Showing papers on "Myoglobin published in 2018"

A Noncanonical Proximal Heme Ligand Affords an Efficient Peroxidase in a Globin Fold.

Abstract: Expanding the range of genetically encoded metal coordination environments accessible within tunable protein scaffolds presents excellent opportunities for the creation of metalloenzymes with augmented properties and novel activities. Here, we demonstrate that installation of a noncanonical Nδ-methyl histidine (NMH) as the proximal heme ligand in the oxygen binding protein myoglobin (Mb) leads to substantial increases in heme redox potential and promiscuous peroxidase activity. Structural characterization of this catalytically modified myoglobin variant (Mb NMH) revealed significant changes in the proximal pocket, including alterations to hydrogen-bonding interactions involving the prosthetic porphyrin cofactor. Further optimization of Mb NMH via a combination of rational modification and several rounds of laboratory evolution afforded efficient peroxidase biocatalysts within a globin fold, with activities comparable to those displayed by nature's peroxidases.

Mechanism Underlying Green Discolouration of Myoglobin Induced by Atmospheric Pressure Plasma

Abstract: In this study, we elucidated the mechanism underlying atmospheric pressure plasma (APP)-induced green discolouration of myoglobin. Green-coloured pigments are produced upon conversion of myoglobin into sulphmyoglobin, choleglobin, verdoheme, nitrihemin, or nitrimyoglobin. We exposed myoglobin dissolved in phosphate buffer to APP for 20 min and found a decrease in a* value (+redness/−greenness) and increase in b* value (+yellowness/−blueness) (P < 0.05). In the ultraviolet absorption spectrum, myoglobin treated with APP for 20 min showed absorption peaks at 503 and 630 nm, a spectrum different from that of sulphmyoglobin or choleglobin. The secondary structure and molecular weight of myoglobin were unaffected by APP treatment, excluding the possibility of verdoheme or nitrihemin formation. After APP treatment, nitrite was produced in myoglobin solution that provided a positive environment for nitrimyoglobin formation. However, the addition of 0.5% sodium dithionite, a strong reducing agent, to myoglobin solution resulted in the formation of deoxymyoglobin, which was subsequently converted to nitrosomyoglobin upon APP treatment to yield a desirable red colour. Thus, APP-induced green colouration in myoglobin solution is associated with nitrimyoglobin formation. The addition of the antioxidant resulted in the production of red colour in myoglobin solution after APP treatment owing to nitrosomyoglobin formation.

Ultrafast carbon monoxide photolysis and heme spin-crossover in myoglobin via nonadiabatic quantum dynamics

Abstract: Light absorption of myoglobin triggers diatomic ligand photolysis and a spin crossover transition of iron(II) that initiate protein conformational change The photolysis and spin crossover reactions happen concurrently on a femtosecond timescale The microscopic origin of these reactions remains controversial Here, we apply quantum wavepacket dynamics to elucidate the ultrafast photochemical mechanism for a heme–carbon monoxide (heme–CO) complex We observe coherent oscillations of the Fe–CO bond distance with a period of 42 fs and an amplitude of ∼1 A These nuclear motions induce pronounced geometric reorganization, which makes the CO dissociation irreversible The reaction is initially dominated by symmetry breaking vibrations inducing an electron transfer from porphyrin to iron Subsequently, the wavepacket relaxes to the triplet manifold in ∼75 fs and to the quintet manifold in ∼430 fs Our results highlight the central role of nuclear vibrations at the origin of the ultrafast photodynamics of organometallic complexes Myoglobin bound to carbon monoxide undergoes an ultrafast light-induced reaction, which ends up in a photolyzed carbon monoxide and a spin transition of the iron center Here, the authors employ quantum wavepacket dynamics to show that photolysis precedes the spin transition, a mechanism dominated by strong electron-nuclear couplings

