N-hydroxyamphetamine (AmphNHOH) is an oxidative metabolite of amphetamine and methamphetamine. It is known to form inhibitory complexes upon binding to heme proteins. However, its interactions with myoglobin (Mb) and hemoglobin (Hb) have not been reported. We demonstrate that the reactions of AmphNHOH with ferric Mb and Hb generate the respective heme-nitrosoamphetamine derivatives characterized by UV-vis spectroscopy. We have determined the X-ray crystal structure of the H64A Mb-nitrosoamphetamine complex to 1.73 Å resolution. The structure reveals the N-binding of the nitroso-d-amphetamine isomer, with no significant H-bonding interactions between the ligand and the distal pocket amino acid residues.

Bison is an alternate meat species gaining increased popularity in North America. Although previous investigations reported that bison meat discolors faster than beef, the molecular basis of this observation has not been investigated. Therefore, the objective of the present study was to determine the redox stability, thermostability, and primary structure of bison myoglobin (Mb), in comparison with beef Mb. Purified bison and beef myoglobins were analyzed for autoxidation, lipid oxidation-induced oxidation, and thermostability. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry was utilized for determining the exact molecular mass of bison Mb, whereas Edman degradation was employed to determine the amino acid sequence. Bison and beef myoglobins behaved similarly in autoxidation, lipid oxidation-induced oxidation, and thermostability. The observed molecular mass of bison and beef myoglobins was 16,949 Da, and the primary structure of bison Mb shared 100% similarity with beef and yak myoglobins. Noticeably, the amino acid sequence of bison Mb was different from other ruminant myoglobins, such as water-buffalo, sheep, goat, and red-deer. The present study is the first to report the primary structure of bison Mb. Same primary structure and similar biochemical attributes of bison and beef myoglobins suggested that the observed rapid discoloration in bison meat could not be attributed to biochemistry of bison Mb.

Globular proteins contain cavities/voids that play specific roles in controlling protein function. Elongated cavities provide migration channels for the transport of ions and small molecules to the active center of a protein or enzyme. Using Monte Carlo and Molecular Dynamics on fully atomistic protein/water models, a new computational methodology is introduced that takes into account the protein's dynamic structure and maps all the cavities in and on the surface. To demonstrate its utility, the methodology is applied to study cavity structure in myoglobin and five of its mutants. Computed cavity and channel size distributions reveal significant differences relative to the wild type myoglobin. Computer visualization of the channels leading to the heme center indicates restricted ligand access for the mutants consistent with the existing interpretations. The new methodology provides a quantitative measure of cavity structure and distributions and can become a valuable tool for the structural characterization of proteins.

The endogenous mechanisms that propagate cardiomyocyte differentiation and prevent de-differentiation remain unclear. While the expression of the heme protein myoglobin increases by over 50% during cardiomyocyte differentiation, a role for myoglobin in regulating cardiomyocyte differentiation has not been tested. Here, we show that deletion of myoglobin in cardiomyocyte models decreases the gene expression of differentiation markers and stimulates cellular proliferation, consistent with cardiomyocyte de-differentiation. Mechanistically, the heme prosthetic group of myoglobin catalyzes the oxidation of the Hippo pathway kinase LATS1, resulting in phosphorylation and inactivation of yes-associated protein (YAP). In vivo, myoglobin-deficient zebrafish hearts show YAP dephosphorylation and accelerated cardiac regeneration after apical injury. Similarly, myoglobin knockdown in neonatal murine hearts shows increased YAP dephosphorylation and cardiomyocyte cycling. These data demonstrate a novel role for myoglobin as an endogenous driver of cardiomyocyte differentiation and highlight myoglobin as a potential target to enhance cardiac development and improve cardiac repair and regeneration.

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.

Myoglobin (Mb), an oxygen-binding heme protein highly expressed in heart and skeletal muscle, has been shown to undergo oxidative modifications on both an inter- and intramolecular level when exposed to hydrogen peroxide (H2O2) in vitro. Here, we show that exposure to H2O2 increases the peroxidase activity of Mb. Reaction of Mb with H2O2 causes covalent binding of heme to the Mb protein (Mb-X), corresponding to an increase in peroxidase activity when ascorbic acid is the reducing co-substrate. Treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. Reaction with H2O2 causes Mb to form dimers, trimers, and larger molecular weight Mb aggregates, and treatment with ascorbic acid regenerates Mb monomers. Reaction of Mb with H2O2 causes formation of dityrosine crosslinks, though the labile nature of the crosslinks broken by treatment with ascorbic acid suggests that the reversible aggregation of Mb is mediated by crosslinks other than dityrosine. Disappearance of a peptide containing a tryptophan residue when Mb is treated with H2O2 and the peptide's reappearance after subsequent treatment with ascorbic acid suggest that tryptophan side chains might participate in the labile crosslinking. Taken together, these data suggest that while exposure to H2O2 causes Mb-X formation, increases Mb peroxidase activity, and causes Mb aggregation, these oxidative modifications are reversible by treatment with ascorbic acid. A caveat is that future studies should demonstrate that these and other in vitro findings regarding properties of Mb have relevance in the intracellular milieu, especially in regard to actual concentrations of metMb, H2O2, and ascorbate that would be found in vivo.

