Blue myoglobin reconstituted with an iron porphycene shows extremely high oxygen affinity

Myoglobin will be a good scaffold for engineering a function into proteins. To modulate the physiological function of myoglobin, almost all approaches have been demonstrated by site-directed mutagenesis, however, there are few studies which show a significant improvement in the function. In contrast, we focused on the replacement of heme in the protein with an artificial prosthetic group. Recently, we prepared a novel myoglobin reconstituted with an iron porphycene as a structural isomer of mesoheme. The bluish colored reconstituted myoglobin is relatively stable and the deoxymyoglobin reversibly binds ligands. Interestingly, the O2 affinity of the reconstituted myoglobin, 1.1 x 109 M-1, is a significant 1,400-fold higher than that of the native myoglobin. Furthermore, the unfavorable autoxidation kinetics show 7-fold decrease in rate for the reconstituted myoglobin relative to the native myoglobin, indicating the stable oxy-form against autoxidation. The net results come from the slow dissociation of the O2 ligand in the reconstituted myoglobin, koff = 0.11 s-1, because of the formation of strong hydrogen bond between His64 and negatively charged dioxygen. The present study indicates that the replacement of native heme with an artificially created prosthetic group will give us a unique function into a hemoprotein.     

Ligand binding properties of myoglobin reconstituted with iron porphycene: unusual O2 binding selectivity against CO binding

Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild-type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O2 affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O2 dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O2 affinity of the reconstituted myoglobin exceeds its CO affinity (M' = K(CO)/K(O2) < 1). The ligand binding studies on H64A mutants support the fact that the slow O2 dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O2 sigma-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O2 affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein.     

Unusual peroxidase activity of a myoglobin mutant with two distal histidines

By retaining the native distal His64 in sperm whale myoglobin(Mb),a second distal histidine was engineered in Mb by mutating Leu29 to His29.The resultant mutant of L29H Mb exhibits an unusual enhanced peroxidase activity with a positive cooperativity in comparison to that of wild type Mb.The new enzyme with two cooperative distal histidines has not been found in native peroxidase, which emphasizes a creation of the rational protein design.     

Preparation and O2 binding study of myoglobin having a cobalt porphycene

Sperm whale myoglobin, an oxygen-storage hemoprotein, was reconstituted with 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatocobalt(II) in order to investigate the reactivities of a cobalt porphycene in a protein matrix. Similar to the previously reported finding for the myoglobin with the iron porphycene, the reconstituted myoglobin with the cobalt porphycene was also found to have an O2 affinity 2 orders of magnitude greater than that of the myoglobin possessing cobalt protoporphyrin IX. The EPR spectra of the deoxy and oxy myoglobins having the cobalt porphycene at 77 K also have features similar to those of the myoglobin with cobalt protoporphyrin IX. These spectra suggest that the porphycene cobalt in the deoxy form is coordinated by one nitrogenous ligand postulated to be the imidazole ring of His93, and that the bond configuration of CoII-O2 is regarded as the CoIII-Omicron2*- species.     

Electrochemical and spectroelectrochemical properties of manganese reconstituted myoglobin

Electrochemical and spectroelectrochemical properties of manganese(III) reconstituted myoglobin (Mn(III)–Mb) have been investigated. No redox wave of Mn(III)–Mb was observed at a highly hydrophilic indium oxide electrode on which rapid direct electron transfer of native myoglobin took place, suggesting the electron transfer reaction of Mn(III)–Mb at an indium oxide electrode is very slow. The rate constant of the chemical reduction of Mn(III)–Mb with dithionite was ca. 20 times smaller than that of native Mb. Using an optically transparent thin layer electrode (OTTLE) cell and Oxazine-170 perchlorate, 5,9-bis(diethylamino)-10-methyl-benzo[a]phenoxazonium perchlorate, as an electron transfer mediator, the redox potential for the Mn(III) Mn(II)–Mb couple was estimated to be −0.32 V versus Ag AgCl (sat. KCl) at 25°C.     

Reductive activation of dioxygen by a myoglobin reconstituted with a flavohemin

We successfully converted myoglobin, an oxygen-storage hemoprotein, into an oxygen-activating hemoprotein like cytochrome P450s by replacing the native hemin with the artificially created flavohemin. The reconstituted myoglobin, rMb(1), was chacterized by ESI-TOF-mass, UV-vis, and fluorescence spectra. The 1H NMR spectrum of cyanomet rMb(1) indicates that two hemin conformers are present in a ratio of 1:1. Upon the addition of NADH to the buffer solution of rMb(1) in the presence of SOD and catalase, the oxymyoglobin was rapidly formed. As compared with the formation of the oxygenated native myoglobin in the presence of 10-N-(acetylaminoethyl)isoalloxazine, the rate constant of the oxyheme formation in rMb(1) is 6 times larger. This is because the flavin covalently linked to the terminal heme propionate functions as an effective mediator of an electron transfer from NADH to the hemin in rMb(1). Furthermore, rMb(1) shows the deformylation activity, when 2-phenylpropionaldehyde (2-PPA) was employed as a substrate. This result indicates that the oxyheme is reductively activated to Fe(III)-peroxoanion (Fe(III)-O22-). The result in this report is the first example of the activation of dioxygen by myoglobin. This study shows the utility of the replacement of the native hemin with a chemically modified one for the functionalization of myoglobin.     