Initial Conformation of Adsorbed Proteins at an Air–Water Interface

Abstract: We present the results of time-resolved X-ray reflectivity measurements carried out to investigate the early stage of protein adsorption and deformation at an air–water interface. Three globular proteins [lysozyme, myoglobin, and bovine serum albumin (BSA)] were studied, and we observed that the proteins adsorbed at the air–water interface initially possessed a thinner conformation than their native structures. The degree of deformation increased in the order myoglobin < lysozyme < BSA, which was inconsistent with the order of molecular flexibility. The initial rate of protein adsorption increased in the order lysozyme < BSA < myoglobin as determined by the dynamic surface tension. More flexible proteins generally adsorb at the interface more rapidly; however, proteins with hydrophobic patches on the protein surface, such as myoglobin, adsorb at the interface with little deformation. These results provide evidence that protein unfolding during adsorption only takes place if the kinetics of adsorption are ...

The Reaction of Oxy Hemoglobin with Nitrite: Mechanism, Antioxidant-Modulated Effect, and Implications for Blood Substitute Evaluation

Abstract: The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.

Myoglobin: Oxygen Depot or Oxygen Transporter to Mitochondria? A Novel Mechanism of Myoglobin Deoxygenation in Cells (review)

Abstract: In this review, we shortly summarize the data of our studies (and also corresponding studies of other authors) on the new mechanism of myoglobin (Mb) deoxygenation in a cell, according to which Mb acts as an oxygen transporter, and its affinity for the ligand, like in other transporting proteins, is regulated by the interaction with the target, in our case, mitochondria (Mch). We firstly found that contrary to previously formulated and commonly accepted concepts, oxymyoglobin (MbO2) deoxygenation occurs only via interaction of the protein with respiring mitochondria (low pO2 values are necessary but not sufficient for this process to proceed). Detailed studies of the mechanism of Mb-Mch interaction by various physicochemical methods using natural and artificial bilayer phospholipid membranes showed that: (i) the rate of MbO2 deoxygenation in the presence of respiring Mch fully coincides with the rate of O2 uptake by mitochondria from a solution irrespectively of their state (native coupled, freshly frozen, or FCCP-uncoupled), i.e. it is determined by the respiratory activity of Mch; (ii) Mb nonspecifically binds to membrane phospholipids of the outer mitochondrial membrane, while any Mb-specific protein or phospholipid sites on it are lacking; (iii) oxygen uptake by Mch from a solution and the uptake of Mb-bound oxygen are two different processes, as their rates are differently affected by proteins (e.g. lysozyme) that compete with MbO2 for binding to the mitochondrial membrane; (iv) electrostatic forces significantly contribute to the Mb-membrane interactions; the dependence of these interactions on ionic strength is provided by the local electrostatic interactions between anionic groups of phospholipids (the heads) and invariant Lys and Arg residues near the Mb heme pocket; (v) interactions of Mb with phospholipid membranes promote conformational changes in the protein, primarily in its heme pocket, without significant alterations in the protein secondary and tertiary structures; and (vi) Mb-membrane interactions lead to decrease in the affinity of myoglobin for O2, which could be monitored by the increase in the MbO2 autooxidation rate under aerobic conditions and under anaerobic ones, by the shift in the MbO2/Mb(2) equilibrium towards the ligand-free protein. The decrease in the affinity of Mb for the ligand should facilitate O2 dissociation from MbO2 at physiological pO2 values in cells.

Heme Dissociation from Myoglobin in the Presence of the Zwitterionic Detergent N,N-Dimethyl-N-Dodecylglycine Betaine: Effects of Ionic Liquids

Abstract: We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.

Selective Recognition of Myoglobin in Biological Samples Using Molecularly Imprinted Polymer-Based Affinity Traps.

Abstract: The current work demonstrates the design, characterization, and preparation of molecularly imprinted microspheres for the selective detection of myoglobin in serum samples. The suspension polymerization approach was applied for the preparation of myoglobin imprinted microspheres. For this purpose, N-methacryloylamino folic acid-Nd3+ (MAFol- Nd3+) was chosen as the complex functional monomer. The optimization studies were performed changing the medium pH, temperature, and myoglobin concentration. pH 7.0 was determined as the optimum value where the prepared imprinted microspheres displayed maximum binding for myoglobin. The maximum binding capacity was achieved as 623 mgg−1. In addition, the selectivity studies were conducted. The results confirmed that the imprinted microspheres showed great selectivity towards myoglobin in the existence of hemoglobin, cytochrome c, and lysozyme which were chosen as potentially competing proteins.

