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PubMed1992

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.

Animals↗
PubMed1975

Electrostatic effects in myoglobin. Application of the modified Tanford-Kirkwood theory to myoglobins from horse, California grey whale, harbor seal, and California sea lion.

The modified Tanford-Kirkwood electrostatic theory (Shire et al., 1974a) was applied to ferrimyoglobins from the following animal species: sperm whale (Physeter catodon), horse, California grey whale (Eschrichtius gibbosus), harbor seal (Phoca vitulina), and California sea lion (Zalophus californianus). Computations were made of the overall hydrogen ion titration curves of the proteins, and of pH and ionic strength variations of ionization equilibria for individual groups in the protein, with particular reference to the hemic acid ionization of the iron bound water molecule. Coordinates and static solvent accessibility were estimated in terms of the sperm whale myoglobin structure. Where possible, theoretical results and experimental data are compared. Some comparative features of charge and ionization properties among the various myoglobins are presented.

Amino Acids↗
PubMed2003

Pseudoperoxidase activity of myoglobin: kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates.

Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) approximately 5.2 at 25.0 degrees C and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M(-1) s(-1), DeltaH# = 66 +/- 4 kJ mol(-1), and DeltaS# = 30 +/- 15 J mol(-1) K(-1) at 25.0 degrees C and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M(-1) s(-1), DeltaH# = 31 +/- 6 kJ mol(-1), and DeltaS# = -53 +/- 19 J mol(-1) K(-1), indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degrees C to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M(-1) s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M(-1) s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.

Benzothiazoles↗
PubMed1995

Functional aspects of ultra-rapid heme doming in hemoglobin, myoglobin, and the myoglobin mutant H93G.

Heme iron out-of-plane displacement following ligand dissociation in hemoglobin, myoglobin, and the proximal cavity mutant H93G is shown to be as rapid as the heme iron out-of-plane vibrational period by sub-picosecond time-resolved resonance Raman spectroscopy. The results demonstrate that the effect of steric repulsion initiated by the spin change of the iron gives rise to heme doming independent of covalent attachment of the proximal ligand to the protein. It is concluded that the protein plays a passive role in the initial ultrafast heme iron motion toward the out-of-plane position observed in the deoxy structure of hemoglobin and myoglobin. The results suggest that the spin change of the heme iron is the primary cause of rapid heme doming and that steric repulsion of the proximal ligand with the heme plays a secondary role in forcing the iron out of the heme plane.

Animals↗
PubMed2005

Monooxygenation of an aromatic ring by F43W/H64D/V68I myoglobin mutant and hydrogen peroxide. Myoglobin mutants as a model for P450 hydroxylation chemistry.

Myoglobin (Mb) is used as a model system for other heme proteins and the reactions they catalyze. The latest novel function to be proposed for myoglobin is a P450 type hydroxylation activity of aromatic carbons (Watanabe, Y., and Ueno, T. (2003) Bull. Chem. Soc. Jpn. 76, 1309-1322). Because Mb does not contain a specific substrate binding site for aromatic compounds near the heme, an engineered tryptophan in the heme pocket was used to model P450 hydroxylation of aromatic compounds. The monooxygenation product was not previously isolated because of rapid subsequent oxidation steps (Hara, I., Ueno, T., Ozaki, S., Itoh, S., Lee, K., Ueyama, N., and Watanabe, Y. (2001) J. Biol. Chem. 276, 36067-36070). In this work, a Mb variant (F43W/H64D/V68I) is used to characterize the monooxygenated intermediate. A modified (+16 Da) species forms upon the addition of 1 eq of H2O2. This product was digested with chymotrypsin, and the modified peptide fragments were isolated and characterized as 6-hydroxytryptophan using matrix-assisted laser desorption ionization time-of-flight tandem mass spectroscopy and 1H NMR. This engineered Mb variant represents the first enzyme to preferentially hydroxylate the indole side chain of Trp at the C6 position. Finally, heme extraction was used to demonstrate that both the formation of the 6-hydroxytryptophan intermediate (+16 Da) and subsequent oxidation to form the +30 Da final product are catalyzed by the heme cofactor, most probably via the compound I intermediate. These results provide insight into the mechanism of hydroxylation of aromatic carbons by heme proteins, demonstrating that non-thiolate-ligated heme enzymes can perform this function. This establishes Mb compound I as a model for P450 type aromatic hydroxylation chemistry.

