aqueous-organic solution (methanol, isopropanol, or acetonitrile) and passed through a microcolumn containing immobilized trypsin. Myoglobin sequence coverage was high (>85%) in all three solvents, and differences in spectra were seen among the different solution conditions. Notably, methanol-based digestions produced fewer missed cleavages while acetonitrile-based digestions produced the most peptides and the most intense mass spectra. Flow rates through the column were varied from 0.5 to 15 micro L/min, corresponding to column residence times of 78 and 2.6 s, respectively. All flow rates produced high sequence coverage of myoglobin, although, at higher flow rates, more missed cleavages were observed. No significant increase in undigested myoglobin was observed with flow rates up to 15 micro L/min. The described method was applied to the digestion of human transferrin (hTf), a proteolytically resistant protein. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analysis detected 42 peptides covering 46% of the hTf sequence. The traditional aqueous method resulted in 12 peptides (8% sequence coverage) only when high concentrations of trypsin were used. Lastly, digestion of low nanomolar myoglobin was shown to produce detectable peptides and resulted in a correct database hit. Thus, we demonstrate a method that is capable of rapid on-line digestion, thereby lending itself to high-throughput identification of proteins.
111 citations
TL;DR: To examine the relevance of these internal docking sites for physiological ligand binding, room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form is performed, providing a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.
Abstract: Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for p...
93 citations
TL;DR: It is reported that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin, and identification of the receptors for tubular uptake ofMyoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.
Abstract: Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in rena...
78 citations
TL;DR: It is demonstrated that myoglobin serves as an important cytoplasmic buffer of iNOS-derived NO, which determines the functional consequences ofiNOS overexpression, and is fully antagonized by the NOS inhibitor S-ethylisothiourea.
Abstract: Elevated cardiac levels of nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) have been implicated in the development of heart failure. The surprisingly benign phenotype of recently generated mice with cardiac-specific iNOS overexpression (TGiNOS) provided the rationale to investigate whether NO scavenging by oxymyoglobin (MbO 2 ) yielding nitrate and metmyoglobin (metMb) is involved in preservation of myocardial function in TGiNOS mice. 1 H nuclear magnetic resonance (NMR) spectroscopy was used to monitor changes of cardiac myoglobin (Mb) metabolism in isolated hearts of wild-type (WT) and TGiNOS mice. NO formation by iNOS resulted in a significant decrease of the MbO 2 signal and a concomitantly emerging metMb signal in spectra of TGiNOS hearts only (ΔMbO 2 : −46.3±38.4 μmol/kg, ΔmetMb: +41.4±17.6 μmol/kg, n=6; P P 31 P NMR spectroscopy (eg, phosphocreatine: 13.3±1.3 mmol/L (TGiNOS) versus 15.9±0.7 mmol/L (WT), n=6; P S -ethylisothiourea. Our findings demonstrate that myoglobin serves as an important cytoplasmic buffer of iNOS-derived NO, which determines the functional consequences of iNOS overexpression.
78 citations
TL;DR: A dynamic model is developed that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.
Abstract: We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, AI and AII, with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, ...
56 citations
TL;DR: In this paper, Gasyna et al. used resonance Raman studies of the annealed samples of oxygenated myoglobin at cryogenic temperatures to identify the key ν(Fe−O) stretching mode of the Fe-OOH fragment.
Abstract: In agreement with previous reports (Gasyna, Z. FEBS Lett. 1979, 106, 213−218 and Leibl, W.; Nitschke, W.; Huettermann, J. Biochim. Biophys. Acta 1986, 870, 20−30) radiolytically reduced samples of oxygenated myoglobin at cryogenic temperatures have been shown by optical absorption and EPR studies to produce directly the peroxo-bound myoglobin at 77 K. Annealing to temperatures near 185 K induces proton transfer, resulting in the formation of the hydroperoxo heme derivative. Resonance Raman studies of the annealed samples has permitted, for the first time, the direct observation of the key ν(Fe−O) stretching mode of the physiologically important Fe-OOH fragment of this ubiquitous intermediate. The assignment of this mode to a feature appearing at 617 cm-1 is strongly supported by documentation of a 25 cm-1 shift to lower energy upon substitution with 18O2 and by a 5 cm-1 shift to lower energy for samples prepared in solutions of deuterated solvent.