Nitrosyl Myoglobins and Their Nitrite Precursors: Crystal Structural and Quantum Mechanics and Molecular Mechanics Theoretical Investigations of Preferred Fe -NO Ligand Orientations in Myoglobin Distal Pockets.

Abstract: The globular dioxygen binding heme protein myoglobin (Mb) is present in several species. Its interactions with the simple nitrogen oxides, namely, nitric oxide (NO) and nitrite, have been known for decades, but the physiological relevance has only recently become more fully appreciated. We previously reported the O-nitrito mode of binding of nitrite to ferric horse heart wild-type (wt) MbIII and human hemoglobin. We have expanded on this work and report the interactions of nitrite with wt sperm whale (sw) MbIII and its H64A, H64Q, and V68A/I107Y mutants whose dissociation constants increase in the following order: H64Q < wt < V68A/I107Y < H64A. We also report their X-ray crystal structures that reveal the O-nitrito mode of binding of nitrite to these derivatives. The MbII-mediated reductions of nitrite to NO and structural data for the wt and mutant MbII–NOs are described. We show that their FeNO orientations vary with distal pocket identity, with the FeNO moieties pointing toward the hydrophobic interior...

Attenuation of Glucose-Induced Myoglobin Glycation and the Formation of Advanced Glycation End Products (AGEs) by (R)-α-Lipoic Acid In Vitro

Abstract: High-carbohydrate containing diets have become a precursor to glucose-mediated protein glycation which has been linked to an increase in diabetic and cardiovascular complications. The aim of the present study was to evaluate the protective effect of (R)-α-lipoic acid (ALA) against glucose-induced myoglobin glycation and the formation of advanced glycation end products (AGEs) in vitro. Methods: The effect of ALA on myoglobin glycation was determined via the formation of AGEs fluorescence intensity, iron released from the heme moiety of myoglobin and the level of fructosamine. The extent of glycation-induced myoglobin oxidation was measured via the levels of protein carbonyl and thiol. Results: The results showed that the co-incubation of ALA (1, 2 and 4 mM) with myoglobin (1 mg/mL) and glucose (1 M) significantly decreased the levels of fructosamine, which is directly associated with the decrease in the formation of AGEs. Furthermore, ALA significantly reduced the release of free iron from myoglobin which is attributed to the protection of myoglobin from glucose-induced glycation. The results also demonstrated a significant protective effect of ALA on myoglobin from oxidative damage, as seen from the decreased protein carbonyls and increased protein thiols. Conclusion: The anti-glycation properties of ALA suggest that ALA supplementation may be beneficial in the prevention of AGEs-mediated diabetic and cardiovascular complications.

Structural Characterization of Myoglobin Molecules Adsorbed within Mesoporous Silicas

Abstract: In the present study, we examined the secondary and tertiary structure of myoglobin (Mb) within folded sheets mesoporous material (FSM)- and Santa Barbara amorphous (SBA)-type mesoporous silicas. The Barrett–Joyner–Halenda pore diameters of SBA-type mesoporous silicas were 39, 70, and 75 A, and that of FSM-type mesoporous silica was 40 A. The secondary and tertiary structures of myoglobin were observed by Fourier transform infrared (FTIR) and small-angle neutron scattering (SANS), respectively. The FTIR and SANS results indicated preservation of the secondary and tertiary structures of myoglobin inside the pores of SBA-type mesoporous silicas. Adsorption of myoglobin within FSM-type mesoporous silica, however, resulted in perturbation of the tertiary structure, accompanied by partial unfolding of the secondary structure. Lower structural stability of myoglobin within the FSM-type mesoporous silica was also confirmed. These findings suggest that the Mb structure is more influenced by the inner pore surface...

