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REC-2006—A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo
Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg –1 body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair.
Methanobactin from methanotrophs: genetics, structure, function and potential applications
ABSTRACT Aerobic methane-oxidizing bacteria of the Alphaproteobacteria have been found to express a novel ribosomally synthesized post-translationally modified polypeptide (RiPP) termed methanobactin (MB). The primary function of MB in these microbes appears to be for copper uptake, but MB has been shown to have multiple capabilities, including oxidase, superoxide dismutase and hydrogen peroxide reductase activities, the ability to detoxify mercury species, as well as acting as an antimicrobial agent. Herein, we describe the diversity of known MBs as well as the genetics underlying MB biosynthesis. We further propose based on bioinformatics analyses that some methanotrophs may produce novel forms of MB that have yet to be characterized. We also discuss recent findings documenting that MBs play an important role in controlling copper availability to the broader microbial community, and as a result can strongly affect the activity of microbes that require copper for important enzymatic transformations, e.g. conversion of nitrous oxide to dinitrogen. Finally, we describe procedures for the detection/purification of MB, as well as potential medical and industrial applications of this intriguing RiPP.
Enhancement of reactive oxygen species production by ultra-short electron pulses
The development of laser-driven accelerators-on-chip has provided an opportunity to miniaturize devices for electron radiotherapy delivery. Laser-driven accelerators produce highly time-compressed electron pulses, on the 100 fs to 1 ps scale. This delivers electrons at high peak power yet low average beam current compared with conventional delivery devices, which generate pulses of approximately 3 µs. The biophysical effects of this time structure, however, are unclear. Here, we use a Monte Carlo simulation approach to explore the effects of the electron beam time structure on the production of reactive oxygen species (ROS) in water. Our results show a power law increase in the generation of hydroxyl ions per deposited electron with decreasing pulse length over the pulse length range of 10 µs to 100 fs. Similar trends were observed for hydrogen peroxide, superoxide, hydroperoxyl, hydronium and solvated electrons. In practical terms, this indicates a fourfold increase in the efficiency of free radical production for sub-picosecond pulses, relative to that of conventional microsecond pulses, for the same number of deposited electrons.
Structure of the cobalamin-binding protein of a putative O-demethylase from Desulfitobacterium hafniense DCB-2
The first crystal structure of the vitamin B12-binding protein from a three-component O-demethylase enzyme system is reported. During O-demethylation methyl groups are transferred from phenyl methyl ethers to tetrahydrofolate via methyl-B12 intermediates. This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense.
Paternal preconception alcohol consumption increased Angiotensin II‐mediated vasoconstriction in male offspring cerebral arteries via oxidative stress‐AT1R pathway
Abstract Alcohol consumption is popular worldwidely and closely associated with cardiovascular diseases. Influences of paternal preconception alcohol consumption on offspring cerebral arteries are largely unknown. Male rats were randomly given alcohol or water before being mated with alcohol‐naive females to produce alcohol‐ and control‐sired offspring. Middle cerebral artery (MCA) was tested with a Danish Myo Technology wire myograph, patch‐clamp, IONOPTIX, immunofluorescence and quantitative PCR. Alcohol consumption enhanced angiotensin II (AngII)‐mediated constriction in male offspring MCA mainly via AT1R. PD123,319 only augmented AngII‐induced constriction in control offspring. AngII and Bay K8644 induced stronger intracellular calcium transient in vascular smooth muscle cells (VSMCs) from MCA of alcohol offspring. L‐type voltage‐dependent calcium channel (L‐Ca 2+ ) current at baseline and after AngII‐stimulation was higher in VSMCs. Influence of large‐conductance calcium‐activated potassium channel (BK C a ) was lower. Caffeine induced stronger constriction and intracellular calcium release in alcohol offspring. Superoxide anion was higher in alcohol MCA than control. Tempol and thenoyltrifluoroacetone alleviated AngII‐mediated contractions, while inhibition was significantly higher in alcohol group. The mitochondria were swollen in alcohol MCA. Despite lower Kcnma1 and Prkce expression, many genes expressions were higher in alcohol group. Hypoxia induced reactive oxygen species production and increased AT1R expression in control MCA and rat aorta smooth muscle cell line. In conclusion, this study firstly demonstrated paternal preconception alcohol potentiated AngII‐mediated vasoconstriction in offspring MCA via ROS‐AT1R. Alcohol consumption increased intracellular calcium via L‐Ca 2+ channel and endoplasmic reticulum and decreased BK Ca function. The present study provided new information for male reproductive health and developmental origin of cerebrovascular diseases.
