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International Journal of Molecular... Aug 2022Myoglobin (Mb), besides its roles as an oxygen (O) carrier/storage protein and nitric oxide NO scavenger/producer, may participate in lipid trafficking and metabolite...
Myoglobin (Mb), besides its roles as an oxygen (O) carrier/storage protein and nitric oxide NO scavenger/producer, may participate in lipid trafficking and metabolite binding. Our recent findings have shown that O is released from oxy-Mb upon interaction with lactate (LAC, anerobic glycolysis end-product). Since pyruvate (PYR) is structurally similar and metabolically related to LAC, we investigated the effects of PYR (aerobic glycolysis end-product) on Mb using isothermal titration calorimetry, circular dichroism, and O-kinetic studies to evaluate PYR affinity toward Mb and to compare the effects of PYR and LAC on O release kinetics of oxy-Mb. Similar to LAC, PYR interacts with both oxy- and deoxy-Mb with a 1:1 stoichiometry. Time-resolved circular dichroism spectra revealed that there are no major conformational changes in the secondary structures of oxy- or deoxy-Mb during interactions with PYR or LAC. However, we found contrasting results with respect to binding affinities and substrate preference, where PYR has higher affinity toward deoxy-Mb when compared with LAC (which prefers oxy-Mb). Furthermore, PYR interaction with oxy-Mb releases a significantly lower amount of O than LAC. Taken together, our findings support the hypothesis that glycolytic end-products play a distinctive role in the Mb-rich tissues by serving as novel regulators of O availability, and/or by impacting other activities related to oxy-/deoxy-Mb toggling in resting vs. exercised or metabolically activated conditions.
Topics: Kinetics; Myoglobin; Oxygen; Pyruvic Acid; Structure-Activity Relationship; Thermodynamics
PubMed: 35955898
DOI: 10.3390/ijms23158766 -
International Journal of Molecular... Oct 2022Computational modeling can provide a mechanistic and quantitative framework for describing intracellular spatial heterogeneity of solutes such as oxygen partial pressure...
Computational modeling can provide a mechanistic and quantitative framework for describing intracellular spatial heterogeneity of solutes such as oxygen partial pressure (pO). This study develops and evaluates a finite-element model of oxygen-consuming mitochondrial bioenergetics using the COMSOL Multiphysics program. The model derives steady-state oxygen (O) distributions from Fickian diffusion and Michaelis-Menten consumption kinetics in the mitochondria and cytoplasm. Intrinsic model parameters such as diffusivity and maximum consumption rate were estimated from previously published values for isolated and intact mitochondria. The model was compared with experimental data collected for the intracellular and mitochondrial pO levels in human cervical cancer cells (HeLa) in different respiratory states and under different levels of imposed pO. Experimental pO gradients were measured using lifetime imaging of a Förster resonance energy transfer (FRET)-based O sensor, Myoglobin-mCherry, which offers in situ real-time and noninvasive measurements of subcellular pO in living cells. On the basis of these results, the model qualitatively predicted (1) the integrated experimental data from mitochondria under diverse experimental conditions, and (2) the impact of changes in one or more mitochondrial processes on overall bioenergetics.
Topics: Humans; Oxygen; Oxygen Consumption; Myoglobin; Computer Simulation; Energy Metabolism
PubMed: 36293452
DOI: 10.3390/ijms232012597 -
Chembiochem : a European Journal of... Sep 2022Myoglobin (Mb) can react with hydrogen peroxide (H O ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as...
Myoglobin (Mb) can react with hydrogen peroxide (H O ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo-peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo-Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands. These Mb variants were fully characterised by UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and native mass spectrometry (nMS). The steady-state kinetics of guaiacol oxidation and 2,4,6-trichlorophenol (TCP) dehalogenation catalysed by Mb variants were determined. Mb variants with iron chlorin e6 (Fe-Ce6) and manganese chlorin e6 (Mn-Ce6) cofactors were found to have improved catalytic efficiency for both guaiacol and TCP substrates in comparison with wild-type Mb, i. e. Fe-protoporphyrin IX-Mb. Furthermore, the selected cofactors were incorporated into the scaffold of a Mb mutant, swMb H64D. Enhanced peroxidase activity for both substrates were found via the reconstitution of Fe-Ce6 into the mutant scaffold.