Unusual ligand discrimination by a myoglobin reconstituted with a hydrophobic domain-linked heme

New, reconstituted horse heart myoglobins possessing a hydrophobic domain at the terminal of the two heme propionate side chains were constructed. The O2 and CO bindings for the reconstituted deoxymyoglobins were examined in detail by laser flash photolysis and stopped-flow rapid mixing techniques. The artificially created domain worked as a barrier against exogenous ligand penetration into the heme pocket, whereas the bound O2 was stabilized in the reconstituted myoglobin as well as in the native one. In contrast, the CO dissociation rate for the reconstituted myoglobin increased by 20-fold compared to the native protein, suggesting that the incorporation of the hydrophobic domain onto the heme pocket perturbs the distal-site structure of the reconstituted myoglobin. As a result, the substantial ligand selectivity for the reconstituted myoglobin significantly increases in favor of O2 over CO with the M' value (= KCO/KO2) of 0.88, whereas, to the best of our knowledge, there is no myoglobin mutant in which the O2 affinity exceeds the CO one. The present work concludes that the O2 selectivity of myoglobin over CO is markedly improved by chemically modifying the heme propionates without any mutation of the amino acid residues in the distal site.     

Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode

Monoazahemin reconstituted myoglobin was prepared and its electrochemical behavior was studied in comparison with native myoglobin. For both myoglobins well-defined voltammograms were clearly obtained at highly hydrophilic surfaces of indium oxide electrodes. Although monoazahemin showed a more positive redox potential than hemin (measured in methanol), monoazahemin reconstituted myoglobin showed a more negative redox potential than native myoglobin in a 50 mM bis-Tris buffer solution (pH 6.5), suggesting that for both native and reconstituted myoglobins the heme environment including proximal histidine as an axial ligand of the redox center plays an important role in determining the redox potential. Also, a unique electrochemical response of cyano-monoazahemin reconstituted myoglobin was demonstrated.     

Synthesis,structure, and chemical property of the first fluorine-containing porphycene

[structure: see text] A pyrrolic macrocycle, beta-tetrakis(trifluoromethyl)porphycene, is the first example of a fluorine-containing porphycene. Four electron-withdrawing CF(3) substituents provide a highly distorted structure and an attractive electron-deficient nature for the porphycene framework. From the electrochemical study, it is found that the LUMO energy level for the beta-trifluoromethylporphycene is 1.24 V more stabilized compared to that for etioporphyrin. Moreover, the deprotonation of the inner N[bond]H proton in the porphycene was observed upon the addition of DBU.     

Molecular structure,quadrupole splitting,and magnetic susceptibility of iron in deoxygenated myoglobin and hemoglobin

For three stereo-structural models of deoxymyoglobin (Mb) and deoxyhemoglobin (Hb) we derive electronic configurations and their mutual spin-orbit coupling. From the temperature dependent molecular electric field gradient (EFG) tensor we calculate temperature dependent quadrupole splittings, E _q(T), asymmetry parameters, (T), and orientations of the EFG component V _zz(T) with respect to the heme group. Comparing theoretical and experimental data we find a molecular electronic structure, which then is used to compute temperature dependent magnetic susceptibilities, (T). Theoretical and experimental (T) data are in reasonable agreement. From the consistency of our model calculations with experimental results we conclude that iron in Mb and Hb probably is pentacoordinated and considerably out of the heme plane by 0.4–0.8 Å.Supported in part by Stiftung Volkswagenwerk, by Deutsche Forschungsgemeinschaft, by the European Molecular Biology Organization, by an award from the Biomedical Sciences Support Grant at the University of Utah, and the National Science Foundation.     

Crystal structure and magnetic properties of dinuclear iron(III) complex with ONNO-donor ligand

A new dinuclear Fe(III) complex, [Fe(5-MeOL1)(OH)_0.86(CH₃O)_0.14]₂?2(CH₃OH), [H₂-5-MeOL1 = N,N′-bis(5-methoxy-2-hydroxybenzylidene)-2,2-dimethylpropane-1,3-diamine], 1 has been synthesized and characterized by single crystal structure analysis. The structure of 1 consists of two Fe(III) centers with one tetradentate schiff base ligand (N₂O₂) which are bridged by dihydroxo/dimethoxo groups to yield a Fe₂O₂ core. Complex 1 exhibits weak antiferromagnetic exchange interaction between Fe(III) ions with J = ?0.21 cm^–1.     