Continuous contractile activity of the heart is essential and the required energy is mostly provided by fatty acid (FA) oxidation. Myocardial lipid accumulation can lead to pathological responses, however the underlying mechanisms remain elusive. The role of myoglobin in dioxygen binding in cardiomyocytes and oxidative skeletal muscle has widely been appreciated. Our recent work established myoglobin as a protector of cardiac function in hypoxia and disease states. We here unravel a novel role of cardiac myoglobin in governing FA metabolism to ensure the physiological energy production through β-oxidation, preventing myocardial lipid accumulation and preserving cardiac functions. In vivo1H magnetic resonance spectroscopy unveils a 3-fold higher deposition of lipids in mouse hearts lacking myoglobin, which was associated with depressed cardiac function compared to wild-type hearts as assessed by echocardiography. Mass spectrometry reveals a marked increase in tissue triglycerides with preferential incorporation of palmitic and oleic acids. Phospholipid levels as well as the metabolome, transcriptome and proteome related to FA metabolism tend to be unaffected by myoglobin ablation. Our results reveal a physiological role of myoglobin in FA metabolism with the lipid accumulation-suppressing effects of myoglobin preventing cardiac lipotoxicity.

Hydroxylamine (HA), which is a natural product of mammalian cells, has been shown to possess vasodilatory properties in several model systems. In this study, HA and methyl-substituted hydroxylamines, N-methylhydroxylamine (NMHA) and N,N-dimethylhydroxylamine (NDMHA), have been tested for their ability to generate free diffusible nitric oxide (NO) in the presence of myoglobin (Mb) and hydrogen peroxide. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI), measured by electron spin resonance (ESR) spectroscopy, along with nitrite and nitrate production, was observed for HA but not for NMHA and NDMHA. ESR measurements at 77 K showed the formation of the ferrous nitrosyl myoglobin, Mb-NO, in the reaction mixtures containing Mb, H2O2 and HA. Our data also demonstrate that Mb-NO is an end product of the reaction pathway involving Mb, H2O2 and HA, rather than a reaction intermediate in the formation of NO. In summary, our results demonstrate a possible pathway of NO formation from HA, however, the significance of this mechanism for bioactivation of HA in vivo is unknown at the present time.

The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO3-) and nitrite (NO2-) has gained increasing attention due to the potential beneficial effects of NO in cardiovascular diseases and exercise performance. We have previously shown in rodents that skeletal muscle is the major nitrate reservoir in the body and that exercise enhances the nitrate reduction pathway in the muscle tissue and have proposed that nitrate in muscle originates from diet, the futile cycle of nitric oxide synthase 1 (NOS1) and/or oxidation of NO by oxymyoglobin. In the present study, we tested the hypothesis that lack of myoglobin expression would decrease nitrate levels in skeletal muscle. We observed a modest but significant decrease of nitrate level in skeletal muscle of myoglobin deficient mice compared to littermate control mice (17.3 vs 12.8 nmol/g). In contrast, a NOS inhibitor, L-NAME or a low nitrite/nitrate diet treatment led to more pronounced decreases of nitrate levels in the skeletal muscle of both control and myoglobin deficient mice. Nitrite levels in the skeletal muscle of both types of mice were similar (0.48 vs 0.42 nmol/g). We also analyzed the expression of several proteins that are closely related to NO metabolism to examine the mechanism by which nitrate and nitrite levels are preserved in the absence of myoglobin. Western blot analyses suggest that the protein levels of xanthine oxidoreductase and sialin, a nitrate transporter, both increased in the skeletal muscle of myoglobin deficient mice. These results are compatible with our previously reported model of nitrate production in muscle and suggest that myoglobin deficiency activates compensatory mechanisms to sustain NO homeostasis.