Topics: Amino Acids; Guaiacol; Heme; Hydrogen Peroxide; Manganese; Myoglobin; Peroxidases
PubMed: 35816250
DOI: 10.1002/cbic.202200197 -
IUBMB Life 2007The pathways for ligand entry and exit in myoglobin have now been well established by a wide variety of experimental results, including pico- to nano- to microsecond... (Review)
Review
The pathways for ligand entry and exit in myoglobin have now been well established by a wide variety of experimental results, including pico- to nano- to microsecond transient absorbance measurements and time-resolved X-ray crystallographic measurements. Trp insertions have been used to block, one at a time, the three major cavities occupied by photodissociated ligands. In this work, we review the effects of the L29(B10)W mutation, which places a large indole ring in the initial 'docking site' for photodissociated ligands. Then, the effects of blocking the Xe4 site with I28W, V68W, and I107W mutations and the Xe1 cavity with L89W, L104W, and F138W mutations are described. The structures of four of these mutants are shown for the first time (Trp28, Trp68, Trp107, and Trp 138 sperm whale metMb). All available results support a 'side path' mechanism in which ligands move into and out of myoglobin by outward rotation of the HisE7 side chain, but after entry can migrate into internal cavities, including the distal Xe4 and proximal Xe1 binding sites. The distal cavities act like the pocket of a baseball glove, catching the ligand and holding it long enough for the histidine gate to close and facilitate internal coordination with the heme iron atom. The physiological role of the proximal Xe1 site is less clear because changes in the size of this cavity have minimal effects on overall O(2) binding parameters.
Topics: Crystallography, X-Ray; Ligands; Mutation; Myoglobin; Recombination, Genetic; Tryptophan
PubMed: 17701550
DOI: 10.1080/15216540701230495 -
The American Journal of Pathology Jul 2009Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging...
Myoglobin is a multifunctional heme protein that is thought to be expressed exclusively in myocytes. Its importance in both oxygen transport and free radical scavenging has been extensively characterized. We hypothesized that solid tumors could take advantage of proteins such as myoglobin to cope with hypoxic conditions and to control the metabolism of reactive oxygen and nitrogen species. We therefore sought to establish whether myoglobin might be expressed and functionally regulated in epithelial tumors that are known to face hypoxia and oxidative stress during disease progression. We analyzed the expression of myoglobin in human epithelial cancers at both transcriptional and protein levels; moreover, we investigated the expression levels of myoglobin in cancer cell lines subjected to different conditions, including hypoxia, oxidative stress, and mitogenic stimuli. We provide evidence that human epithelial tumors, including breast, lung, ovary, and colon carcinomas, express high levels of myoglobin from the earliest stages of disease development. In human cancer cells, myoglobin is induced by a variety of signals associated with tumor progression, including mitogenic stimuli, oxidative stress, and hypoxia. This study provides evidence that myoglobin, previously thought to be restricted to myocytes, is expressed at high levels by human carcinoma cells. We suggest that myoglobin expression is part of a cellular program aimed at coping with changed metabolic and environmental conditions associated with neoplastic growth.
Topics: Biomarkers, Tumor; Cell Line, Tumor; Gene Expression; Gene Expression Profiling; Humans; Immunohistochemistry; Myoglobin; Neoplasms, Glandular and Epithelial; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 19541931
DOI: 10.2353/ajpath.2009.081124 -
Molecules (Basel, Switzerland) May 2023Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical... (Review)
Review
Protein folding is essential for a polypeptide chain to acquire its proper structure and function. Globins are a superfamily of ubiquitous heme-binding α-helical proteins whose function is principally to regulate oxygen homoeostasis. In this review, we explore the hierarchical helical formation in the globin proteins apomyoglobin and leghemoglobin, and we discuss the existence of non-native and misfolded structures occurring during the course of folding to its native state. This review summarizes the research aimed at characterizing and comparing the equilibrium and kinetic intermediates, as well as delineating the complete folding pathway at a molecular level, in order to answer the following questions: "What is the mechanism of misfolding via a folding intermediate? Does the non-native structure stabilize the contemporary intermediate structure? Does the non-native structure induce slower folding?" The role of the non-native structures in the folding intermediate related to misfolding is also discussed.
Topics: Myoglobin; Apoproteins; Protein Folding; Leghemoglobin; Kinetics
PubMed: 37175379
DOI: 10.3390/molecules28093970 -
Journal of Animal Science Jul 2020The emerging market of frozen meat emphasizes the need to better understand beef surface discoloration and the ideal parameters of freezing beef to retain an acceptable...
