Manganese(II) zero-field interaction in cambialistic and manganese superoxide dismutases and its relationship to the structure of the metal binding site

The Mn(II) high-magnetic-field electron paramagnetic resonance (HFEPR) spectra of five different superoxide dismutases (SODs) were measured at 190 and 285 GHz. The native E. coli manganese SOD was found to be distinct from the other SODs by virtue of its large zero-field E-value. The two wild-type cambialistic proteins from Porphyromonas gingivalis and Rhodobacter capsulatus were also distinct. However, the Gly155Thr mutant of the P. gingivalis SOD changed the Mn(II) spectrum so that it closely resembled the spectrum of manganese reconstituted E. coli iron SOD. This observation paralleled enzyme activity measurements that show that this mutation causes the loss of activity with manganese and enhanced activity with iron indicating a conversion from a cambialistic to an iron-specific protein. The Mn(II) magnetic parameters were determined by simultaneously fitting the multifrequency data. Simulations were carried out by numerically diagonalizing the spin Hamiltonian and explicitly calculating all possible transition probabilities. The relationship between the Mn(II) zero-field interaction and structure of the metal binding site is also discussed.     

Comparative Analysis and Modeling of Superoxide Dismutases (SODs) in Brachypodium distachyon L.

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme catalyzing the dismutation of superoxide radical to hydrogen peroxide and dioxygen. To date, four types of SODs — Cu/ZnSOD, MnSOD, FeSOD, and NiSOD — have been identified. In this study, SOD proteins of Brachypodium distachyon (L.) Beauv. were screened by utilization of bioinformatics approaches. According to our results, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found to be in basic and acidic character, respectively. Domain analyzes of SOD proteins revealed that iron/manganese SOD and copper/zinc SOD were within studied SOD proteins. Based on the seconder structure analyzes, Mn/FeSODs and Cu/ZnSODs of B. distachyon were found as having similar sheets, turns and coils. Although helical structures were noticed in the types of Mn/FeSODs, no the type of Cu/ZnSODs were identified having helical structures. The predicted binding sites of Fe/MnSODs and Cu/ZnSODs were confirmed for having His-His-Asp-His and His-His-His-Asp-Ser residues with different positions, respectively. The 3D structure analyzes of SODs revealed that some structural divergences were observed in patterns of SODs domains. Based on phylogenetic analysis, Mn/FeSODs were found to have similarities whereas Cu/ZnSODs were clustered independently in phylogenetic tree.     

Superoxide Dismutase Administration: A Review of Proposed Human Uses

Superoxide dismutases (SODs) are metalloenzymes that play a major role in antioxidant defense against oxidative stress in the body. SOD supplementation may therefore trigger the endogenous antioxidant machinery for the neutralization of free-radical excess and be used in a variety of pathological settings. This paper aimed to provide an extensive review of the possible uses of SODs in a range of pathological settings, as well as describe the current pitfalls and the delivery strategies that are in development to solve bioavailability issues. We carried out a PubMed query, using the keywords “SOD”, “SOD mimetics”, “SOD supplementation”, which included papers published in the English language, between 2012 and 2020, on the potential therapeutic applications of SODs, including detoxification strategies. As highlighted in this paper, it can be argued that the generic antioxidant effects of SODs are beneficial under all tested conditions, from ocular and cardiovascular diseases to neurodegenerative disorders and metabolic diseases, including diabetes and its complications and obesity. However, it must be underlined that clinical evidence for its efficacy is limited and consequently, this efficacy is currently far from being demonstrated.     

Superoxide dismutase activity of iron(II)TPEN complex and its derivatives

Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis. Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen. Thus, SODs and their functional mimics have potential value as pharmaceuticals. We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J. Biol. Chem., 264, 9243-9249 (1989)). Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat. In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes. Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics. In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J. Organometal. Chem., in press). Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity. Therefore our findings should be significant for the development of clinically useful SOD mimics.     

Rational De Novo Design of a Cu Metalloenzyme for Superoxide Dismutation

Superoxide dismutases (SODs) are highly efficient enzymes for superoxide dismutation and the first line of defense against oxidative stress. These metalloproteins contain a redox-active metal ion in their active site (Mn, Cu, Fe, Ni) with a tightly controlled reduction potential found in a close range around the optimal value of 0.36 V versus the normal hydrogen electrode (NHE). Rationally designed proteins with well-defined three-dimensional structures offer new opportunities for obtaining functional SOD mimics. Here, we explore four different copper-binding scaffolds: H₃ (His₃), H₄ (His₄), H₂DH (His₃Asp with two His and one Asp in the same plane) and H₃D (His₃Asp with three His in the same plane) by using the scaffold of the de novo protein GRα₃D. EPR and XAS analysis of the resulting copper complexes demonstrates that they are good Cu^II-bound structural mimics of Cu-only SODs. Furthermore, all the complexes exhibit SOD activity, though three orders of magnitude slower than the native enzyme, making them the first de novo copper SOD mimics.     