5-Hydroxytryptophan↗
PubMed1991

Fluorescence study of the conformational properties of myoglobin structure. 3. pH-dependent changes in porphyrin and tryptophan fluorescence of the complex of sperm whale apomyoglobin with protoporphyrin IX; the role of the porphyrin macrocycle and iron in formation of native myoglobin structure.

The porphyrin and tryptophan fluorescence of sperm whale apomyoglobin complexed with protoporphyrin IX has been studied in the pH range 2-13. It has been shown that the fluorescence and absorption spectra of protoporphyrin incorporated into the heme crevice remain constant in the pH range 5.5-10.8 but change significantly at pH less than 5.5 and pH greater than 10.8, due to the acid and alkaline denaturation, respectively, of the complex accompanied by dissociation of protoporphyrin IX. At the same pH ranges, the quantum yield of tryptophanyl fluorescence increases sharply as a result of removal of protoporphyrin, acting as a quencher, from the complex. Other parameters of tryptophanyl fluorescence (maximum position, halfwidth and spectrum shape) change in the alkaline region as well. In the acidic pH range, these parameters change only at pH less than 4.3, indicating that the Trp surroundings are more stable to denaturation than the heme crevice region. Between pH 5.5 and 10.9, where the complex of apomyoglobin with protoporphyrin IX is in its native state, the main parameters of tryptophan fluorescence remain unchanged except for the ratio I325/I350 which diminishes at pH greater than 9.5. Its alteration precedes the alkaline denaturation of the complex and can be explained by a local conformational change induced by the break of the 'salt bridges' essential for the maintenance of the native Mb structure in the N-terminal region. The fluorescence data obtained for apomyoglobin, myoglobin and the complex between protoporphyrin IX and apomyoglobin enable one to compare their structures and to evaluate the role of the porphyrin macrocycle and the iron atom in the formation of the native myoglobin structure and its functioning.

Animals↗
PubMed1996

Localization and amount of myoglobin and myoglobin mRNA in ragged-red fiber of patients with mitochondrial encephalomyopathy.

The localization and amounts of myoglobin (Mb) and Mb mRNA in ragged-red fibers (RRF) in skeletal muscle of 6 patients with mitochondrial encephalomyopathy were examined immunohistochemically and by in situ hybridization. The amounts of Mb and Mb mRNA were expressed in terms of optical densities (ODs) of Mb immunostaining and Mb mRNA signals. In nonatrophic RRF, Mb was predominantly seen in the ragged-red region and Mb mRNA signals were increased throughout the sarcoplasm. The amounts of Mb and Mb mRNA in nonatrophic RRF were greater than those in nonatrophic non-RRF. In contrast, the localization and amount in atrophic RRF were similar to those in atrophic non-RRF. Thus, Mb synthesis in nonatrophic RRF may increase to compensate for mitochondrial dysfunction and to supply sufficient oxygen to mitochondria, but this compensatory function may be impaired in atrophic RRF.

Adult↗
PubMed1996

Protein radicals in myoglobin dimerization and myoglobin-catalyzed styrene epoxidation.

Two mechanisms have been identified for the H2O2-dependent epoxidation of styrenes by sperm whale myoglobin (Mb) [S. Rao, A. Wilks, and P. R. Ortiz de Montellano, J. Biol. Chem. 268, 803-908 (1993)]: (a) ferryl (FeIV = O) oxygen transfer with retention of stereochemistry and incorporation of an oxygen from H2O2, and (b) protein peroxy radical cooxidation with loss of stereochemistry and incorporation of an oxygen from O2. As shown here, cis-beta-methylstyrene is preferentially oxidized to the trans-epoxide when the H2O2:Mb ratio is <0.5 but increasingly to the cis-isomer as the ratio increases to and above 1. At a high (4:1) H2O2:Mb ratio, both the absolute yield and the cis:trans-epoxide ratio increase in proportion to the cis-beta-methylstyrene concentration. A protein radical formed in the Mb-H2O2 reaction also causes dimer and trimer formation, maximum dimer formation (approximately 30%) being obtained with 1 equivalent of H2O2. At low H2O2:Mb ratios, the oxidation equivalents utilized for protein oligomerization and styrene oxidation account for the available H2O2. Previous studies have shown that His-64 is important for protein-mediated olefin cooxidation and Tyr-151/Tyr-103 for Mb dimerization. The W7F, W14F, and W7F/W14F Mb mutants have now been prepared and the W14F, but not W7F, mutation shown to modestly decrease cooxidation of cis-beta-methylstyrene to the trans-epoxide. Neither tryptophan mutation alters dimer formation. Dimer formation is modestly increased rather than decreased by styrene, suggesting that styrene cooxidation and dimerization do not compete. The results indicate that (a) cis-beta-methylstyrene cooxidation and protein dimerization, both of which are mediated by protein radicals, are favored at low H2O2:Mb ratios, (b) as the H2O2:Mb ratio increases, the ferryl epoxidation pathway surpasses the cooxidation mechanism, (c) Trp-14 but not Trp-7 influences olefin cooxidation, and (d) different, possibly nonequilibrating, radicals mediate olefin cooxidation and protein dimerization.