56 citations
TL;DR: This study demonstrates the use of electrospray mass spectrometry in conjunction with rapid online mixing ("time-resolved" ESI-MS) for monitoring protein conformational dynamics under equilibrium conditions.
Abstract: This study demonstrates the use of electrospray mass spectrometry in conjunction with rapid online mixing (“time-resolved” ESI-MS) for monitoring protein conformational dynamics under equilibrium conditions. The hydrogen/deuterium exchange (HDX) kinetics of mildly denatured myoglobin (Mb) at pD 9.3, in the presence of 27% acetonitrile, were studied with millisecond time resolution. Analytical ultracentrifugation indicates that the average protein compactness under these solvent conditions is similar to that of native holomyoglobin (hMb). The mass spectrum shows protein ions in a wide array of charge and heme binding states, indicating the presence of multiple coexisting conformations. The experimental approach used allows the HDX kinetics of all of these species to be monitored separately. A combination of EX1 and EX2 behavior was observed for hMb ions in charge states 7+ to 9+, which predominantly represent nativelike hMb in solution. The EX1 kinetics are biphasic, indicating the presence of two protein ...
51 citations
TL;DR: This paper investigated the vibrational dynamics of proteins in amorphous hydrated films of lysozyme and myoglobin using polarization-selective time-domain Raman spectroscopy.
TL;DR: The crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein, and suggests distal pocket influences on FeNO geometry.
Abstract: The interactions of nitric oxide (NO) and organic nitroso compounds with heme proteins are biologically important, and adduct formation between NO-containing compounds and myoglobin (Mb) have served as prototypical systems for studies of these interactions. We have prepared crystals of horse heart (hh) MbNO from nitrosylation of aqua-metMb crystals, and we have determined the crystal structure of hh MbNO at a resolution of 1.9 A. The Fe-N-O angle of 147° in hh MbNO is larger than the corresponding 112° angle previously determined from the crystal structure of sperm whale MbNO (Brucker et al., Proteins 1998;30:352–356) but is similar to the 150° angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al., J Am Chem Soc 1998;120:10827–10836). The Fe-N(O) bond length of 2.0 A (this work) is longer than the 1.75 A distance determined from the XAFS study and suggests distal pocket influences on FeNO geometry. The nitrosyl N atom is located 3.0 A from the imidazole Nϵ atom of the distal His64 residue, suggesting electrostatic stabilization of the FeNO moiety by His64. The crystal structure of the nitrosoethane adduct of ferrous hh Mb was determined at a resolution of 1.7 A. The nitroso O atom of the EtNO ligand is located 2.7 A from the imidazole Nϵ atom of His64, suggesting a hydrogen bond interaction between these groups. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein. Proteins 2003. © 2003 Wiley-Liss, Inc.
TL;DR: Structural pictures of the Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, are obtained and the distance of the water molecule is obtained even after dissociation from the iron, with a 0.16-A statistical error.
TL;DR: It is suggested that in addition to the measurement of the concentration of MA, immunohistochemical staining of myoglobin, HSP70, 8-OH-dG, 4-HNE, and SOD offers important information for the diagnosis of MA poisoning.
15 Jul 2003
TL;DR: A new method for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins.
Abstract: A new method is presented for monitoring the conformational stability of various parts of a protein that is physically adsorbed onto nanometer-sized silica particles. The method employs hydrogen/deuterium (H/D) exchange of amide hydrogens, a process that is extremely sensitive to structural features of proteins. The resulting mass increase is analyzed with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Higher structural specificity is obtained by enzymatically cleaving the adsorbed proteins prior to mass spectrometric analysis. The mass increases of four peptic fragments of myoglobin are followed as a function of the H/D exchange time. The four peptic fragments cover 90% of the myoglobin structure. Two of the peptic fragments, located in the middle of the myoglobin sequence and close to the heme group, do not show any adsorption-induced changes in their structural stability, whereas the more stable C- and N-terminal fragments are destabilized. Interestingly, for the N-terminal fragment, comprising residues 1-29, two distinct and equally large conformational populations are observed. One of these populations has a stability similar to that in solution (-23 kJ/mol), whereas the other population is highly destabilized upon adsorption (-11 kJ/mol).