( R )-α-Lipoic acid inhibits fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro

Abstract: Fructose-mediated protein glycation (fructation) has been linked to an increase in diabetic and cardiovascular complications due to over consumption of high-fructose containing diets in recent times. The objective of the present study is to evaluate the protective effect of (R)-α-lipoic acid (ALA) against fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro. The anti-glycation activity of ALA was determined using the formation of AGEs fluorescence intensity, iron released from the heme moiety of myoglobin and the level of fructosamine. The fructation-induced myoglobin oxidation was examined using the level of protein carbonyl content and thiol group estimation. The results showed that co-incubation of myoglobin (1 mg/mL), fructose (1 M) and ALA (1, 2 and 4 mM) significantly inhibited the formation of AGEs during the 30 day study period. ALA markedly decreased the levels of fructosamine, which is directly associated with the reduction of AGEs formation. Furthermore, ALA significantly reduced free iron release from myoglobin which is attributed to the protection of myoglobin from fructose-induced glycation. The results also demonstrated a significant protective effect of ALA on myoglobin oxidative damages, as seen from decreased protein carbonyl content and increased protein thiols. These findings provide new insights into the anti-glycation properties of ALA and emphasize that ALA supplementation is beneficial in the prevention of AGEs-mediated diabetic and cardiovascular complications.

Aggregation and conformational stability evaluation of myoglobin in the presence of ionic surfactant.

Abstract: Sodium lauroyl sarcosinate (SLS) is frequently used for the solubilization of inclusion bodies in vitro due to its structural similarity to lipid plasma membrane. There are many factors that could influence protein aggregation propensity, including overall protein surface charge and hydrophobicity. Here, the aggregation pathway of myoglobin protein was studied under different conditions (pH 3.5 and 7.4) in the presence of varying concentrations of SLS to evaluate the underlying forces dictating protein aggregation. Data obtained from Rayleigh light scattering, ThT binding assay, and far-UV CD indicated that SLS have different effects on the protein depending on its concentration and environmental conditions. In the presence of low concentrations of SLS (0.05–0.1 mM), no aggregation was detected at both pH conditions tested. Whereas, as we reach higher SLS concentrations (0.5–10.0 mM), myoglobin started forming larger-sized aggregates at pH 3.5 and not pH 7.4. These results suggest that electrostatics interactions as well as hydrophobic forces play an important role in SLS-induced myoglobin aggregation.

First Principles Calculation of the Reaction Rates for Ligand Binding to Myoglobin: The Cases of NO and CO.

Abstract: Ligand binding by proteins is among the most fundamental processes in nature. Among these processes the binding of small gas molecules, such as O2 , CO and NO to heme proteins has traditionally received vivid interest, which was further boosted by their recently recognized significant role in gas sensing in the body. At the heart of the binding of these ligands to the heme group is the spinforbidden reaction between high-spin iron(II) and the ligand yielding a low-spin adduct. We use computational means to address the complete mechanism of CO and NO binding by myoglobin. Considering that it involves several steps occurring on different time scales, molecular dynamics simulations were performed to address the diffusion of the ligand through the enzyme, and DFT calculations in combination with statistical rate calculation to investigate the spin-forbidden reaction. The calculations yielded rate constants in qualitative agreement with experiments and revealed that the bottleneck of NO and CO binding is different; for NO, diffusion was found to be rate-limiting, whereas for CO, the spin-forbidden step is the slowest.