Animals↗
PubMed2001

Reaction of human myoglobin and peroxynitrite: characterizing biomarkers for myoglobin-derived oxidative stress.

Mixtures of human myoglobin (Mb) (or the Y103F variant of human Mb), authentic peroxynitrite (ONOO(-), ONOO(-):protein 2 mol/mol), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave radicals adducts at cysteine-110 (DMPO-C110) that are detected directly by electron paramagnetic magnetic spectroscopy (EPR). DMPO-C110 was detected exclusively over a range of DMPO concentrations (DMPO:protein ratios 25-100 mol/mol). Treatment of human Mb (or Y103F Mb) with the ONOO(-) generator 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium (SIN-1) chloride (ONOO(-):protein 5 mol/mol) yielded a cross-linked Mb dimer as judged by SDS-PAGE analyses. Addition of DMPO or carbonate effectively eliminated the cross-linked product. Mass analyses of samples containing human Mb (or Y103F Mb), carbonate, and ONOO(-) indicated that nitration occurs exclusively at Y103. Thus, reaction of human Mb and ONOO(-) yields specific products that depend on the presence or absence of physiological concentrations of carbonate. These products may serve as biomarkers for the participation of Mb-derived radicals in the oxidative damage associated with myocardial reperfusion injury.

Biomarkers↗
PubMed1998

A novel vertebrate myoglobin gene containing three A+T-rich introns is conserved among Antarctic teleost species which differ in myoglobin expression.

This report presents the first teleost myoglobin (Mb) genomic DNA sequence, several features of which are distinct from mammalian Mb genes. We have isolated and compared genomic clones of three closely related Antarctic icefish: Chionodraco rastrospinosus, which expresses Mb mRNA and functional polypeptide; Champsocephalus gunnari, which transcribes the Mb gene but does not produce polypeptide due to a 5-base pair (bp) frameshift insertion; and Chaenocephalus aceratus, which lacks both Mb protein and mRNA. The single-copy icefish Mb gene contains three introns: two at positions identical to those found in mammalian Mb and a novel intron located in the 5' untranslated region three nucleotides upstream from the initiator codon. All three introns are shorter than those found in mammalian Mb genes and exhibit a considerably higher A+T content. The entire Mb transcriptional unit is intact in C. aceratus, indicating that the failure to express this gene is not due to aberrations in the coding region, splice junctions, polyadenylation signals, or core promoter elements. The three icefish Mb sequences display an extreme degree of identity in the transcriptional unit and putative promoter region. In contrast, sequences 65 bp downstream from the polyadenylation site bear no homology among the three species, demonstrating that rapid sequence change has occurred in the 1 million years since the divergence of these species.

Amino Acid Sequence↗
PubMed1983

Variation of serum myoglobin levels in normal individuals. With reference to the use of myoglobin measurements for the detection of women carrying genes for the X-linked muscular dystrophies.