TL;DR: Perfusion with 5% red blood cells in the perfusate increases the baseline mean myoglobin saturation and better preserves cardiac function at low oxygen tension relative to buffer perfusion, suggesting that caution should be used in extrapolating intracellular oxygen dynamics from buffer-perfused to blood-perFused hearts.
Abstract: Myocardial mean myoglobin oxygen saturation was determined spectroscopically from isolated guinea pig hearts perfused with red blood cells during increasing hypoxia. These experiments were undertaken to compare intracellular myoglobin oxygen saturation in isolated hearts perfused with a modest concentration of red blood cells (5% hematocrit) with intracellular myoglobin saturation previously reported from traditional buffer-perfused hearts. Studies were performed at 37 degrees C with hearts paced at 240 beats/min and a constant perfusion pressure of 80 cmH2O. It was found that during perfusion with a hematocrit of 5%, baseline mean myoglobin saturation was 93% compared with 72% during buffer perfusion. Mean myoglobin saturation, ventricular function, and oxygen consumption remained fairly constant for arterial perfusate oxygen tensions above 100 mmHg and then decreased precipitously below 100 mmHg. In contrast, mean myoglobin saturation, ventricular function, and oxygen consumption began to decrease even at high oxygen tension with buffer perfusion. The present results demonstrate that perfusion with 5% red blood cells in the perfusate increases the baseline mean myoglobin saturation and better preserves cardiac function at low oxygen tension relative to buffer perfusion. These results suggest that caution should be used in extrapolating intracellular oxygen dynamics from buffer-perfused to blood-perfused hearts.
TL;DR: For a bulkier protein, hemoglobin, adsorption onto the quartz surface was enhanced in the presence of a phosphate buffer, while the opposite effect was observed for the smaller cytochrome c and myoglobin molecules.
Abstract: Slab optical waveguide (SOWG) spectroscopy was used to observe the adsorption behavior of three important heme proteins, namely cytochrome c, myoglobin and hemoglobin, in a quartz surface. Using prism-coupled polychromatic visible light propagated into a quartz waveguide by internal total reflection, the real-time monitoring of evanescent wave absorption revealed a strong dependence of the protein-surface interaction on the protein concentration, the solution pH and the ionic strength. For the three proteins studied, the absorbance-bulk concentration ratio was higher at low bulk concentrations, and decreased at higher concentrations. For cytochrome c and myoglobin, the absorbance approached a limiting value, but buffered hemoglobin surprisingly did not show any indication of forming a signal plateau. Moreover, the slow introduction of protein into the solution lessened the total adsorbed amount per unit area. These observations suggested a possible conformational transition of the protein molecules at the quartz surface after adsorption. For a bulkier protein, hemoglobin, adsorption onto the quartz surface was enhanced in the presence of a phosphate buffer, while the opposite effect was observed for the smaller cytochrome c and myoglobin molecules. The results of pH studies concurred with the electrostatic interactions predicted from the isoelectric data of proteins and the quartz surface.
TL;DR: Spectroscopic data strongly suggest that O(2) and CO binding to TCPs is controlled mainly by the two different electrostatic effects exerted by the overhanging OH groups: destabilization of CO binding by decreasing back-bonding and stabilization of O( 2) binding by hydrogen bonding.