The role of aldo‐keto reductase 1 C 3 ( AKR 1 C 3)‐mediated prostaglandin D 2 ( PGD 2) metabolism in keloids
Abstract Keloids are progressively expanding scars, mostly prevalent in individuals of African descent. Previous data identified increased mast cell number and activation state in keloids suggesting a role in disease progression. The major eicosanoid secreted by mast cells is prostaglandin D2 ( PGD 2), a relatively unstable pro‐inflammatory mediator which can be spontaneously converted to 15‐deoxy‐(Delta12,14)‐prostaglandin J2(15d‐PGJ2) or enzymatically metabolized to 9α,11β‐ PGF 2 by aldo‐keto reductase 1C3 ( AKR 1C3). In this work, we investigated the possible role of PGD 2 and its metabolites in keloids using CRL 1762 keloid fibroblasts ( KF ) and immunohistochemical staining. Our data suggested approximately 3‐fold increase of tryptase‐positive mast cell count in keloids compared with normal skin. Furthermore, AKR 1C3 was overexpressed in the fibrotic area of keloids while relatively weak staining detected in normal skin. Metabolism of PGD 2 to 9 α ,11 β ‐ PGF 2 by both, KF and normal fibroblasts, was dependent on AKR 1C3 as this reaction was attenuated in the presence of the AKR 1C3 inhibitor, 2′‐hydroxyflavanone, or in cells with decreased AKR 1C3 expression. 15d‐ PGJ 2, but not the other tested PG s, inhibited KF proliferation, attenuated KF ‐mediated collagen gel contraction and increased caspase‐3 activation. In addition, treatment with 15d‐ PGJ 2 activated P38‐ MAPK , induced reactive oxygen species and upregulated superoxide dismutase‐1 ( SOD ‐1). Finally, inhibition of P38‐ MAPK further augmented 15d‐ PGJ 2‐induced caspase‐3 cleavage and attenuated its effect on SOD ‐1 transcription. This work suggests that localized dual inhibition of AKR 1C3 and P38‐ MAPK may inhibit keloid progression. Inhibiting AKR 1C3 activity may generate oxidative environment due to redirection of PGD 2 metabolism towards 15d‐ PGJ 2 while inhibition of P38‐ MAPK will sensitize keloid cells to ROS ‐induced apoptosis.
Oxygen isotope analysis of bacterial and fungal manganese oxidation
Abstract The ability of micro‐organisms to oxidize manganese (Mn) from Mn( II ) to Mn( III / IV ) oxides transcends boundaries of biological clade or domain. Many bacteria and fungi oxidize Mn( II ) to Mn( III / IV ) oxides directly through enzymatic activity or indirectly through the production of reactive oxygen species. Here, we determine the oxygen isotope fractionation factors associated with Mn( II ) oxidation via various biotic (bacteria and fungi) and abiotic Mn( II ) reaction pathways. As oxygen in Mn( III / IV ) oxides may be derived from precursor water and molecular oxygen, we use a twofold approach to determine the isotope fractionation with respect to each oxygen source. Using both 18 O‐labeled water and closed‐system Rayleigh distillation approaches, we constrain the kinetic isotope fractionation factors associated with O atom incorporation during Mn( II ) oxidation to −17.3‰ to −25.9‰ for O 2 and −1.9‰ to +1.8‰ for water. Results demonstrate that stable oxygen isotopes of Mn( III / IV ) oxides have potential to distinguish between two main classes of biotic Mn( II ) oxidation: direct enzymatic oxidation in which O 2 is the oxidant and indirect enzymatic oxidation in which superoxide is the oxidant. The fraction of Mn( III / IV ) oxide‐associated oxygen derived from water varies significantly (38%–62%) among these bio‐oxides with only weak relationship to Mn oxidation state, suggesting Mn( III ) disproportionation may account for differences in the fraction of mineral‐bound oxygen from water and O 2 . Additionally, direct incorporation of molecular O 2 suggests that Mn( III / IV ) oxides contain a yet untapped proxy of of environmental O 2 , a parameter reflecting the integrated influence of global respiration, photorespiration, and several other biogeochemical reactions of global significance.
