Showing papers on "Myoglobin published in 2008"

Nitrite reductase activity of myoglobin regulates respiration and cellular viability in myocardial ischemia-reperfusion injury.

Abstract: The nitrite anion is reduced to nitric oxide (NO•) as oxygen tension decreases. Whereas this pathway modulates hypoxic NO• signaling and mitochondrial respiration and limits myocardial infarction in mammalian species, the pathways to nitrite bioactivation remain uncertain. Studies suggest that hemoglobin and myoglobin may subserve a fundamental physiological function as hypoxia dependent nitrite reductases. Using myoglobin wild-type (+/+) and knockout (−/−) mice, we here test the central role of myoglobin as a functional nitrite reductase that regulates hypoxic NO• generation, controls cellular respiration, and therefore confirms a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. We find that myoglobin is responsible for nitrite-dependent NO• generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO• by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In isolated myoglobin+/+ but not in myoglobin−/− hearts, nitrite treatment resulted in an improved recovery of postischemic left ventricular developed pressure of 29%. In vivo administration of nitrite reduced myocardial infarction by 61% in myoglobin+/+ mice, whereas in myoglobin−/− mice nitrite had no protective effects. These data support an emerging paradigm that myoglobin and the heme globin family subserve a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation. myoglobin knockout mice

Atomic level computational identification of ligand migration pathways between solvent and binding site in myoglobin

Abstract: Myoglobin is a globular protein involved in oxygen storage and transport. No consensus yet exists on the atomic level mechanism by which oxygen and other small nonpolar ligands move between the myoglobin's buried heme, which is the ligand binding site, and surrounding solvent. This study uses room temperature molecular dynamics simulations to provide a complete atomic level picture of ligand migration in myoglobin. Multiple trajectories—providing a cumulative total of 7 μs of simulation—are analyzed. Our simulation results are consistent with and tie together previous experimental findings. Specifically, we characterize: (i) Explicit full trajectories in which the CO ligand shuttles between the internal binding site and the solvent and (ii) pattern and structural origins of transient voids available for ligand migration. The computations are performed both in sperm whale myoglobin wild-type and in sperm whale V68F myoglobin mutant, which is experimentally known to slow ligand-binding kinetics. On the basis of these independent, but mutually consistent ligand migration and transient void computations, we find that there are two discrete dynamical pathways for ligand migration in myoglobin. Trajectory hops between these pathways are limited to two bottleneck regions. Ligand enters and exits the protein matrix in common identifiable portals on the protein surface. The pathways are located in the “softer” regions of the protein matrix and go between its helices and in its loop regions. Localized structural fluctuations are the primary physical origin of the simulated CO migration pathways inside the protein.

Globins and hypoxia adaptation in the goldfish, Carassius auratus

Abstract: Goldfish (Carassius auratus) may survive in aquatic environments with low oxygen partial pressures. We investigated the contribution of respiratory proteins to hypoxia tolerance in C. auratus. We determined the complete coding sequence of hemoglobin α and β and myoglobin, as well as partial cDNAs from neuroglobin and cytoglobin. Like the common carp (Cyprinus carpio), C. auratus possesses two paralogous myoglobin genes that duplicated within the cyprinid lineage. Myoglobin is also expressed in nonmuscle tissues. By means of quantitative real-time RT-PCR, we determined the changes in mRNA levels of hemoglobin, myoglobin, neuroglobin and cytoglobin in goldfish exposed to prolonged hypoxia (48 h at Po2 ∼ 6.7 kPa, 8 h at Po2 ∼ 1.7 kPa, 16 h at Po2 ∼ 6.7 kPa) at 20 °C. We observed small variations in the mRNA levels of hemoglobin, neuroglobin and cytoglobin, as well as putative hypoxia-responsive genes like lactate dehydrogenase or superoxide dismutase. Hypoxia significantly enhanced only the expression of myoglobin. However, we observed about fivefold higher neuroglobin protein levels in goldfish brain compared with zebrafish, although there was no significant difference in intrinsic myoglobin levels. These observations suggest that both myoglobin and neuroglobin may contribute to the tolerance of goldfish to low oxygen levels, but may reflect divergent adaptive strategies of hypoxia preadaptation (neuroglobin) and hypoxia response (myoglobin).