Abstract: Inspired by the observation of polar interactions between CO and O 2 ligands and the peptide residues at the active site of hemoglobin and myoglobin, we synthesized two kinds of superstructured porphyrins: TCP-IM, which contains a linked imidazole ligand, and TCP-PY, which contains a linked pyridine ligand, and examined the thermodynamic, kinetic, and spectroscopic (UV/Vis, IR, NMR, and resonance Raman) properties of their CO and O 2 complexes. On both sides of each porphyrin plane, bulky binaphthyl bridges form hydrophobic cavities that are suitable for the binding of small molecules. In the proximal site, an imidazole or pyridine residue is covalently fixed and coordinates axially to the central iron atom. In the distal site, two naphtholic hydroxyl groups overhang toward the center above the heme. The CO affinities of TCPs are significantly lower than those of other heme models. In contrast, TCPs have moderate O 2 binding ability. Compared with reported model hemes, the binding selectivity of O 2 over CO in TCP-IM and TCP-PY complexes is greatly improved. The high O 2 selectivity of the TCPs is mainly attributable to a low CO affinity. The comparison of k o n (CO) values of TCPs with those of unhindered hemes indicates the absence of steric hindrance to the intrinsically linear CO coordination to Fe I I in TCP-IM and TCP-PY. The abnormally large k o f f (CO) values are responsible for the low CO affinities. In contrast, k o f f (O 2 ) of TCP-PY is smaller than those of other pyridine-coordinated model hemes. For the CO adducts of TCPs, unusually low v(Fe-CO) and unusually high v(C-O) frequencies are observed. These results can be ascribed to decreased back-bonding from the iron atom to the bound CO. The lone pairs of the oxygen atoms of the hydroxyl groups prevent back-bonding by exertion of a strong negative electrostatic interaction. On the other hand, high v(Fe-O 2 ) frequencies are observed for the O 2 adducts of TCPs. In the resonance Raman (RR) spectrum of oxyTCP-IM, we observed simultaneous enhancement of the Fe-O 2 and O-O stretching modes. Furthermore, direct evidence for hydrogen bonding between the hydroxyl groups and bound dioxygen was obtained by RR and IR spectroscopy. These spectroscopic data strongly suggest that O 2 and CO binding to TCPs is controlled mainly by the two different electrostatic effects exerted by the overhanging OH groups: destabilization of CO binding by decreasing back-bonding and stabilization of O 2 binding by hydrogen bonding.
TL;DR: The iron complex of hemiporphycene was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid and anomalies in the ferric and ferrous derivatives were ascribed to the modified iron-N(pyrrole) interactions in the asymmetric metallo core of hemidazole.
Abstract: The iron complex of hemiporphycene, a molecular hybrid of porphyrin with porphycene, was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid. Apomyoglobin was successfully coupled with a stoichiometric amount of ferric hemiporphycene to afford the reconstituted myoglobin equipped with the iron coordination structure of native protein. Cyanide, imidazole, and fluoride coordinated to the ferric protein with affinities comparable with those for native myoglobin. The ferrous myoglobin was functionally active to bind O(2) and CO reversibly at pH 7.4 and 20 degrees C. The O(2) affinity is 12-fold higher than that of native myoglobin while the CO affinity is slightly lower, suggesting decreased discrimination between O(2) and CO in the heme pocket. The functional anomaly was interpreted to reflect increased sigma-bonding character in the Fe(II)-O(2) bond. In contrast with 6-coordinate native NO protein, the NO myoglobin containing ferrous hemiporphycene is in a mixed 5- and 6-coordinate state. This observation suggests that the in-plane configuration of the iron atom in hemiporphycene is destabilized by NO. Influence of the core deformation was also detected with both the infrared absorption for the ferrous CO derivative and electron paramagnetic resonance for ferric imidazole complex. Anomalies in the ferric and ferrous derivatives were ascribed to the modified iron-N(pyrrole) interactions in the asymmetric metallo core of hemiporphycene.
TL;DR: A better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.
Abstract: Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.
TL;DR: The data show that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.
TL;DR: In this paper, the authors showed that the pigment in dry-cured Parma ham at no stage is a nitrosyl complex of ferrous myoglobin as found in brinecured ham and Spanish Serrano hams.