Mechanistic assessment of tolerance to iron deficiency mediated by Trichoderma harzianum in soybean roots
Abstract Aims Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. Methods and Results Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2•–) and hydrogen peroxide (H2O2) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. Conclusions TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. Significance and Impact of the Study These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.
Astragaloside IV improved antioxidative stress capacity and related gene expression of the Keap1‐Nrf2 pathway in grass carp ( Ctenopharyngodon idella ) hepatocytes under heat stress
Abstract This study aims to investigate the protective effects of astragaloside IV (AS‐IV) on the hepatocytes of grass carp ( Ctenopharyngodon idella ) on heat stress. Cultured cells were treated with AS‐IV (0, 50, 100 and 200 μg/ml) at 28°C for 24 h and then exposed to heat stress by increasing the culturing temperature (32 ± 0.5°C) for 6 h. The increased temperatures significantly reduced cell viability and superoxide dismutase (SOD) activity, and increased malondialdehyde (MDA) levels in the 0 μg/ml AS‐IV treatment group at 32°C, but the grass carp hepatocytes treated with 100 and 200 μg/ml AS‐IV had significantly increased cell viability and SOD activity and decreased MDA levels. The mRNA levels of keap1a , keap1b , nrf2 , gsh‐px , cat , cu‐zn sod , mgst1 and il‐6 were significantly lower in the 0 μg/ml AS‐IV treatment group at 32°C, while those of keap1a , nrf2 , gsh‐px , cat , cu‐zn sod , gstp1 , ho‐1 and il‐6 were significantly higher in cells treated with 100 or 200 μg/ml AS‐IV. Our findings indicate that AS‐IV could enhance the antioxidative stress capacity of grass carp hepatocytes under heat stress, and its mechanism may be associated with the activation of the Keap1‐Nrf2 pathway. Thus, these results provide new insights into how to alleviate heat stress in grass carp.
Development and testing of techniques for in-ground stabilization, size reduction, and safe removal of radioactive wastes stored in containments buried in ground
Since the 1950's radioactive wastes from a number of laboratories have been stored below ground at the Hanford site, Washington State, USA, in vertical pipe units (VPUs) made of five 200 litre drums without tops or bottoms, and in caissons, made out of corrugated pipe, or concrete and typically 2,500 mm in diameter. The VPU's are buried of the order of 2,100 mm below grade, and the caissons are buried of the order of 6,000 mm below grade. The waste contains fuel pieces, fission products, and a range of chemicals used in the laboratory processes. This can include various energetic reactants such as un-reacted sodium potassium (NaK), potassium superoxide (KO{sub 2}), and picric acid, as well as quantities of other liquids. The integrity of the containments is considered to present unacceptable risks from leakage of radioactivity to the environment. This paper describes the successful development and full scale testing of in-ground augering equipment, grouting systems and removal equipment for remediation and removal of the VPUs, and the initial development work to test the utilization of the same basic augering and grouting techniques for the stabilization, size reduction and removal of caissons. (authors)
A high-energy-density lithium-oxygen battery based on a reversible four-electron conversion to lithium oxide
Lithium-oxygen (Li-O 2 ) batteries have attracted much attention owing to the high theoretical energy density afforded by the two-electron reduction of O 2 to lithium peroxide (Li 2 O 2 ). We report an inorganic-electrolyte Li-O 2 cell that cycles at an elevated temperature via highly reversible four-electron redox to form crystalline lithium oxide (Li 2 O). It relies on a bifunctional metal oxide host that catalyzes O–O bond cleavage on discharge, yielding a high capacity of 11 milliampere-hours per square centimeter, and O 2 evolution on charge with very low overpotential. Online mass spectrometry and chemical quantification confirm that oxidation of Li 2 O involves transfer of exactly 4 e – /O 2 . This work shows that Li-O 2 electrochemistry is not intrinsically limited once problems of electrolyte, superoxide, and cathode host are overcome and that coulombic efficiency close to 100% can be achieved.
