Wild-type Myoglobin Research Articles

Myoglobin mutant with enhanced nitrite reductase activity regulates intracellular oxidative stress in human breast cancer cells

Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.

Engineered myoglobin as a catalyst for atom transfer radical cyclisation.

Myoglobin was subjected to site-directed mutagenesis and transformed into a catalyst able to perform atom transfer radical cyclisation reactions, i.e. intramolecular atom transfer radical additions. Replacing the iron-coordinating histidine with serine, or introducing small changes inside or at the entrance of the active site, transformed the completely inactive wild-type myoglobin into an artificial metalloenzyme able to catalyse the 5-exo cyclisation of halogenated unsaturated compounds for the synthesis of γ-lactams. This new-to-nature activity was achieved not only with purified protein but also in crude cell lysate and in whole cells.

An Enzymatic Platform for the Highly Enantioselective and Stereodivergent Construction of Cyclopropyl-δ-lactones.

Abiological enzymes offers new opportunities for sustainable chemistry. Herein, we report the development of biological catalysts derived from sperm whale myoglobin that exploit a carbene transfer mechanism for the asymmetric synthesis of cyclopropane-fused-δ-lactones, which are key structural motifs found in many biologically active natural products. While hemin, wild-type myoglobin, and other hemoproteins are unable to catalyze this reaction, the myoglobin scaffold could be remodeled by protein engineering to permit the intramolecular cyclopropanation of a broad spectrum of homoallylic diazoacetate substrates in high yields and with up to 99 % enantiomeric excess. Via an alternate evolutionary trajectory, a stereodivergent biocatalyst was also obtained for affording mirror-image forms of the desired bicyclic products. In combination with whole-cell transformations, the myoglobin-based biocatalyst was used for the asymmetric construction of a cyclopropyl-δ-lactone scaffold at a gram scale, which could be further elaborated to furnish a variety of enantiopure trisubstituted cyclopropanes.

Nonbonded Atomic Contacts Drive Ultrafast Helix Motions in Myoglobin.

The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.

Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.

Myoglobin-Catalyzed Efficient In Situ Regeneration of NAD(P)+and Their Synthetic Biomimetic for Dehydrogenase-Mediated Oxidations

On the basis of its catalytic promiscuity, wild-type myoglobin (Mb) from horse heart was used for in situ regeneration of oxidized nicotinamide cofactors. Mb proved to be a versatile catalyst, since it was capable of efficient oxidation of both natural cofactors NAD(P)H and their synthetic biomimetic BNAH with H2O2. Mb-catalyzed oxidation of reduced nicotinamide cofactors was promoted significantly in the presence of mediators such as scopoletin and acetaminophen. A cofactor total turnover number (TTN) up to 50 000 was obtained in glucose dehydrogenase-catalyzed oxidation of glucose coupled with the Mb/scopoletin regeneration system. Approximately 4 mol of glucose was enzymatically oxidized with 1 mol of H2O2. Besides, this system is well compatible with various dehydrogenases. The desired products were afforded with yields of 93–97% in dehydrogenase-catalyzed oxidations. A radical mechanism was proposed for Mb/scopoletin-catalyzed recycling of NAD(P)+. The Mb-based recycling system may create opportuniti...

Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor.

The orientation of a CF3-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using 19F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5-15 meso axis, with respect to the protein moiety. Crystal structure of the met-aquo form of the wild-type myoglobin reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein.

Dynamic void distribution in myoglobin and five mutants

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.

Computational Infrared and Two-Dimensional Infrared Photon Echo Spectroscopy of Both Wild-Type and Double Mutant Myoglobin-CO Proteins