Review: Lipid and myoglobin oxidations in muscle foods

Abstract: Lipid oxidation and myoglobin oxidation in muscle foods occur in a concurrent manner and each process appears to enhance the other. During oxidation of oxymyoglobin, both superoxide anion and hydrogen peroxide are produced and further react with iron to produce hydroxyl radical. The hydroxyl radical has the ability to penetrate into the hydrophobic lipid region and hence facilitates lipid oxidation. The prooxidant effect of oxymyoglobin on lipid oxidation is concentrationdependent. At equimolar concentrations, oxymyoglobin shows higher prooxidative activity towards lipid than metmyoglobin. However, the catalytic activity of metmyoglobin is promoted by hydrogen peroxide. The reaction between hydrogen peroxide and metmyoglobin results in the formation of two active hypervalent myoglobin species, perferrylmyoglobin and ferrylmyoglobin, which are responsible for lipid oxidation. Additionally, lipid oxidation results in a wide range of aldehyde products, which are reported to induce the oxidation of oxymyoglobin. Metmyoglobin formation is generally greater in the presence of unsaturated aldehydes than their saturated counterparts of equivalent carbon chain length. In addition, increasing chain length of aldehydes, from hexenal through nonenal, results in the increased metmyoglobin formation. Moreover, aldehydes alter myoglobin redox stability by increasing oxymyoglobin oxidation, decreasing the metmyoglobin reduction via enzymatic process, and enhance the prooxidant activity of metmyoglobin. Therefore, the oxidation of both lipid and myoglobin directly affect the quality and acceptability of muscle foods and the lowering of such a phenomenon can enhance the shelf-life stability of those foods.

Oxygen-binding haem proteins.

Abstract: Myoglobin and haemoglobin, the respiratory pigments of mammals and some molluscs, annelids and arthropods, belong to an ancient superfamily of haem-associated globin proteins. Members of this family share common structural and spectral features. They also share some general functional characteristics, such as the ability to bind ligands, e.g. O2, CO and NO, at the iron atom and to undergo redox changes. These properties are used in vivo to perform a wide range of biochemical and physiological roles. While it is acknowledged that the major role of haemoglobin is to bind oxygen reversibly and deliver it to the tissues, this is not its only function, while the often-stated role of myoglobin as an oxygen storage protein is possibly a misconception. Furthermore, haemoglobin and myoglobin express enzymic activities that are important to their function, e.g. NO dioxygenase activity or peroxidatic activity that may be partly responsible for pathophysiology following haemorrhage. Evidence for these functions is described, and the discussion extended to include proteins that have recently been discovered and that are expressed at low levels within the cell. These proteins are hexaco-ordinate, unlike haemoglobin and myoglobin, and are widely distributed throughout the animal kingdom (e.g. neuroglobins and cytoglobins). They may have specialist roles in oxygen delivery to particular sites within the cell but may also perform roles associated with O2 sensing and signalling and in responses to stress, e.g. protection from reactive oxygen and nitrogen species. Haemoglobins are also widespread in plants and bacteria and may serve similar protective functions.

Structure and dynamics of the water around myoglobin

Abstract: The interplay between simulations at various levels of hydration and experimental observables has led to a picture of the role of solvent in thermodynamics and dynamics of protein systems. One of the most studied protein-solvent systems is myoglobin, which serves as a paradigm for the development of structure-function relationships in many biophysical studies. We review here some aspects of the solvation of myoglobin and the resulting implications. In particular, recent theoretical and simulation studies unify much of the diverse set of experimental results on water near proteins.

Atomistic Simulation of Adiabatic Reactive Processes Based on Multi-State Potential Energy Surfaces.