A room temperature rechargeable Li 2 O-based lithium-air battery enabled by a solid electrolyte
Lithium-air batteries have scope to compete with gasoline in terms of energy density. However, in most systems, the reaction pathways either involve one- or two-electron transfer, leading to lithium peroxide (Li 2 O 2 ) or lithium superoxide (LiO 2 ), respectively. Kondori et al. investigated a lithium-air battery that uses a ceramic-polyethylene oxide–based composite solid electrolyte and found that it can undergo a four-electron redox reaction through lithium oxide (Li 2 O) formation and decomposition (see the Perspective by Dong and Lu). The composite electrolyte embedded with Li 10 GeP 2 S 12 nanoparticles shows high ionic conductivity and stability and high cycle stability through a four-electron transfer process.
Electrophotocatalytic perfluoroalkylation by LMCT excitation of Ag(II) perfluoroalkyl carboxylates
Molecular Ag(II) complexes are superoxidizing photoredox catalysts capable of generating radicals from redox-reticent substrates. In this work, we exploited the electrophilicity of Ag(II) centers in [Ag(bpy) 2 (TFA)][OTf] and Ag(bpy)(TFA) 2 (bpy, 2,2′-bipyridine; OTf, CF 3 SO 3 – ) complexes to activate trifluoroacetate (TFA) by visible light–induced homolysis. The resulting trifluoromethyl radicals may react with a variety of arenes to forge C(sp 2 )–CF 3 bonds. This methodology is general and extends to other perfluoroalkyl carboxylates of higher chain length (R F CO 2 – ; R F , CF 2 CF 3 or CF 2 CF 2 CF 3 ). The photoredox reaction may be rendered electrophotocatalytic by regenerating the Ag(II) complexes electrochemically during irradiation. Electrophotocatalytic perfluoroalkylation of arenes at turnover numbers exceeding 20 was accomplished by photoexciting the Ag(II)–TFA ligand-to-metal charge transfer (LMCT) state, followed by electrochemical reoxidation of the Ag(I) photoproduct back to the Ag(II) photoreactant.
Abundance and Biogeography of Picoprasinophyte Ecotypes and Other Phytoplankton in the Eastern North Pacific Ocean
Here, eukaryotic algae within the picoplankton size class (≤2 μm in diameter) are important marine primary producers, but their spatial and ecological distributions are not well characterized. Here, we studied three picoeukaryotic prasinophyte genera and their cyanobacterial counterparts, Prochlorococcus and Synechococcus , during two cruises along a North Pacific transect characterized by different ecological regimes. Picoeukaryotes and Synechococcus reached maximum abundances of 1.44 × 10 5 and 3.37 × 10 5 cells · ml –1 , respectively, in mesotrophic waters, while Prochlorococcus reached 1.95 × 10 5 cells · ml –1 in the oligotrophic ocean. Of the picoeukaryotes, Bathycoccus was present at all stations in both cruises, reaching 21,368 ± 327 18S rRNA gene copies · ml –1 . Micromonas and Ostreococcus clade OI were detected only in mesotrophic and coastal waters and Ostreococcus clade OII only in the oligotrophic ocean. To resolve proposed Bathycoccus ecotypes, we established genetic distances for 1,104 marker genes using targeted metagenomes and the Bathycoccus prasinos genome. The analysis was anchored in comparative genome analysis of three Ostreococcus species for which physiological and environmental data are available to facilitate data interpretation. We established that two Bathycoccus ecotypes exist, named here BI (represented by coastal isolate Bathycoccus prasinos ) and BII. These share 82% ± 6% nucleotide identity across homologs, while the Ostreococcus spp. share 75% ± 8%. We developed and applied an analysis of ecomarkers to metatranscriptomes sequenced here and published -omics data from the same region. The results indicated that the Bathycoccus ecotypes cooccur more often than Ostreococcus clades OI and OII do. Exploratory analyses of relative transcript abundances suggest that Bathycoccus NRT2.1 and AMT2.2 are high-affinity NO 3 – and low-affinity NH 4 + transporters, respectively, with close homologs in multiple picoprasinophytes. Additionally, in the open ocean, where dissolved iron concentrations were low (0.08 nM), there appeared to be a shift to the use of nickel superoxide dismutases (SODs) from Mn/Fe/Cu SODs closer inshore. Our study documents the distribution of picophytoplankton along a North Pacific ecological gradient and offers new concepts and techniques for investigating their biogeography.