The CO stretching mode of both wild-type and double mutant ( T67R / S92D ) MbCO (carbonmonoxymyoglobin) proteins is an ideal infrared (IR) probe for studying the local electrostatic environment inside the myoglobin heme pocket. Recently, to elucidate the conformational switching dynamics between two distinguishable states, extensive IR absorption, IR pump-probe, and two-dimensional (2D) IR spectroscopic studies for various mutant MbCO's have been performed by the Fayer group. They showed that the 2D IR spectroscopy of the double mutant, which has a peroxidase enzyme activity, reveals a rapid chemical exchange between two distinct states, whereas that of the wild-type does not. Despite the fact that a few simulation studies on these systems were already performed and reported, such complicated experimental results have not been fully reproduced nor described in terms of conformational state-to-state transition processes. Here, we first develop a distributed vibrational solvatochromic charge model for describing the CO stretch frequency shift reflecting local electric potential changes. Then, by carrying out molecular dynamic simulations of the two MbCO's and examining their CO frequency trajectories, it becomes possible to identify a proper reaction coordinate consisting of His64 imidazole ring rotation and its distance to the CO ligand. From the 2D surfaces of the resulting potential of mean forces, the spectroscopically distinguished A1 and A3 states of the wild-type as well as two more substates of the double mutant are identified and their vibrational frequencies and distributions are separately examined. Our simulated IR absorption and 2D IR spectra of the two MbCO's are directly compared with the previous experimental results reported by the Fayer group. The chemical exchange rate constants extracted from the two-state kinetic analyses of the simulated 2D IR spectra are in excellent agreement with the experimental values. On the basis of the quantitative agreement between the simulated spectra and experimental ones, we further examine the conformational differences in the heme pockets of the two proteins and show that the double mutation, T67R / S92D , suppresses the A1 population, restricts the imidazole ring rotation, and increases hydrogen-bond strength between the imidazole Nε-H and the oxygen atom of the CO ligand. It is believed that such delicate change of distal His64 imidazole ring dynamics induced by the double mutation may be responsible for its enhanced peroxidase catalytic activity as compared to the wild-type myoglobin.

Effects of local protein environment on the binding of diatomic molecules to heme in myoglobins. DFT and dispersion-corrected DFT studies.

The heme-AB binding energies (AB = CO, O2) in a wild-type myoglobin (Mb) and two mutants (H64L, V68N) of Mb have been investigated in detail with both DFT and dispersion-corrected DFT methods, where H64L and V68N represent two different, opposite situations. Several dispersion correction approaches were tested in the calculations. The effects of the local protein environment were accounted for by including the five nearest surrounding residues in the calculated systems. The specific role of histidine-64 in the distal pocket was examined in more detail in this study than in other studies in the literature. Although the present calculated results do not change the previous conclusion that the hydrogen bonding by the distal histidine-64 residue plays a major role in the O2/CO discrimination by Mb, more details about the interaction between the protein environment and the bound ligand have been revealed in this study by comparing the binding energies of AB to a porphyrin and the various myoglobins. The changes in the experimental binding energies from one system to another are well reproduced by the calculations. Without constraints on the residues in geometry optimization, the dispersion correction is necessary, since it improves the calculated structures and energetic results significantly.

Implications of active site orientation in myoglobin for direct electron transfer and electrocatalysis based on monolayer and multilayer covalent immobilization on gold electrodes

Cyclic voltammetry (CV) and atomic force microscopy (AFM) were used to investigate the importance of active site orientation of an immobilized protein for direct electron transfer (DET) and electrocatalysis. While the bioconjugated wild-type myoglobin (WT Mb) was immobilized on the modified gold electrode surface in a random multilayered fashion, the Ser3 replaced with unnatural amino acid, 3-amino-l-tyrosine, (NH2Tyr) mutant Mb was immobilized via a Diels–Alder reaction specific to NH2Tyr residue to form a homogeneous monolayer. Electrochemical calculations for the number of surface exposed redox-active molecules on the electrode surface (Γ) and heterogeneous rate constant for DET were 1.29×10−10molcm−2; 2.3s−1 for the WT Mb and 1.54×10−10molcm−2; 1.3s−1 for the S3NH2Tyr Mb mutant, respectively. Electro-catalytic conversion of thioanisole to sulfoxide products showed similar turnover frequencies (TOF) around 1.9×103s−1 (with 87% conversion) for the WT Mb, and 1.5×103s−1 for the mutant S3NH2Tyr Mb (with 81% conversion). These results indicate that site-directed single monolayer immobilization affords almost the same number of surface exposed Mb active sites as the random multilayer immobilization strategy, though the latter contains a greater number of protein molecules on the electrode surface, as observed from the AFM data.