Abstract: The adiabatic reactive molecular dynamics (ARMD) method provides a framework to study chemical reactions using molecular dynamics simulations with minimal computational overhead. Here, ARMD is generalized to an arbitrary reactive process between two states in which reactants and products can be treated by an atomistic force field. The implementation is described, and the method is applied to two systems: the kinetics of NO rebinding to myoglobin (Mb) as a validation system and the conformational transition in neuroglobin (Ngb) which explores the full functionality of ARMD. For MbNO, the nonexponential kinetics observed both in experiment and earlier ARMD studies is reproduced. Furthermore, the sensitivity of the results with respect to the asymptotic separation between the two potential energy surfaces (NO bound and unbound) is studied.

Optoacoustic monitoring of multiple parameters

Abstract: An optoacoustic technique for absolute, accurate, continuous, and real-time measurement of a variety of important diagnostic variables is disclosed, where the variables include: (1) noninvasive measurements of circulating blood volume (BV) and cardiac output (CO); (2) calculation from the measured variables of cardiac index (CI) and systemic oxygen delivery (DO2); (3) concentrations of hemoglobin derivatives (e.g., carboxyhemoglobin [HbCO], reduced hemoglobin [Hb], oxygenated hemoglobin [HbOxy], and methemoglobin [HbMet]), total hemoglobin concentration [THb], concentrations of lactate, myoglobin, cholesterol, body pigments, and exogenous dyes; (4) content in tissues of water, fat, protein, calcium, and blood; as well as density of hard and soft tissues; and (5) accurate noninvasive measurement of blood pressure (or vascular pressure) in arteries, arterioles, veins, capillaries, using occlusion-induced changes in optoacoustic signal induced in blood circulating in the vessels. The optoacoustic technique can be used for single measurement, continuous measurement, or continuous monitoring of these variables.

Oxidation of porcine Myosin by hypervalent myoglobin: the role of thiol groups.

Abstract: Oxidation of the myofibrillar muscle protein myosin from pork by hypervalent myoglobin species (MbFe(III)/H2O2 radical generating system) was investigated in aqueous solution in the pH range of 5.0–7.8 by electron spin resonance (ESR) spectroscopy using N-tert-butyl-α-phenylnitrone (PBN) as spin trap and indirectly by determination of the rate of reduction of hypervalent myoglobin species by UV spectroscopy. Cross-linking of myosin was examined by SDS-PAGE. The target for oxidative modification of myosin was studied by thiol blocking by N-acetylmaleimide (NEM) and by determining oxidative modification of myosin thiols. The reaction between myosin and hypervalent myoglobin was fast and showed little dependence on pH. The myosin radicals formed were observed to be short-lived. Myosin thiols are suggested to be the main target for oxidative modification, as NEM-treated myosin did not form radicals in the presence of hypervalent myoglobin. A significant decrease in thiol content was already demonstrated 25 s ...

Nitric Oxide Complex of Iron(II) Myoglobin Converted from Metmyoglobin by Staphylococcus xylosus

Abstract: With Staphylococcus xylosus FAX-1, metmyoglobin in MRS broth (pH 5.8) was found to undergo conversion to hexacoordinate nitric oxide (NO) complex of Fe(II) myoglobin. When the pH of the MRS culture containing myoglobin changed from 5.8 to 4.0, it affected the conversion from hexacoordinate to pentacoordinate NO complex of Fe(II) myoglobin. This conversion process was reversible. Salami without nitrite or nitrate addition was prepared by inoculating S. xylosus FAX-1, and pentacoordinate NO complex of Fe(II) myoglobin (nitrosylmyoglobin formed in cured meat) was formed in the salami.