Identification of microRNAs responsive to arbuscular mycorrhizal fungi in Panicum virgatum (switchgrass)
Background: MicroRNAs (miRNAs) are important post-transcriptional regulators involved in the control of a range of processes, including symbiotic interactions in plants. MiRNA involvement in arbuscular mycorrhizae (AM) symbiosis has been mainly studied in model species, and our study is the first to analyze global miRNA expression in the roots of AM colonized switchgrass ( Panicum virgatum ), an emerging biofuel feedstock. AM symbiosis helps plants gain mineral nutrition from the soil and may enhance switchgrass biomass production on marginal lands. Our goals were to identify miRNAs and their corresponding target genes that are controlling AM symbiosis in switchgrass. Results: Through genome-wide analysis of next-generation miRNA sequencing reads generated from switchgrass roots, we identified 122 mature miRNAs, including 28 novel miRNAs. By comparing miRNA expression profiles of AM-inoculated and control switchgrass roots, we identified 15 AM-responsive miRNAs across lowland accession “Alamo”, upland accession “Dacotah”, and two upland/lowland F 1 hybrids. We used degradome sequencing to identify target genes of the AM-responsive miRNAs revealing targets of miRNAs residing on both K and N subgenomes. Notably, genes involved in copper ion binding were targeted by downregulated miRNAs, while upregulated miRNAs mainly targeted GRAS family transcription factors. Conclusion: Through miRNA analysis and degradome sequencing, we revealed that both upland and lowland switchgrass genotypes as well as upland-lowland hybrids respond to AM by altering miRNA expression. We demonstrated complex GRAS transcription factor regulation by the miR171 family, with some miR171 family members being AM responsive while others remained static. Copper miRNA downregulation was common amongst the genotypes tested and we identified superoxide dismutases and laccases as targets, suggesting that these Cu-miRNAs are likely involved in ROS detoxification and lignin deposition, respectively. Other prominent targets of the Cu miRNAs were blue copper proteins. Overall, the potential effect of AM colonization on lignin deposition pathways in this biofuel crop highlights the importance of considering AM and miRNA in future biofuel crop development strategies.
Mariprofundus ferrooxydans PV-1 the First Genome of a Marine Fe(II) Oxidizing Zetaproteobacterium
Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO2, carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria.
Tetramerization Reinforces the Dimer Interface of MnSOD
Two yeast manganese superoxide dismutases (MnSOD), one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), have most biochemical and biophysical properties in common, yet ScMnSOD is a tetramer and CaMnSODc is a dimer or ‘‘loose tetramer’’ in solution. Although CaMnSODc was found to crystallize as a tetramer, there is no indication from the solution properties that the functionality of CaMnSODc in vivo depends upon the formation of the tetrameric structure. To elucidate further the functional significance of MnSOD quaternary structure, wild-type and mutant forms of ScMnSOD (K182R, A183P mutant) and CaMnSODc (K184R, L185P mutant) with the substitutions at dimer interfaces were analyzed with respect to their oligomeric states and resistance to pH, heat, and denaturant. Dimeric CaMnSODc was found to be significantly more subject to thermal or denaturant-induced unfolding than tetrameric ScMnSOD. The residue substitutions at dimer interfaces caused dimeric CaMnSODc but not tetrameric ScMnSOD to dissociate into monomers. We conclude that the tetrameric assembly strongly reinforces the dimer interface, which is critical for MnSOD activity.