EPR and ligand field studies of iron superoxide dismutases and iron-substituted manganese superoxide dismutases: relationships between electronic structure of the active site and activity

The problem of metal selectivity of iron/manganese superoxide dismutases (SODs) is addressed through the electronic structures of active sites using electron paramagnetic resonance and ligand field calculations. Studies of wild-type iron(III) SOD (FeSOD) from Escherichia coli and from Methanobacterium thermoautotrophicum and iron-substituted manganese(III) SOD (Fe(sub)MnSOD) from E. coli and from Serratia marcescens are reported. EPR spectroscopy of wild-type enzymes shows transitions within all three Kramers doublets identified by their g values. From the temperature dependence of the observed transitions, the zero-field splitting is found to be negative, D = -2 +/- 0.2 cm-1. The electronic structure is typical of a distorted trigonal bipyramid, all the EPR features being reproduced by ligand field analysis. This unique and necessary electronic structure characterizes wild-type enzymes whatever their classification from the amino acid sequence into iron or manganese types, as E. coli FeSOD or M. thermoautotrophicum FeSOD. In iron-substituted manganese SODs, reduced catalytic activity is found. We describe how inhomogeneity of all reported substituted MnSODs might explain the activity decrease. EPR spectra of substituted enzymes show several overlapping components. From simulation of these spectra, one component is identified which shares the same electronic structure of the wild-type FeSODs, with the proportion depending on pH. Ligand field calculations were performed to investigate distortions of the active site geometry which induce variation of the excitation energy of the lowest quartet state. The corresponding coupling between the ground state and the excited state is found to be maximum in the geometry of the native SODs. We conjecture that such coupling should be considered in the electron-transfer process and in the contribution of the typical electronic structure of FeSOD to the activity.     

Resolution of three forms of superoxide dismutase by immobilised metal affinity chromatography.

A new method for separation of three forms of superoxide dismutase (SOD) using immobilised metal affinity chromatography (IMAC) is reported. Fe-, Mn- and Cu/Zn-SODs were eluted sequentially from Cu(2+)-IMAC column with an increasing gradient of a counter ion (NH+4) run in combination with an increasing pH gradient (6.8-7.8). The combined gradient elution method resulted in separation of SODs with high resolution, the three proteins being eluted in electrophoretically homogeneous forms. Similar preparation could not be achieved by either increasing gradient of a counter ion or decreasing pH gradients used separately. The described methodology has been successfully applied for separation of three SODs from a protozoan parasite, indicating that this combined gradient elution system for IMAC offers new possibilities for the high-resolution separation of proteins exhibiting only minor differences in their amino acid composition and structure.     

无花果叶超氧化物歧化酶的分离、纯化及性质研究

以我国山东无花果树(Brunswike, 原产法国Ficus carica)树叶为原料, 采用缓冲液抽提、硫酸铵分级、DEAE-Sepharose Fast Flow阴离子交换层析和Sephacryl S-100 HR分子筛层析分离纯化得到电泳纯CuZn-SOD, 并对其酶学性质进行研究.     

Purification and Properties of Superoxide Dismutase（SOD） in Allium Sativum

Superoxide dismutases(SODs) were purified to homogeneity from Allium Sativum by means of ammoni-um sulfate precipitation and column chromatography with DEAE--cellulose (DE52) and Sephadex G-75. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-AGE), Allium Sativum is predicted to contain four SODs. The molecular weights of the native SODs are 41.3 kD, 37. 0 kD, 35.2 kD and 31.0 kD, which consist of subunits of 20. 7 kD, 18. 4 kD, 17. 7 kD and 15.4 kD respectively. Because of their specific sensitivity to hydrogen peroxide, cyanogens potassium and chloroform-alcohol, the SODs in Allium Sativum appear to be Cu, Zn-SOD isoenzymes. The isoelectric analysis indicates that three of the four isoermymes are acidic proteins with isoelectric points at pH 3.5, 3.7 and 4. 0, respectively, and the fourth one is a basic pro-tein with isoeletric point at pH 8. 5.     