The extent of variation in the serum myoglobin (Mb) levels of normal healthy females were investigated so that the value of Mb measurements for carrier detection could be properly assessed. Factors considered were sex, age, menstrual status, pregnancy, diurnal variation and daily activity. In each instance, Mb levels were compared with those for serum creatine kinase (CK). A significant difference (P less than 0.001) was found between the Mb levels of men and women but no relationship could be found between several measurements of body build and the Mb or CK levels of either sex. An apparent correlation of Mb concentrations with age was found in control groups of both sexes up to 85 years of age, but this was not significant below 60 years of age. No differences in Mb levels were found among premenarchal, menstruating, postmenopausal or hysterectomised women and neither smoking nor the use of oral contraceptives seemed to affect Mb levels. Serum CK levels were very much higher in premenarchal (P less than 0.001) and postmenarchal (P less than 0.05) teenagers than in adult controls. No difference was found between the mean Mb levels of non-pregnant and pregnant (6-40 weeks) women. The mean value for CK activity was significantly lower than normal in the pregnant women (P less than 0.001). A marked circadian rhythm of serum MB levels was found with samples taken at 5 p.m. being approximately 20% lower than those taken at 9 a.m. This unexpected decrease during the day persisted with 48 hours of bedrest suggesting that this rhythm is inherent and unrelated to muscular activity.

Adolescent↗
PubMed1992

Effect of NaCl addition on nanosecond O2 escaping reaction of myoglobin: evidences for the transition of myoglobin dynamic structure at 20 degrees C.

We studied the nanosecond (ns) geminate O2 escape reaction from the protein interior of myoglobin (Mb) to the solvent phase in the temperature range of 5-40 degrees C containing 0-0.1 M NaCl. In the flash photolysis experiments, we found that both the rate constant, kout, and its Arrhenius plot changed upon the variation of the NaCl concentration. In particular, it was noteworthy that the Arrhenius plot of kout dramatically changed in its slope, keeping the break at 20 degrees C, upon the addition of NaCl, indicating that the thermodynamic parameters such as an enthalpy of activation (delta H not equal to) and an entropy of activation (delta S not equal to) are different between above and below 20 degrees C, and that they are further altered upon the NaCl addition to the sample solution. From these results, we suggested that the Mb dynamic structure in the ns geminate O2 escape reaction is sensitively regulated by the interaction of the protein surface and the salt. The present study also showed that an inconsistency of the Arrhenius plot of kout between Chatfield et al. ((1990) J. Am. Chem. Soc. 112, 4680-4687) and us ((1990) J. Biol. Chem. 265, 18823-18828) is probably due to the difference in the solution condition.

Myoglobin↗
PubMed1994

Spectroscopic study of Ser92 mutants of human myoglobin: hydrogen bonding effect of Ser92 to proximal His93 on structure and property of myoglobin.

Neutron diffraction studies have demonstrated that the hydroxyl group oxygen of Ser92(F7) is hydrogen bonded to the proximal His93(48) N epsilon H proton in myoglobin (Mb) [Cheng, X., & Shoenborn, B. P. (1991) J. Mol. Biol. 220, 381-399]. In order to examine the importance of this hydrogen bond, Ser92 was replaced with Ala and Asp in human Mb. By comparing the optical, 1H-NMR, resonance Raman, and IR spectra of Mb(S92A) in several spin and oxidation states with those of wild-type Mb, it was found that the mutation causes a structural change on the heme proximal side but not on the distal side. Comparison of the NMR spectra of the cyanomet form of Mb(S92A) and Mb(WT) suggests that the imidazole plane of His93 rotates somewhat around the Fe-N delta (His93) bond upon loss of the hydrogen bond between His93 and Ser92. The 2D 1H-NMR measurements of the CO complexes show that mutation of Ser92 to Ala changes the relative position of the His97 imidazole group to the heme plane, but the change is not so drastic as reported in the crystal data of Ser92 mutant of pig Mb [Smerdon et al. (1993) Biochemistry 32, 5132-5138]. On the other hand, ligand (CO, O2) binding is only slightly affected by this mutation. From these results, we conclude that the Ser92-His93 hydrogen bond maintains the protein structure of the proximal heme pocket, but it does not strongly affect the electronic structure of the heme as well as of the His93 imidazole ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗
PubMed1984

Modification of the heme distal side in myoglobin by cyanogen bromide. Heme environmental structures and ligand binding properties of the modified myoglobin.