Assessment of dispersion corrections in DFT calculations on large biological systems

The performance of empirical dispersion corrections in DFT calculations has been assessed for several large, genuine biological systems that include MbAB, H64L(AB), and V68N(AB) (AB = CO, O2), where Mb stands for a wild-type myoglobin, H64L is the (histidine64 → leucine) mutated myoglobin, and V68N is the (valine68 → asparagine) mutated myoglobin. The effects of the local protein environment are accounted for by including the five nearest surrounding residues in the calculated systems and they are examined by comparing the binding energies of AB to the myoglobin and to the porphyrin (Por) without residues. Three versions of Grimme's dispersion correction methods, labeled as DFT-D1, DFT-D2, and DFT-D3, were all tested. In the first version (-D1), the dispersion correction (Edisp) is calculated only for noncovalent interactions between molecular fragments and Edisp within a covalent molecule is not calculated. For the DFT functionals, for which the calculated Por–AB binding energies are already too large, only further overestimation occurs when a dispersion correction is made. The geometry optimizations show that the DFT-D2 and DFT-D3 approaches give too short distances between the residues and the heme moiety in the myoglobins and their calculated relative binding energies ΔEbind(myoglobin-AB/Por–AB) are in poor agreement with experiment in most cases. DFT-D1 performs very well, ensuring structural and energetic features in close agreement with experiment.

The effect of point mutation on the equilibrium structural fluctuations of ferric Myoglobin

The ultrafast equilibrium fluctuations of the Fe(III)-NO complex of a single point mutation of Myoglobin (H64Q) have been studied using Fourier Transform 2D-IR spectroscopy. Comparison with data from wild type Myoglobin (wt-Mb) shows the presence of two conformational substates of the mutant haem pocket where only one exists in the wild type form. One of the substates of the mutant exhibits an almost identical NO stretching frequency and spectral diffusion dynamics to wt-Mb while the other is distinctly different in both respects. The remarkably contrasting dynamics are largely attributable to interactions between the NO ligand and a nearby distal side chain which provides a basis for understanding the roles of these side chains in other ferric haem proteins.

Amyloid fibril formation by circularly permuted and C-terminally deleted mutants

The natural N- and C-termini, i.e., the given order of secondary structure segments, are critical for protein folding and stability, as shown by several studies using circularly permuted proteins, mutants that have their N- and C-termini linked and are then digested at another site to create new termini. A previous work showed that circularly permuted mutants of sperm whale myoglobin (Mb) are functional, have native-like folding and bind heme, but are less stable than the wild-type protein and aggregate. The ability of wild-type myoglobin to form amyloid fibrils has been established recently, and because circularly permuted mutations are destabilizing, we asked whether these permutations would also affect the rate of amyloid fibril formation. Our investigations revealed that, indeed, the circularly permuted mutants formed cytotoxic fibrils at a rate higher than that of the wild-type. To further investigate the role of the C-terminus in the overall stability of the protein, we investigated two C-terminally deleted mutant, Mb1–123 and Mb1–99, and found that Mb1–123 formed cytotoxic fibrils at a higher rate than that of the wild-type while Mb1–99 formed cytotoxic fibrils at a similar rate than that of the wild-type. Collectively, our findings show that the native position of both the N-and C-termini is important for the precise structural architecture of myoglobin.

The effects of the L29F mutation on the ligand migration kinetics in crystallized myoglobin as revealed by molecular dynamics simulations

By using multiple molecular dynamics (MD) trajectories, a quantitative description of carbon monoxide (CO) migration within crystal of L29F myoglobin mutant (L29F-Mb) was obtained. The aim was to provide a detailed model for ligand diffusion in the protein to be compared to the available L29F-Mb experimental-computational data and to the corresponding model kinetics we previously obtained for photolyzed CO within crystallized wild-type myoglobin (wt-Mb). Results suggest a clear migration pathway from distal pocket to the proximal site, similar to the one observed in wt-Mb, with a relaxation kinetics differing from the wt-Mb one essentially for the escape rate which is much higher in the mutant. Moreover MD data indicated a clear correlation between CO location within the protein and the conformation adopted by Phe29, well matching the available experimental data as obtained by time-resolved X-ray density maps. Such data, further validating the model used in the simulations, point out the subtle mutual effect between ligand diffusion and protein functional motions possibly explaining the observed dramatic variation of CO exit rate in L29F-Mb.

