Excess Copper Induces A Cytosolic Cu,Zn-Superoxide Dismutase in Soybean Root

The induction of several proteins in soybean roots in responseto copper was investigated. The major Cu²⁺-induced protein of16 kDa was purified by SDS-PAGE and 2D-PAGE. This Cu²⁺-inducedprotein cross-reacted with antibodies against cytosolic Cu,Zn-SODand the sequence of 40 amino-terminal residues of this proteinwas found to be 70% and 62.5% homologous to the sequences ofcytosolic Cu,Zn-SOD from rice and tomato, respectively. An assayof enzymatic activity revealed that SOD activity of Cu²⁺-treatedsoybean roots was more than twice that in roots of non-treatedcontrol plants. Thus, treatment with copper of soybean roots appears to inducean increase in SOD activity via the synthesis of cytosolic Cu,Zn-SOD.The induction of SOD by such treatment may be the result eitherof a direct effect of copper on the gene for SOD or of an indirecteffect via an increase in levels of O₂^– (Received July 12, 1991; Accepted January 22, 1992)     

Cu,Zn-Superoxide Dismutase Increases Toxicity of Mutant and Zinc-deficient Superoxide Dismutase by Enhancing Protein Stability

When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive “partially unfolded intermediate” responsible for the toxic gain of function conferred by ALS mutant SOD.     

Occurrence of Cu,Zn-Superoxide Dismutase in the Intrathylakoid Space of Spinach Chloroplasts

Thylakoids obtained from intact spinach chloroplasts showedno superoxide dismutase (SOD) activity, but Cu,Zn- and Mn-SODactivities were detected in the presence of Triton X-100. Thylakoidmembranes and the lumen fraction were separated by centrifugationafter treatment of the thylakoids with a Yeda pressure cell.Cu,Zn-SOD was found in the lumen fraction. Mn-SOD was detectedin the thylakoid fraction only after addition of 1% Triton X-100.Antibody against spinach Cu,Zn-SOD did not interact with thelatent Cu,Zn-SOD in the thylakoids unless Triton was added.These results indicate that Cu,Zn-SOD occurs in the lumen inaddition to the stroma of spinach chloroplasts, and Mn-SOD bindsto the thylakoid membranes. (Received February 29, 1984; Accepted May 28, 1984)     

Cu,Zn-Superoxide Dismutase Gene Dosage and Cell Resistance to Oxidative Stress: A Review

It is well established that superoxide dismutase (SOD) is the irresplaceable enzyme for aerobic lifestyle. Our understanding of its role has made strides recently as the result of gene transfection approach. Available data on consequences of Cu,Zn-SOD gene transfection in cell resistance to oxygen toxicity are reviewed. There are data that increasing only Cu,Zn-SOD can be toxic, and the balance between Cu,Zn-SOD and peroxide-removing enzymes is supposed to be of prime importance in the antioxidant defence. Role of Cu,Zn-SOD deregulation in carcinogenesis is discussed.     

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.     

Mutation-dependent Polymorphism of Cu,Zn-Superoxide Dismutase Aggregates in the Familial Form of Amyotrophic Lateral Sclerosis

More than 100 different mutations in Cu,Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (fALS). Pathogenic mutations facilitate fibrillar aggregation of SOD1, upon which significant structural changes of SOD1 have been assumed; in general, however, a structure of protein aggregate remains obscure. Here, we have identified a protease-resistant core in wild-type as well as fALS-causing mutant SOD1 aggregates. Three different regions within an SOD1 sequence are found as building blocks for the formation of an aggregate core, and fALS-causing mutations modulate interactions among these three regions to form a distinct core, namely SOD1 aggregates exhibit mutation-dependent structural polymorphism, which further regulates biochemical properties of aggregates such as solubility. Based upon these results, we propose a new pathomechanism of fALS in which mutation-dependent structural polymorphism of SOD1 aggregates can affect disease phenotypes.     

The Rate of Cu,Zn Superoxide Dismutase Evolution

The rate of amino acid replacement in Cu, Zn SOD greatly departs from the expectations of the molecular clock. We examine 27 Cu, Zn SOD sequences available and conclude that: (I) the SOD enzymes from different mammal families differ from each other by roughly the same number of replacements, which is consistent with a simultaneous mammalian radiation; (2) over the most recent 60 million years (MY) the rate of SOD evolution is fairly high (15aa/100aa/100MYR) and may be considered constant; (3) the rate of accumulation of amino acid replacements since the divergence of fungi. plants and animals to the present is inconstant along different branches of the evolutionary tree; moreover it steadily decreases with time, to the same extent in all lineages; (4) some comparisons exhibit divergences that are in any case greater than expected from a Poisson process on the assumption of a molecular clock; (5) plant chloroplast enzymes display fewer differences from each other than cytoplasmic ones; (6) bacteriocuprein (from Photobacterium leiognathi), fluke and human extracellular SOD are all three extremely remotely related to one another and to the SOD of other eukaryotes.