Abstract: Spectroscopic studies of Parma ham during processing revealed a gradual transformation of muscle myoglobin, initiated by salting and continuing during ageing. Electron spin resonance spectra did, however, conclusively show that the pigment in dry-cured Parma ham at no stage is a nitrosyl complex of ferrous myoglobin as found in brine-cured ham and Spanish Serrano hams. Both near-infra red reflectance spectra of sliced ham and UV/visible absorption spectra of extract of hams, obtained with aqueous buffer or acetone, showed the presence of different red pigments at varying processing stages for both solvents. Especially, the pigment extracted with aqueous buffer exhibited unique spectral features different from those of well-known myoglobin derivatives. At the end of processing, the pigment(s) becomes less water extractable, while the fraction of red pigment(s) extractable with acetone/water (75%/25%) increases throughout the processing time up to full maturation at 18 months. The chemical identity of the 6th ligand of myoglobin could not be conclusively established, but possible candidates are discussed. The partition of the pigment(s) between pentane and acetone/water showed a strong preference for pentane, suggesting that only the heme moiety is present in the acetone/water extract, and that Parma ham pigment is gradually transformed from a myoglobin derivative into a non-protein heme complex, which was found to be thermally stable in acetone/water solution
TL;DR: The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn2+ and Cd2+.
Abstract: A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1H NMR spectroscopy indicates that Mn2+ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn2+ is 1.28(4) × 104 M−1 (pH 6.96, ionic strength I = 17.2 μM, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pKa for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn2+-peroxidase activity of the variant are both increased ≈3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn2+. The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn2+ and Cd2+. Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.
TL;DR: Results strongly suggest that the heme-iron absorption pathway is saturable and should be considered as a safe source of iron for women.
Abstract: In developed countries where meat is an important constituent of the diet, much of the dietary iron is in the heme-iron form as hemoglobin and myoglobin. Heme-iron is absorbed more efficiently than inorganic iron by the human intestine. Thus, it is important to know how the dose of heme-iron affects iron absorption. The purpose of this study was to establish the dose-effect of heme-iron on the percentage and absolute amount of iron absorbed. Twenty-seven healthy women (28- to 50-y-old) were selected to participate in two iron absorption studies. Through the use of iron isotopes ((59)Fe and (55)Fe), the studies were performed to characterize the dose-response curve of non-heme-iron absorption (ferrous sulfate), and to establish the dose-response curve of heme-iron absorption (hemoglobin). The labeled hemoglobin was prepared by use of red blood cells from rabbits. The geometric means (+/-1 SEM range) of non-heme iron absorbed were 0.2 (0.2-0.3), 1.2 (1.0-1.5), 6.7 (5.7-8.0) and 13.0 (11.5-14.6) mg of iron for doses of 0.5, 5, 50 and 100 mg of iron as ferrous sulfate, respectively; and 0.1 (0.1-0.2), 0.4 (0.3-0.4), 2.2 (2.0-2.4) and 2.2 (1.7-3.0) mg of iron for doses of 0.5, 3, 15 and 30 mg of heme-iron as hemoglobin, respectively. The fitted curves for heme and non-heme iron differed (P < 0.04). These results strongly suggest that the heme-iron absorption pathway is saturable.
TL;DR: In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, it was found that the replacement of His-64 of myoglobin with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively.
Abstract: In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H2O2 to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2‘-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-...
TL;DR: First report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic characterization of the relevant redox forms of myoglobin in dimethyl sulfoxide is reported.