Myoglobin is a cytoplasmic hemoprotein, expressed solely in cardiac myocytes and oxidative skeletal muscle fibers, that reversibly binds O2 by its heme residue. Myoglobin is an essential oxygen-storage hemoprotein capable of facilitating oxygen transport and modulating nitric oxide homeostasis within cardiac and skeletal myocytes. Functionally, myoglobin is well accepted as an O2- storage protein in muscle, capable of releasing O2 during periods of hypoxia or anoxia. There is no evidence available regarding active sites, ligand binding sites, antigenic determinants and the ASA value of myoglobin in Channa striata. We further document the predicted active sites in the structural model with solvent exposed ASA residues. During this study, the model was built by CPH program and validated through PROCHECK, Verify 3D, ERRAT and ProSA for reliability. The active sites were predicted in the model with further ASA analysis of active site residues. The discussed information thus provides the predicted active sites, ligand binding sites, antigenic determinants and ASA values of myoglobin model in Channa striata.

High performance liquid chromatography (HPLC) coupled with a Fraction Collector was employed to isolate Maillard reaction products (MRPs) formed in model systems comprising of asparagine and monosaccharides in the 60-180°C range. The primary MRP which is detected at 60°C is important for Acrylamide content and color/aroma development in foods and also in the field of food biotechnology for controlling the extent of the Maillard reaction with temperature. The discrete fractions of the reaction products were reacted with Hemoglobin (Hb) and Myoglobin (Mb) at physiological conditions and the reaction adducts were monitored by UV-vis and Attenuated Total Reflection-Fourier transform infrared (FTIR) spectrophotometry. The UV-vis kinetic profiles revealed the formation of a Soret transition characteristic of a low-spin six-coordinated species and the ATR-FTIR spectrum of the Hb-MRP and Mb-MRP fractions showed modifications in the protein Amide I and II vibrations. The UV-vis and the FTIR spectra of the Hb-MRPs indicate that the six-coordinated species is a hemichrome in which the distal E7 Histidine is coordinated to the heme Fe and blocks irreversibly the ligand binding site. Although the Mb-MRPs complex is a six-coordinated species, the 1608 cm-1 FTIR band characteristic of a hemichrome was not observed.

The muscle‑associated respiratory protein myoglobin (MB) is expressed in multiple types of cancer, including breast and prostate tumors. In Kaplan‑Meier analyses of the two tumor types, MB positivity is associated with favorable prognoses. Despite its well‑characterized function in myocytes, the role of MB in cancer remains unclear. To study the impact of endogenous MB expression, small interfering RNA MB‑knockdown cells were engineered using breast, prostate and colon cancer cell lines (MDA‑MB468, LNCaP, DLD‑1), and their transcriptomes were investigated using RNA‑Seq at different oxygen levels. In MB‑positive cells, increased expression of glycolytic genes was observed, which was possibly mediated by a higher activity of hypoxia‑inducible factor 1α. In addition, the results of the gene set enrichment analysis suggested that MB contributed to fatty acid transport and turnover. MB‑positive, wild‑type‑p53 LNCaP cells also exhibited increased expression of p53 target genes involved in cell cycle checkpoint control and prevention of cell migration. MB‑positive cells expressing mutant p53 exhibited upregulation of genes associated with prolonged cancer cell viability and motility. Therefore, it was hypothesized that these transcriptomic differences may result from MB‑mediated generation of nitric oxide or reactive oxygen species, thus employing established enzymatic activities of the globin. In summary, the transcriptome comparisons identified potential molecular functions of MB in carcinogenesis by highlighting the interaction of MB with key metabolic and regulatory processes.

Complexes were prepared of horse heart myoglobin with derivatives of (bacterio)chlorophylls and the linear tetrapyrrole, phycocyanobilin. Structural factors important for binding are (i) the presence of a central metal with open ligation site, which even induces binding of phycocyanobilin, and (ii) the absence of the hydrophobic esterifying alcohol, phytol. Binding is further modulated by the stereochemistry at the isocyclic ring. The binding pocket can act as a reaction chamber: with enolizable substrates, apo-myoglobin acts as a 13(2)-epimerase converting, e.g., Zn-pheophorbide a' (13(2)S) to a (13(2)R). Light-induced reduction and oxidation of the bound pigments are accelerated as compared to solution. Some flexibility of the myoglobin is required for these reactions to occur; a nucleophile is required near the chromophores for photoreduction (Krasnovskii reaction), and oxygen for photooxidation. Oxidation of the bacteriochlorin in the complex and in aqueous solution continues in the dark.

Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.