Development of a myoglobin impedimetric immunosensor based on mixed self-assembled monolayer onto gold

Abstract: Immunosensors rely on antibody-antigen binding with a range of possible detection methodologies. In this study, electrochemical impedance spectroscopy was used to monitor the sensor surface assembly and recognition of the analyte (myoglobin). Myoglobin is rapidly released into the circulatory system after an acute myocardial infarction and rapidly rising levels make it the first biochemical marker of myocardial damage. The immunosensor fabrication steps comprised the steps of (a) formation of mixed self-assembled monolayers (mSAM) on gold electrodes using a mixture of biotinyl-phospholipid and mer captohexadecanoic acid; (b) neutravidin functionalisation and (c) attachment of biotinyl anti-myoglobin antibodies. A range of analyte concentration (10−12–10−6 M) was successfully detected in phosphate buffered saline and in serum concentration ranging from 10% (v/v) to 100% (v/v) serum. Quartz crystal microbalance and atomic force microscopy studies were carried out to study each step of fabrication to elucidate binding characteristics and surface topography.

Physiology: Myoglobin's new clothes.

Abstract: Nitric oxide generated from the nitrite ion limits the tissue damage caused by restricted blood flow. Gene knockout experiments in mice now reveal that myoglobin is the mediator of this effect.

The Crystal Structure of Peroxymyoglobin Generated Through Cryoradiolytic Reduction of Myoglobin Compound III During Data Collection.

Abstract: Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.

Nitrite binding sites on myoglobin

Abstract: 1 5 N-labelled nitrite was used to determine the amount of nitric oxide bound to unheated compared to heated myoglobin. Heated samples contained twice the amount of 15N as unheated samples. The globin portion was likely detached from the myoglobin by heating, and two sites for binding of nitric oxide were therefore made available.

Structural Dynamics of Myoglobin: FTIR-TDS Study of NO Migration and Binding†

Abstract: By using Fourier transform infrared photolysis difference spectroscopy combined with temperature derivative spectroscopy at cryogenic temperatures, we have measured infrared spectra of the stretching absorption on nitric oxide (NO) in the heme-bound and photodissociated states of ferrous and ferric nitrosyl myoglobin (MbNO) and a few site-specific Mb mutants. The NO absorption was utilized as a sensitive local probe of ligand interactions with active-site residues and movements within the protein. By comparison with results obtained in previous spectroscopic and structural studies of carbonmonoxy myoglobin (MbCO), the MbNO data were interpreted in structural terms. In the NO-bound state, conformational heterogeneity was inferred from the appearance of multiple bands, arising from different electrostatic interactions with active site residues, most importantly, His-64. In ferrous MbNO, a primary photoproduct site similar to site B of MbCO was found, as indicated by a characteristic NO stretching spectrum. ...

Toward quantitative simulations of carbon monoxide escape pathways in myoglobin.

Abstract: Straightforward molecular dynamics trajectories have been computed to explore the diffusion of carbon monoxide through myoglobin. The classical equations of motion were integrated for 2 ns and the resulting pathways analyzed. Two types of runs were examined. Type i: Myoglobin and a ligand embedded in a periodic box with 9996 water molecules; the water molecules are rigid but the bonds of the protein are flexible. Type ii: Myoglobin with a solvation shell (153 water molecules) in which all bond lengths are fixed. In trajectories of type i, the diffusing ligand visits a significant part of the protein matrix and was not constrained to the proximity of the heme pocket before escaping. The maximum time of the trajectories was 2 ns. It was shorter if the ligand escaped earlier. Two ligands (from a total of 88) escape to the solvent from nonclassical gates (non-E-helix gates). In trajectories of type ii, the overall fluctuations of the protein are smaller and the ligand explores significantly smaller internal space. The escape rate from type ii trajectories (11 of 400) is comparable to type i and is not dramatically different from experiment (1 of 100). Interestingly, the two simulations with comparable rates sampled different pathways. In trajectories of type ii, we observe escapes from the classical gate (His 64) and from the Xe4 cavity. Further studies (that are underway) are required to define the escape pathways and the overall rate.

Identification of protein radicals formed in the human neuroglobin-H2O2 reaction using immuno-spin trapping and mass spectrometry.