Redox properties of engineered ruthenium myoglobin

Ruthenium (II) complex of mesoporphyrin-IX was incorporated into apomyoglobin to prepare artificial ruthenium myoglobin (RuMb) containing the ruthenium porphyrin in place of protoheme. The electrochemical and spectral characteristics (i.e., UV and CD spectra) of RuMb were investigated in comparison with wild type myoglobin. The effect of the metal center on the redox properties of myoglobin is directly observed by electrochemical analysis, all of which may be compared with similar measurements of the wild type myoglobin. Unlike other metal reconstituted myoglobins, i.e., cobalt myoglobin and manganese myoglobin, fast and reversible electron transfer properties were observed for RuMb, which is comparable with wild type myoglobin. The formal potential of 170 (± 10) mV vs. Ag|AgCl (sat. KCl) of RuMb was directly determined for the first time by cyclic voltammetry, where the k 0′ value was estimated to be about 3(± 0.2) × 10 − 4 cm s − 1 at pH 6.8. Mediatorless and reversible spectroelectrochemical behaviors were also observed using an optically transparent thin-layer electrode cell (OTTLE). The present results suggest that the major redox properties of the protein result from both the metal porphyrin center and globin environment. The novel redox properties predict that the engineered RuMb has analogous biofunctionalities to the wild type myoglobin in contrast to other metal reconstituted myoglobins.

Different Modes of Binding of Mono-, Di-, and Trihalogenated Phenols to the Hemoglobin Dehaloperoxidase from Amphitrite ornata

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.

Mechanisms of Heme Protein-Mediated Lipid Oxidation Using Hemoglobin and Myoglobin Variants in Raw and Heated Washed Muscle

The hemoglobin variant rHb 0.1, which possesses a decreased ability to form subunits, stimulated lipid oxidation in washed fish muscle less effectively as compared to wild-type hemoglobin (rHb 0.0). This could be due to the lower hemin affinity and more rapid autoxidation rate of subunits as compared to tetramers. To differentiate between hemin affinity and autoxidation effects, ferrous V68T Mb was compared to ferrous wild-type myoglobin (WT Mb). WT Mb has a more rapid hemin loss rate (25-fold) than does V68T, while V68T autoxidized more rapidly than did WT Mb (60-fold). Ferrous WT Mb promoted TBARS and lipid peroxide formation more rapidly than did ferrous V68T (p < 0.01). This indicated hemin loss rate was more critical in determining onset of lipid oxidation as compared to autoxidation rate. Hemin alone was capable of stimulating lipid oxidation. Albumin enhanced the ability of hemin to promote lipid oxidation. MetMb promoted lipid oxidation more effectively than did ferrous Mb, which could be due to the lower hemin affinity of metMb as compared to that of ferrous Mb. EDTA, an iron chelator, had no effect on the rate or extent of lipid oxidation mediated by Mb in the cooked system. Variants with a 975-fold range of hemin affinities promoted lipid oxidation with equivalent efficacy in cooked washed cod contrary to results in uncooked washed cod. The cooking temperatures apparently denature the globin and release hemin reactant to such an extent that the impact of hemin affinity on lipid oxidation observed in the raw state is negated in the cooked state. These studies collectively suggest released hemin is of primary importance in promoting lipid oxidation in raw and cooked washed fish muscle.

Role of heme propionates of myoglobin in vibrational energy relaxation

The role of heme propionates of myoglobin in vibrational energy relaxation was studied by time-resolved resonance Raman spectroscopy. Time-resolved anti-Stokes spectra were measured to monitor the vibrational energy relaxation of the heme. The decay rates of the band intensities were compared between wild-type myoglobin and etioheme-substituted myoglobin where the heme lacks hydrogen-bonding side chains. The decay rates of the anti-Stokes intensities of the latter were less than those of the former, providing strong support for a theoretical proposal that the propionates and their coupling to solvent bath play an important role in the dissipation of excess energy of the excited heme in solvated wild-type myoglobin.

Catalytic Reduction of NO to N2O by a Designed Heme Copper Center in Myoglobin: Implications for the Role of Metal Ions

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV-vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO.mol CuBMb-1.min-1, close to 3 mol NO.mol enzyme-1.min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV-vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV-vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron-proximal histidine toward a five-coordinate key intermediate in NO reduction.