The contribution of superoxide-mediated injury to oxidative stress is not fully understood. A potential mechanism is the reaction of superoxide with tyrosyl radicals, which either results in repair of the tyrosine or formation of tyrosine hydroperoxide by addition. Whether these reactions occur with protein tyrosyl radicals is of interest because they could alter protein structure or modulate enzyme activity. Here, we have used a xanthine oxidase/acetaldehyde system to generate tyrosyl radicals on sperm whale myoglobin in the presence of superoxide. Using mass spectrometry we found that superoxide prevented myoglobin dimer formation by repairing the protein tyrosyl radical. An addition product of superoxide at Tyr151 was also identified, and exogenous lysine promoted the formation of this product. In our system, reaction of tyrosyl radicals with superoxide was favored over dimer formation with the ratio of repair to addition being approximately 10:1. Our results demonstrate that reaction of superoxide with protein tyrosyl radicals occurs and may play a role in free radical-mediated protein injury.

Extant cetaceans, such as sperm whale, acquired the great ability to dive into the ocean depths during the evolution from their terrestrial ancestor that lived about 50 million years ago. Myoglobin (Mb) is highly concentrated in the myocytes of diving animals, in comparison with those of land animals, and is thought to play a crucial role in their adaptation as the molecular aqualung. Here, we resurrected ancestral whale Mbs, which are from the common ancestor between toothed and baleen whales (Basilosaurus), and from a further common quadrupedal ancestor between whale and hippopotamus (Pakicetus). The experimental and theoretical analyses demonstrated that whale Mb adopted two distinguished strategies to increase the protein concentration in vivo along the evolutionary history of deep sea adaptation; gaining precipitant tolerance in the early phase of the evolution, and increase of folding stability in the late phase.

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump-probe experiments for the investigation of electronic and nuclear changes during light-induced reactions. On timescales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1,2. However, all ultrafast TR-SFX studies to date have employed such high pump laser energies that nominally several photons were absorbed per chromophore3-17. As multiphoton absorption may force the protein response into non-physiological pathways, it is of great concern18,19 whether this experimental approach20 allows valid conclusions to be drawn vis-à-vis biologically relevant single-photon-induced reactions18,19. Here we describe ultrafast pump-probe SFX experiments on the photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics21) are seen to depend strongly on pump laser energy, in line with quantum chemical analysis. Our results confirm both the feasibility and necessity of performing ultrafast TR-SFX pump-probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing both the design and the interpretation of ultrafast TR-SFX pump-probe experiments20 such that mechanistically relevant insight emerges.

This work investigates the role of myoglobin in mediating the vascular relaxation induced by nitrite. Nitrite, previously considered an inert by-product of nitric oxide metabolism, is now believed to play an important role in several areas of pharmacology and physiology. Myoglobin can act as a nitrite reductase in the heart, where it is plentiful, but it is present at a far lower level in vascular smooth muscle-indeed, its existence in the vessel wall is controversial. Haem proteins have been postulated to be important in nitrite-induced vasodilation, but the specific role of myoglobin is unknown. The current study was designed to confirm the presence of myoglobin in murine aortic tissue and to test the hypothesis that vascular wall myoglobin is important for nitrite-induced vasodilation.

[Purpose] Atrophy is a common phenomenon caused by prolonged muscle disuse associated with bed-rest, aging, and immobilization. However, changes in the expression of atrophy-related myoglobin are still poorly understood. In the present study, we examined whether or not myoglobin expression is altered in the gastrocnemius muscles of rats after seven days of cast immobilization. [Methods] We conducted a protein expression and high-resolution differential proteomic analysis using, two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry, and western blotting. [Results] The density and expression of myoglobin increased significantly more in atrophic gastrocnemius muscle strips than they did in the control group. [Conclusion] The results suggest that cast immobilization-induced atrophy may be related to changes in the expression of myoglobin in rat gastrocnemius muscles.

Research focused on determining the fundamental mechanisms by which lactate influences color stability has not considered a direct effect of lactate on myoglobin. Thus, the objective of this study was to use Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry to examine lactate adduction to myoglobin. Equine oxymyoglobin and equine carboxymyoglobin (0.15mM) were incubated with sodium lactate (200mM) at 4 degrees C, pH 5.6 in 50mM sodium citrate buffer or at 37 degrees C, pH 7.4 in 50mM sodium phosphate buffer, simulating typical meat storage and physiological conditions, respectively. Controls consisted of myoglobin plus a volume of deionized water equivalent to that used to deliver the lactate treatments. No peaks corresponding to lactate-Mb adducts could be detected in the mass spectra of samples incubated up to 360min at pH 7.4, 37 degrees C or 8days at pH 5.6 and 4 degrees C. Our results suggest that lactate did not form covalent adducts with equine oxy- and carboxy-myoglobin.