SOD模拟及其抗氧化和抗炎症功能的研究进展*

超氧化物歧化酶(superoxide dismutase, 简称SOD) 作为生物体内超氧离子自由基的清除剂, 具备有效抗氧化、抗炎症、抗衰老之功效, 并用于临床, 其化学模拟引起了人们的广泛研究兴趣。本文将简要介绍近年来SOD 人工模拟酶及其生物医学活性研究所取得的重要进展, 特别是Cu,Zn-SOD 模拟物结构与功能的相互关系、Mn-SOD 模拟物的生物活性及其医学应用。     

Spectroscopic and computational investigation of second-sphere contributions to redox tuning in Escherichia coli iron superoxide dismutase

In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461-467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe(3+/2+)reduction potentials that vary by approximately 1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe(3+/2+) reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.     

Stereochemistry of silicon tetrafluoride complexes with N- and O-donor ligands

Known experimental and theoretical data used in the analysis of stereoisomeric structures of penta-and hexacoordinate silicon tetrafluoride complexes with the molecular N-and O-donor ligands are discussed.     

Tuning the redox properties of manganese(II) and its implications to the electrochemistry of manganese and iron superoxide dismutases

Superoxide dismutases (SODs) catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. The active metal sites of iron and manganese superoxide dismutases are structurally indistinguishable from each other. Despite the structural homology, these enzymes exhibit a high degree of metal selective activity suggesting subtle redox tuning of the active site. The redox tuning model, however, up to now has been challenged by the existence of so-called cambialistic SODs that function with either metal ion. We have prepared and investigated two sets of manganese complexes in which groups of varying electron-withdrawing character, as measured by their Hammett constants sigma Para, have been introduced into the ligands. We observed that the Mn(III)/Mn(II) reduction potential for the series based on 4'-X-terpyridine ligands together with the corresponding values for the iron-substituted 4'-X-terpyridine complexes changed linearly with sigma Para. The redox potential of the iron and manganese complexes could be varied by as much as 600 mV by the 4'-substitution with the manganese complexes being slightly more sensitive to the substitution than iron. The difference was such that in the case where the 4'-substituent was a pyrrolidine group both the manganese and the iron complex were thermodynamically competent to catalytically disproportionate superoxide, making this particular ligand "cambialistic". Taking our data and those available from the literature together, it was found that in addition to the electron-withdrawing capacity of the 4'-substituents the overall charge of the Mn(II) complexes plays a major role in tuning the redox potential, about 600 mV per charge unit. The ion selectivity in Mn and FeSODs and the occurrence of cambialistic SODs are discussed in view of these results. We conclude that the more distant electrostatic contributions may be the source of metal specific enzymatic activity.     

Synthesis and Properties of 2,2-Dichlorovinyl Trifluoromethyl Ketone

Highly reactive 2,2-dichlorovinyl trifluoromethyl ketone was synthesized. Its reactions with N,N-and N,S-nucleophiles gave 1,3-thiazine, pyrazole, and imidazole derivatives containing a trifluoromethyl substituent.     

Effects of a Single Exposure to UVB Radiation on the Activities and Protein Levels of Copper-Zinc and Manganese Superoxide Dismutase in Cultured Human Keratinocytes

Abstract— Ultraviolet B irradiation has been believed to decrease or impair the activity of reactive oxygen species (ROS) scavenging enzymes such as superoxide dismutase (SOD) in the skin. It has been recently reported that two isozymes of SOD, namely copper-zinc SOD (Cu-Zn SOD) and manganese SOD (Mn SOD), exist in mammalian cells and that the two enzymes play different roles in living systems. The aim of this study was to investigate changes in SOD activities and protein levels in cultured human keratinocytes after acute UVB irradiation. In addition, the protein levels of Cu-Zn SOD and Mn SOD were quantified separately. A single exposure to UVB irradiation produced an increase in SOD activity and protein level that peaked immediately after UVB irradiation, after which a decline was observed, with subsequent recovery to baseline levels 24 h after irradiation. In individual assays of Mn SOD and Cu-Zn SOD, the amount of Mn SOD protein decreased and then gradually recovered 24 h after irradiation. In contrast, the amount of Cu-Zn SOD protein increased immediately after UVB irradiation, and then gradually declined. To evaluate the mechanisms of these changes, we examined the effects of the cytokines, interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), which can be secreted from keratinocytes after UVB irradiation, on the SOD activity and protein levels in keratinocytes. Interleukin-la and TNF-α enhanced both the SOD activity and protein level of Mn SOD, while these cytokines had no effect on Cu-Zn SOD protein levels in cultured human keratinocytes after incubation for 24 h. Furthermore, when neutralizing antibodies against IL-1α and TNF-α were added separately or together to the culture medium before UVB irradiation, the recovery of total SOD activity and Mn SOD protein level were markedly inhibited 24 h after irradiation. Our results suggest that significant increases in SOD activity and protein level occur as a cutaneous antioxidant defense mechanism that protects against the cytotoxicity as a result of UVB irradiation, and that this increase in SOD is attributed to Cu-Zn SOD. The Cu-Zn SOD and Mn SOD protein levels changed in a different manner after UVB irradiation. The former may participate in an early phase and the latter in a late phase defense mechanism directed against oxidant cytotoxicity through UVB irradiation. In addition, the recovery of Mn SOD to baseline levels 24 h after UVB irradiation seems to be mediated through cytokines such as IL-1α and TNF-α, which are secreted from keratinocytes.     