Abstract: Neuroglobin (Ngb) is a recently discovered protein that shows only minor sequence similarity with myoglobin and hemoglobin but conforms to the typical 3-over-3 α-helical fold characteristic of vertebrate globins. An intriguing feature of Ngb is its heme hexacoordination in the absence of external ligands, observed both in the ferrous and in the ferric (met) forms. In Ngb, the imidazole of a histidine residue (His-64) in the distal position, above the heme plane, provides the sixth coordination bond. In this work, a valine residue was introduced at position 64 (H64V variant) to clarify the possible role(s) of the distal residue in protecting the heme iron of Ngb from attack by strong oxidants. SDS-PAGE analyses revealed that the oxidation of the H64V variant of metNgb by H 2 O 2 resulted in the formation of dimeric and trimeric products in contrast to the native protein. Dityrosine cross-links were shown by their fluorescence to be present in the oligomeric products. When the spin trap 5,5-dimethyl-1 -pyrroline N-oxide (DMPO) was included in the reaction mixture, nitrone adducts were detected by immuno-spin trapping. The specific location of the DMPO adducts on the H64V variant protein was determined by a mass spectrometry method that combines off-line immuno-spin trapping and chromatographic procedures. This method revealed Tyr-88 to be the site of modification by DMPO. The presence of His-64 in the wild-type protein results in the nearly complete loss of detectable radical adducts. Together, the data support the argument that wild-type Ngb is protected from attack by H 2 O 2 by the coordinated distal His.

Porphyrin-cyclodextrin supramolecular complexes as myoglobin model in water

Abstract: This mini-review reports supramolecular system composed of O-methylated β-cyclodextrins and metalloporphyrins that mimic the functions of myoglobin (Mb) in aqueous solution. Although many Mb functional models have been demonstrated so far, most models can bind dioxygen only in organic solvents such as toluene. Recently, we prepared the model systems composed of O-methylated β-cyclodextrin dimers having pyridine and imidazole linkers and tetrakis(4-sulfonatophenyl)porphinato iron(II) (hemoCD and Fe(II)PImCD). HemoCD binds dioxygen reversibly in aqueous solution, and the dioxygen adduct of hemoCD is very stable (a half-lifetime is 30 h at pH 7). Although the dioxygen affinity of Fe(II)PImCD is much higher than hemoCD, the stability and the reversibility of this system is lower. This review compares the functions of these model systems with those in biological systems.

Myoglobin Modification by Enzyme-Generated Dopamine Reactive Species

Abstract: The generation of reactive quinone species (DAQ) from oxidation of dopamine (DA) is involved in neurodegenerative pathologies like Parkinson's disease (A. Borta, G. U. Hoglinger, J. Neurochem. 2007, 100, 587-595). The oxidation of DA to DAQ can occur either in a single two-electron process or in two consecutive one-electron steps, through semiquinone radicals, giving rise to different patterns of reactions. The former type of reaction can be promoted by tyrosinase, the latter by peroxidases in the presence of H(2)O(2), which can be formed under oxidative stress conditions. Both enzymes were employed for the characterization of the thiol-catechol adducts formed by reaction of DA and cysteine or glutathione, and for the identification of specific amino acid residues modified by DAQs in two representative target proteins, human and horse heart myoglobin. Our results indicate that the cysteinyl-DA adducts are formed from the same quinone intermediate independently of the mechanism of DA oxidation, and that the hallmark of a radical mechanism is the formation of the cystine dimer. The reactivity of quinone species also controls the DA-promoted derivatization of histidine residues in proteins. However, for the modification of the cysteine residue in human myoglobin, a radical intramolecular mechanism has been proposed, in which the protein acts both as the catalyst and target of the reaction. Most importantly, the modification of myoglobins through DAQ linkages, and in particular by DA oligomers, has dramatic effects on their stability, as it induces protein unfolding and incorporation into insoluble melanic precipitates.