Cardiac myoglobin participates in the metabolic pathway of selenium in rats

Abstract: As an essential micronutrient, selenium deficiency is a leading cause of cardiovascular diseases. The heart is continuously beating to deliver blood to the entire body, and this requires a high amount of energy. An adult heart normally obtains 50-70% of its adenosine 5'-triphosphate from fatty acid β-oxidation. An increase in fatty acid oxidation activity induces the generation of larger amounts of by-products (reactive oxygen species, ROS) from mitochondrial oxidative phosphorylation. Selenium-dependent glutathione peroxidases play a critical role in the removal of these ROS, especially organic hydroperoxides, from the heart. The definitive transport and/or detailed metabolic pathways from the selenium-source compounds to the selenoproteins in the heart still remain unclear. We explored the selenium-binding proteins in a rat cardiac cell lysate using its reactive metabolic intermediate, selenotrisulfide (STS), and MALDI TOF-mass spectrometry. Several proteins with a free cysteine (Cys) thiol were found to be reactive with STS through a thiol-exchange reaction. The most distinctive Cys-containing protein in the cardiac cell lysate was identified as myoglobin (Mb) from a rat protein database search and tryptic fragmentation experiments. When separately examined in selenium adequate rats, selenium-binding to the cardiac Mb was verified using selenium-specific fluorometry. Cardiac Mb is thought to participate in the selenium metabolic pathway in the heart.

Characterization of Myoglobin Adsorption into Mesoporous Silica Pores by Differential Scanning Calorimetry.

Abstract: Adsorption of protein molecules into the pores of a porous material is an important process for chromatographic separation of proteins and synthesis of nanoscale biocatalyst systems; however, there are barriers to developing a method for analyzing the process quantitatively. The purpose of this study is to examine the applicability of differential scanning calorimetry (DSC) for quantitative analysis of protein adsorption into silica mesopores. For this purpose myoglobin, a globular protein (diameter: 35.2 A) was selected, and its adsorption onto mesoporous silica powders with uniform pore diameters (pore diameters: 39 and 64 A) was measured by adsorption assay and DSC experiments. Our results confirmed that the adsorption of myoglobin into the silica mesopores induced significant changes in the positions and areas of freezing/melting peaks of the pore water. The decrease in heat of fusion of the pore water after myoglobin adsorption could be utilized to quantify the amount of myoglobin inside the silica mesopores. The advantages of DSC include its applicability to small wet mesoporous silica samples.

Synergistic Effect of Distal Polar Interactions in Myoglobin and Their Structural Consequences

Abstract: In the L29F variant of myoglobin (Mb), the coordination of oxygen (O2) to the heme Fe atom is stabilized by favorable electrostatic interactions between the polar Fe–O2 moiety and the multipole of the phenyl ring of the Phe29 side chain (Phe29 interaction), in addition to the well-known hydrogen bond (H-bond) between the Fe-bound O2 and the 64th residue (distal H-bond; Carver, T. E.; Brantley, R. E., Jr.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. J. Biol. Chem. 1992, 267, 14443–14450). The O2 and carbon monoxide (CO) binding properties and autoxidation of the L29F/H64L and L29F/H64Q variants reconstituted with a series of chemically modified heme cofactors were analyzed and then compared with those of native Mb, and the L29F, H64Q, and H64L variants similarly reconstituted with the chemically modified heme cofactors in order to elucidate the relationship between the Phe29 interaction and the distal H-bond that critically contributes to stabilization of Fe-bound O...

Dynamical comparison between myoglobin and hemoglobin.

Abstract: Myoglobin and hemoglobin are globular hemeproteins, when the former is a monomer and the latter a heterotetramer. Despite the structural similarity of myoglobin to α and β subunits of hemoglobin, there is a functional difference between the two proteins, owing to the quaternary structure of hemoglobin. The effect of the quaternary structure of hemoglobin on the intrinsic dynamics of its subunits is explored by dynamical comparison of the two proteins. Anisotropic Network Model modes of motion were calculated for hemoglobin and myoglobin. Dynamical comparison between the proteins was performed using global and local Anisotropic Network Model mode alignment algorithms based on the algorithms of Smith-Waterman and Needleman-Wunsch for sequence comparison. The results indicate that the quaternary structure of hemoglobin substantially alters the intrinsic dynamics of its subunits, an effect that may contribute to the functional difference between the two proteins. Local dynamics similarity between the proteins is still observed at the major exit route of the ligand.