Ditopic Ligands. The Synthesis of a Series of Phosphine-Functionalised Macrocycles

The synthesis of a series of phosphine-functionalised macrocycles, 1–6 , is described. The combination of N-and O-sites with P- and S-sites provides ligands which may bind transition or non-transition metal ions; as a consequence, they give access to dinuclear complexes containing both a Lewis acid and a redox metallic site. Compounds 1,2 and 6 are heterodinucleating ligands capable of binding two dissimilar metals in proximity. Macrocycles 3–5 are homotopic ligands which may form homodinuclear complexes of transition metals.     

Electronic and Functional Structure of Copper in Plant Cu/Zn Superoxide Dismutase with Combined Site-directed Mutagenesis and Electron Paramagnetic Resonance

Arabidopsis thaliana copper-zinc superoxide dismutase 1 (AtSOD1) is a typical metalloenzyme conferring cellular protection against the excessive accumulation of toxic reactive oxygen species, and is therefore considered as a critical protein. However, the structure and function of the vital amino acids around the active site of AtSOD1 remain poorly understood. Herein, the coordinated geometry of the catalytic center in AtSOD1 was reconstructed by electron paramagnetic resonance (EPR) technique, and it was found to be composed of copper and four histidine (H) residues using site-directed mutagenesis. Analysis of the mutants showed that H45 and H62 play essential roles in the catalytic reaction, and H119 plays an accessary role in facilitating substrate or proton transfer. The results indicated that the redox change of the Cu ion and the overall enzymatic activity of the protein were sustained by the H45-Cu-H62 core structure. In contrast, the residue H47 showed nearly no effect on the SOD catalytic activity. These data should contribute to a deeper understanding of the catalytic mechanism of the enzyme, and provide a new approach for the effective molecular modification of copper/zinc SODs to facilitate further research in this field.     

Selective inhibition of Fe- versus Cu/Zn-superoxide dismutases by 2,3-dihydroxybenzoic acid derivatives

A series of catechol derivatives were synthesised and tested for their ability to inactivate the iron-containing superoxide dismutase (Fe-SOD) from Escherichia coli and the bovine erythrocytes Cu/Zn-SOD. Incubation of catechols with Fe- or Cu/Zn SODs resulted in a time-dependent loss of enzyme activity with highly selective inhibition for the iron-dependent enzyme. Catechol-induced inactivation of SODs was correlated with the auto-oxidation of the catechol compounds to their corresponding ortho-quinone derivatives, which was found to be non-dependent on the presence of enzymes. Mass electrospray experiments on catechol-incubated Fe-SOD provided evidence for the irreversible nature of the inhibition process, yielding to a complex mixture of modified proteins.     

子宫肌瘤患者血清SOD,MDA的观察

检测了６６例子宫肌瘤患者血清中超氧化物歧化酶（ＳＯＤ）和丙二醛（ＭＤＡ）的含量,探讨其与子宫肌瘤发病的关系。结果表明,子宫肌瘤患者血清中ＳＯＤ的活性较健康妇女明显降低,而ＭＤＡ含量明显增加,两者呈负相关,提示自由基在子宫肌瘤的发病中起着重要作用．     

季铵盐中N-手性在不对称烷基化反应中的作用研究

为了探明手性季铵盐中N-手性在不对称烷基化反应中的作用,从天然氨基酸出发合成了4个仅含C-手性的季铵盐.将它们与4个同时含有C-和N-手性的季铵盐用于催化二苯亚胺甘氨酸叔丁酯的不对称烷基化反应,通过对烷基化产物的ee值进行比较,结果表明简单的链状季铵盐中的N-手性不能对烷基化产物的ee值产生明显影响.