邻菲咯啉镉配合物的合成、晶体结构及抑菌活性

合成了以邻菲咯啉为配体的金属镉的配合物,利用红外光谱、元素分析、单晶衍射和热重分析等手段对其结构及性质进行了表征和分析,并研究了金属配合物对金黄色葡萄球菌、大肠杆菌、酵母菌的抑菌活性.结果表明,此类配合物对三类菌种均有不同程度的抑制作用.     

Semisynthetic tellurosubtilisin with glutathione peroxidase activity

Reaction of the hydroxyl group of serine-221 of subtilisin with phenylmethanesulfonylfluoride followed by nucleophilic substitution with sodium hydrogen telluride, a semisynthetic telluroprotein, tellurosubtilisin, was prepared. Tellurosubtilisin, which displays high substrate specificity for aromatic thiols, exhibits remarkable peroxidase activity and catalyzes the reduction of hydrogen peroxide by 3-carboxy-4-nitrobenzenethiol 20 000 times more efficiently than diphenyl diselenide.     

Crystal structure and theoretical calculations of Julocrotine,a natural product with antileishmanial activity

Julocrotine, N‐(2,6‐dioxo‐1‐phenethyl‐piperidin‐3‐yl)‐2‐methyl‐butyramide, is a potent antiproliferative agent against the promastigote and amastigote forms of Leishmania amazonensis (L.). In this work, the crystal structure of Julocrotine was solved by X‐ray diffraction, and its geometrical parameters were compared with theoretical calculations at the B3LYP and HF level of theory. IR and NMR spectra also have been obtained and compared with theoretical calculations. IR absorptions calculated with the B3LYP level of theory employed together with the 6‐311G+(d,p) basis set, are close to those observed experimentally. Theoretical NMR calculations show little deviation from experimental results. The results show that the theory is in accordance with the experimental data. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Molecular structures of five-coordinated halide ligated iron(III) porphyrin,porphycene, and corrphycene complexes

Molecular structures of 12 porphyrin analogues, Fe(III)(EtioP)X(1(a)-1(d)), Fe(III)(EtioCn)X(2(a)-2(d)), and Fe(III)(Etio-Pc)X(3(a)-3(d)), where X = F (a), Cl (b), Br (c), and I (d), are determined on the basis of X-ray crystallography. Combined analyses using M?ssbauer, (1)H NMR, and EPR spectroscopy as well as SQUID magnetometry have revealed that 3(d) exhibits a quite pure S = 3/2 spin state with a small amount of an S = 5/2 spin admixture. In contrast, all the other complexes show the S = 5/2 spin state with a small amount of the S = 3/2 spin admixture. The structural and spectroscopic data indicate a strong correlation between the spin states of the complexes and the core geometries such as Fe-N bond lengths, cavity areas, and DeltaFe values.     

Cobalt complexes of terpyridine ligands: Crystal structure and nuclease activity

Cobalt(II) (1) and cobalt(III) (2) complexes of tridentate ligand, imidazole terpyridine (Itpy), have been synthesized and characterized by both spectroscopic and electrochemical techniques. Single crystal X-ray diffraction studies of complexes 1 and 2 shows that the complexes belong to monoclinic crystal system, with the two Itpy ligands coordinated to the central metal ion. The binding behavior of both the cobalt complexes to calf thymus DNA has been investigated by UV–Vis, fluorescence spectroscopy, viscosity and electrochemical measurements. The results suggest that complexes 1 and 2 bind to DNA through intercalation. The intrinsic DNA binding constant values obtained from absorption spectral titration studies were found to be (5.07 ± 0.12) × 10³ M⁻¹ and (7.46 ± 0.16) × 10³ M⁻¹, respectively, for complexes 1 and 2. Gel electrophoresis studies with the cobalt complexes show that while complex 1 cleaves DNA in the presence of hydrogen peroxide, complex 2 cleaves DNA in the presence of ascorbic acid and hydrogen peroxide.     

N,N′-亚水杨基皮考林酰肼合铁(Ⅱ)的晶体结构和荧光性质

合成了N,N′-亚水杨基皮考林酰肼(HL)及其铁配合物[FeL2](C26H20FeN6O4,Mr=536 33).X射线衍射实验结果表明,标题配合物晶体属于正交晶系,空间群为Pbcn,晶体学参数:a=1 4970(1)nm,b=1 51556(9)nm,c=2.0920(2)nm,V=4 7462(6)nm3,Z=8,Dc=1 501Mg·m-3,F(000)=2208,μ(MoKα)=0 682mm-1,R=0 0695,Rw=0 1502.在配合物[FeL2]中,铁(Ⅱ)原子具有扭曲的八面体配位构型,晶体通过分子间氢键作用形成缔合分子对.红外光谱表明,配体在形成配合物后,ν(CO)和ν(CN)红移.电子光谱表明存在π-π 和d-π 的跃迁;荧光光谱表明,配合物金属对配体n-π 激发引起的荧光发射峰有较大的影响.