Met, deoxy, and CO forms of myoglobin (Mb) react with a stoichiometric amount of cyanogen bromide (BrCN) to cause substantial changes in the 1H NMR, optical absorption, and infrared spectra. These spectral changes were interpreted as arising from the substantial alterations in the heme environments, most probably due to the modification of the histidine residue at the heme distal side. It is also revealed that the modified Mb does not combine with some exogenous ligands such as CN-, CH3NH2, and O2, although it does with N-3 or CO. These unique ligand binding properties are also discussed with relevance to a role of the distal histidine in stabilizing the coordinated ligand through a hydrogen bond and to a steric constraint.

Animals↗
PubMed2002

Role of tyrosine-103 in myoglobin peroxidase activity: kinetic and steady-state studies on the reaction of wild-type and variant recombinant human myoglobins with H(2)O(2).

Myoglobin (Mb) catalyzes a range of oxidation reactions in the presence of hydrogen peroxide (H(2)O(2)) through a peroxidase-like cycle. C110A and Y103F variants of human Mb have been constructed to assess the effects of removing electron-rich oxidizable amino acids from the protein on the peroxidase activity of Mb: a point mutation at W14 failed to yield a viable protein. Point mutations at C110 and Y103 did not result in significant changes to structural elements of the heme pocket, as judged by low-temperature electron paramagnetic spectroscopy (EPR) studies on the ground-state ferric proteins. However, compared to the native protein, the yield of globin radical (globin*) was significantly decreased for the Y103F but not the C110A variant Mb upon reaction of the respective proteins with H(2)O(2). In contrast with our expectation that inhibiting pathways of intramolecular electron transfer may lead to enhanced Mb peroxidase activity, mutation of Y103 marginally decreased the rate constant for reaction of Mb with H(2)O(2) (1.4-fold) as judged by stopped-flow kinetic analyses. Consistent with this decrease in rate constant, steady-state analyses of Y103F Mb-derived thioanisole sulfoxidation indicated decreased V(max) and increased K(m) relative to the wild-type control. Additionally, thioanisole sulfoxidation proceeded with lower stereoselectivity, suggesting that Y103 plays a significant role in substrate binding and orientation in the heme pocket of Mb. Together, these results show that electron transfer within the globin portion of the protein is an important modulator of its stability and catalytic activity. Furthermore, the hydrogen-bonding network involving the residues that line the heme pocket of Mb is crucial to both efficient peroxidase activity and stereospecificity.

Amino Acid Sequence↗
PubMed2003

Molecular engineering of myoglobin: influence of residue 68 on the rate and the enantioselectivity of oxidation reactions catalyzed by H64D/V68X myoglobin.

In the elucidation of structural requirements of heme vicinity for hydrogen peroxide activation, we found that the replacement of His-64 of myoglobin (Mb) with a negatively charged aspartate residue enhanced peroxidase and peroxygenase activities by 78- and 580-fold, respectively. Since residue 68 is known to influence the ligation of small molecules to the heme iron, we constructed H64D/V68X Mb bearing Ala, Ser, Leu, Ile, and Phe at position 68 to improve the oxidation activity. The Val-68 to Leu mutation of H64D Mb accelerates the reaction with H(2)O(2) to form a catalytic species, called compound I, and improves the one-electron oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (i.e., peroxidase activity) approximately 2-fold. On the other hand, H64D/V68I Mb oxygenates thioanisole 2.7- and 1600-fold faster than H64D and wild-type Mb, respectively. In terms of the enantioselectivity, H64D/V68A and H64D/V68S Mb were good chiral catalysts for thioanisole oxidation and produced the (R)-sulfoxide dominantly with 84% and 88% ee, respectively [Kato, S., et al. (2002) J. Am. Chem. Soc. 124, 8506-8507]. On the contrary, the substitution of Val-68 in H64D Mb with an isoleucine residue alters the dominant sulfoxide product from the (R)- to the (S)-isomer. The crystal structures of H64D/V68A and H64D/V68S Mb elucidated in this study do not clearly indicate residues interacting with thioanisole. However, comparison of the active site structures provides the basis to interpret the changes in oxidation activity: (1) direct steric interactions between residue 68 and substrates (i.e., H(2)O(2), ABTS, thioanisole) and (2) the polar interactions between tightly hydrogen-bonded water molecules and substrates.

Amino Acid Substitution↗
PubMed2003

Structural dynamics of myoglobin: spectroscopic and structural characterization of ligand docking sites in myoglobin mutant L29W.

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.

Amino Acid Substitution↗