Abstract: This paper reports the first report of rapid, reversible direct electron transfer between a redox protein, specifically, horse myoglobin, and a solid electrode substrate in nonaqueous media and the spectroscopic (UV-vis, fluorescence, and resonance Raman) characterization of the relevant redox forms of myoglobin (Mb) in dimethyl sulfoxide (DMSO). In DMSO, the heme active site of metmyoglobin (metMb) appears to remain six-coordinate high-spin, binding water weakly. Changes in the UV-fluorescence spectra for metMb in DMSO indicate that the protein secondary structure has been perturbed and suggest that helix A has moved away from the heme. UV-vis and RR spectra for deoxyMb in DMSO suggest that the heme iron is six-coordinate low-spin, most likely coordinating DMSO. Addition of CO to deoxyMb in DMSO produces a single, photostable six-coordinate CO adduct. UV-vis and RR for Mb-CO in DMSO are consistent with a six-coordinate low-spin heme iron binding His93 weakly, if at all. The polarity of the distal heme pocket is comparable to that of the closed form of horse Mb-CO in aqueous solution, pH 7. Direct electron transfer between horse Mb and Au in DMSO solution was investigated by cyclic voltammetry. Mb exhibits stable and well-defined electrochemical responses that do not appear to be affected by the water content (1.3–7.5%). The electrochemical characteristics are consistent with a one-electron, quasi-reversible, diffusion-controlled charge transfer process at Au. E° for horse Mb in DMSO at Au is –0.241±0.005 V vs. NHE. The formal heterogeneous electron transfer rate constant, calculated from ΔE
p at 20 mV/s, is 1.7±0.5×10–4 cm/s. The rate, which is unaffected by the presence of 1.3–7.5% water, is competitive with that previously reported for horse Mb in aqueous solution.
TL;DR: The data presented in this study show that Cu(II)-mediated cleavage of myoglobin is able to proceed at neutral pH, more selectively than Pd(II), and buffer solution of phosphate and NH4HCO3 accelerates the cleavage reaction.
Abstract: Selective hydrolytic cleavage of myoglobin was studied with CuCl2, Cu(ClO4)2, Cu(AC)2, and binuclear Cu(II) complexes of 3,6,9,16,19,22-hexaaza-6,19-bis (2-hydroxyethyl)-tricyclo-[22,2,2,211,14]-triaconta-1,11,13,24,27,29-hexaene (1) and 3,6,9,16,19,22-hexaaza-tricyclo-[22,2,2,211,14]-triaconta-1,11,13,24,27,29-hexaene (2). The sites of cleavage were precisely determined by LC-ESIMS and further confirmed by an MS/MS method through fragmentation from both the N-terminal and C-terminal. The peptide bonds of Gln91-Ser92 and Ala94-Thr95 were remarkably cleaved by Cu(II) anchored to the side chain of the His93 residue. The data presented in this study show that Cu(II)-mediated cleavage of myoglobin is able to proceed at neutral pH, more selectively than Pd(II)-mediated cleavage, and buffer solution of phosphate and NH4HCO3 accelerates the cleavage reaction.
TL;DR: In this paper, the structure and dynamics of the Fe-CO bond in carbonmonoxy myoglobin (MbCO) were investigated using density functional theory, Car-Parrinello molecular dynamics and hybrid quantum mechanics/molecular mechanics approaches.
Abstract: This paper is a review of our recent work on the structure and dynamics of the Fe–CO bond in carbonmonoxy myoglobin (MbCO), performed using density functional theory, Car–Parrinello molecular dynamics and hybrid quantum mechanics/molecular mechanics approaches. The results of these investigations have served to shed light onto one of the long standing questions in myoglobin research: whether the protein discriminates the CO ligand with respect to O2 by distorting the FeCO bond. The calculations show that both in the gas phase and in the protein the Fe–CO bond is essentially linear and therefore exclude the hypothesis that the CO in MbCO is sterically hindered. In contrast, hydrogen bonding between the O2 ligand and the His64 residue easily explains the protein discrimination for CO.
TL;DR: This study characterized myoglobin in vitro toxicity using renal cortical slices isolated from male Fischer 344 rats and examined the effect of deferoxamine (DFX) and glutathione on myoglobin toxicity to determine the role of radicals and iron.
TL;DR: The iron heme protein myoglobin (Mb) coadsorbs with surfactant onto glassy carbon, pyrolytic graphite, and platinum electrodes from microemulsions of sodium dodecyl sulfate or cetyltrimethylammoniu as mentioned in this paper.
Abstract: The iron heme protein myoglobin (Mb) coadsorbs with surfactant onto glassy carbon, pyrolytic graphite, and platinum electrodes from microemulsions of sodium dodecyl sulfate or cetyltrimethylammoniu...