The emerging market of frozen meat emphasizes the need to better understand beef surface discoloration and the ideal parameters of freezing beef to retain an acceptable color. The objectives of this study were to determine the impacts of myoglobin oxygenation level prior to freezing and frozen storage duration on frozen beef color. USDA Choice strip loins (n = 36) were aged for 4 d or 20 d. Steaks were randomly assigned to a myoglobin oxygenation level [deoxygenated (DeOxy; immediately packaged after cutting), oxygenated (Oxy; oxygenated in air for 30 min), or highly oxygenated (HiOxy; packaged for 24 h in 80% O2)]. Steaks were then vacuum packaged in oxygen permeable or impermeable film and immediately frozen (-5 °C). Following either 0, 2, 4, or 6 mo of frozen storage, steaks were removed from the packaging and immediately analyzed for instrumental color (L*, a*, and b*), percent oxymyoglobin, metmyoglobin, and deoxymyoglobin, delta E, redness ratio, a*:b* ratio, hue angle, subjective discoloration, and lipid oxidation. The HiOxy steaks had greater oxygen penetration and the greatest a* values compared with DeOxy and Oxy steaks, regardless of packaging (P < 0.0005). With 4 d of aging, HiOxy steaks had greater a* values than DeOxy and Oxy at all storage times (P = 0.0118). The HiOxy steaks aged for 20 d and frozen for 6 mo had significantly higher delta E values than all other myoglobin oxygenation levels and postmortem aging periods (P < 0.0001). Redness and percent oxymyoglobin were highest for HiOxy steaks within each storage period (P < 0.0002). The HiOxy steaks had the highest percent oxymyoglobin and DeOxy had the lowest percent oxymyoglobin within each aging and storage period (P < 0.01). Conversely, DeOxy steaks had the highest percent metmyoglobin and HiOxy had the lowest percent metmyoglobin when packaged in impermeable film (P < 0.0001). The HiOxy steaks from 20 d of aging had the highest discoloration compared with 4 d aging and more discoloration than all other myoglobin treatments at 6 mo of storage (P < 0.0001). The HiOxy 20 d aged steaks exhibited the highest lipid oxidation values at 2, 4, and 6 mo (P = 0.0224) and HiOxy steaks exhibited a brighter and deeper cherry red color compared with the DeOxy steaks. The HiOxy steaks were greater in redness or similar when compared with Oxy steaks, but experienced more detrimental effects when frozen storage was extended.
Topics: Animals; Cattle; Color; Food Packaging; Food Storage; Freezing; Metmyoglobin; Muscle, Skeletal; Myoglobin; Oxidation-Reduction; Oxygen; Red Meat
PubMed: 32516410
DOI: 10.1093/jas/skaa193 -
Angewandte Chemie (International Ed. in... Jul 2018Functionalized indoles are recurrent motifs in bioactive natural products and pharmaceuticals. While transition metal-catalyzed carbene transfer has provided an...
Functionalized indoles are recurrent motifs in bioactive natural products and pharmaceuticals. While transition metal-catalyzed carbene transfer has provided an attractive route to afford C3-functionalized indoles, these protocols are viable only in the presence of N-protected indoles, owing to competition from the more facile N-H insertion reaction. Herein, a biocatalytic strategy for enabling the direct C-H functionalization of unprotected indoles is reported. Engineered variants of myoglobin provide efficient biocatalysts for this reaction, which has no precedents in the biological world, enabling the transformation of a broad range of indoles in the presence of ethyl α-diazoacetate to give the corresponding C3-functionalized derivatives in high conversion yields and excellent chemoselectivity. This strategy could be exploited to develop a concise chemoenzymatic route to afford the nonsteroidal anti-inflammatory drug indomethacin.
Topics: Biocatalysis; Catalysis; Indoles; Molecular Structure; Myoglobin
PubMed: 29905974
DOI: 10.1002/anie.201804779 -
Biomolecules Oct 2019Recent electronic transport experiments using metallic contacts attached to proteins identified some "stylized facts", which contradict conventional wisdom that...
Recent electronic transport experiments using metallic contacts attached to proteins identified some "stylized facts", which contradict conventional wisdom that increasing either the spatial distance between the electrodes or the temperature suppresses conductance exponentially. These include nearly temperature-independent conductance over the protein in the 30 to 300 K range, distance-independent conductance within a single protein in the 1 to 10 nm range and an anomalously large conductance in the 0.1 to 10 nS range. In this paper, we develop a generalization of the low temperature Landauer formula, which can account for the joint effects of tunneling and decoherence and can explain these new experimental findings. We use novel approximations, which greatly simplify the mathematical treatment and allow us to calculate the conductance in terms of a handful macroscopic parameters, instead of the myriads of microscopic parameters describing the details of an atomic level quantum chemical computation. The new approach makes it possible to get predictions for the outcomes of new experiments without relying solely on high performance computing and can distinguish important and unimportant details of the protein structures from the point of view of transport properties.
Topics: Cytochromes c; Electron Transport; Electrons; Myoglobin; Streptavidin
PubMed: 31614584
DOI: 10.3390/biom9100599 -
Scientific Reports Jul 2019We develop an effective theory approach to investigate the phase properties of globular proteins. Instead of interactions between individual atoms or localized...
We develop an effective theory approach to investigate the phase properties of globular proteins. Instead of interactions between individual atoms or localized interaction centers, the approach builds directly on the tertiary structure of a protein. As an example we construct the phase diagram of (apo)myoglobin with temperature (T) and acidity (pH) as the thermodynamical variables. We describe how myoglobin unfolds from the native folded state to a random coil when temperature and acidity increase. We confirm the presence of two molten globule folding intermediates, and we predict an abrupt transition between the two when acidity changes. When temperature further increases we find that the abrupt transition line between the two molten globule states terminates at a tricritical point, where the helical structures fade away. Our results also suggest that the ligand entry and exit is driven by large scale collective motions that destabilize the myoglobin F-helix.
Topics: Models, Molecular; Myoglobin; Protein Folding; Protein Structure, Tertiary
PubMed: 31346242
DOI: 10.1038/s41598-019-47317-y