The process of consistent decline of the rate of evolution of Cu. Zn SOD can be described by a number of mathematical functions. We explore simple models that assume constant rates and might be applicable to other proteins or genes that apparently evolve at disparate rates.     

Activation of Cu,Zn-Superoxide Dismutase in the Absence of Oxygen and the Copper Chaperone CCS

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.Oxygen is essential for aerobic respiration, but reactive byproducts of oxygen metabolism, such as the superoxide anion, can damage cellular molecules, including proteins, DNA, and lipids (1–3). SOD1s (copper- and zinc-containing superoxide dismutases) provide the primary defense against superoxide damage by catalytically removing it through a disproportionation reaction (4). This reaction involves redox cycling at the copper active site (5). SOD1s require several post-translational modifications to form an active molecule. Copper and zinc are bound by the enzyme, and an intramolecular disulfide bond is formed between two conserved cysteine residues. Although the zinc ion and disulfide bond are not directly involved in the disproportionation reaction, these modifications are required for proper stability and formation of the active site (6–10). The presence of an intramolecular disulfide bond is intriguing, given the fact that the cytosol favors reduced thiols.The activity of SOD1s in vivo is largely controlled through the aforementioned post-translational modifications. Most of what is currently known about activation of SOD1 in vivo has emerged through studies of the bakers'' yeast Saccharomyces cerevisiae SOD1. Here insertion of the catalytic copper requires the action of the copper chaperone for SOD³ (CCS) (11). CCS physically interacts with SOD1 to deliver the copper ion and catalyze the disulfide bond formation in an oxygen-dependent manner (12–15). In fact, S. cerevisiae SOD1 (ySOD1) is completely dependent on CCS for insertion of the catalytic copper and oxidation of the disulfide bond (11, 15, 16).Although ySOD1 is dependent on CCS for activity, other eukaryotic SOD1s are not. Mouse and human SOD1 (hSOD1), when expressed in CCS^−/− mouse fibroblasts and in ccs1Δ yeast, still retain some SOD1 activity (17–19). Moreover, the genome for the nematode Caenorhabditis elegans does not contain a CCS-like gene, yet harbors several Cu,Zn-SODs. Previous studies with C. elegans SOD-1 (wSOD-1) have shown that this SOD is activated completely independently of CCS (20). Together, these studies present a strong case for a second SOD1 activation mechanism independent of CCS.There must be inherent differences in SOD1 sequences that dictate whether the enzyme uses CCS or the CCS-independent pathway or both. Through targeted mutagenesis, sequences near the C terminus have been previously identified as being important (19). Yeast SOD1 contains dual prolines at positions 142 and 144, which when mutated in combination allow for CCS-independent activation. Conversely, hSOD1 and wSOD-1 contain non-proline residues at these positions, and if dual prolines are introduced, then CSS-independent activation is blocked (19, 20). How this pair of prolines influences SOD1 activation is not understood.It is interesting that nature has developed two activation mechanisms for such a key enzyme in oxidative stress protection, and these are not likely to be redundant. It was previously predicted that the two pathways draw upon distinct sources of copper (19), since the addition of the catalytic copper ion is limiting for enzyme activation. However, since disulfide oxidation is also limiting for enzyme activity, it is possible that the two pathways diverge at this level. In the current study, we investigate the requirements and regulation of the CCS-dependent and -independent SOD1 activation pathways. Our results strongly indicate that the two pathways do not diverge at the level of upstream copper transporter sources or the kinetics of copper incorporation into SOD1 but rather at the level of disulfide bond formation. Copper is required for CCS-mediated disulfide bond oxidation in yeast SOD1, whereas SOD1s that can be activated without CCS show no such requirement for copper in disulfide oxidation. Moreover, oxygen is required for enzyme activation through CCS, but the CCS-independent pathway is able to bypass the need for molecular oxygen. This allows for significant SOD1 activity to be found at a variety of oxygen concentrations by utilizing two activation pathways.     

Chloroplast and Cytosol Isozymes of Cu,Zn-Superoxide Dismutase: Their Characteristic Amino Acid Sequences

lsozymes of CuZn-superoxide dismutase (SOD) were purified from angiosperms (spinach and rice), fern (horsetail) and green alga (Spirogyra). Occurrence of CuZn-SOD was confirmed by its purification in the group of green algae which shows the phragmoplast type of cell division. Purified CuZn-SODS are divided to chloroplast and cytosol types by their cellular localization and immunological properties. Their amino acid compositions, absorption spectra, CD spectra, and sensitivity to hydrogen peroxide also are distinguished from each other. All organisms including Spirogyra contain both types of isozyme. Thus, the divergence of the two types of CuZn-SOD isozyme occurred immediately after its acquisition by the most evolved green algae.

Amino acid sequences of amino-terminal regions of CuZn-SOD isozyrnes from spinach, rice and horsetail were determined and compared with those of CuZn-SODS from other plants. The chloroplast and cytosol isozymes of CuZn-SOD show each characteristic sequences. Sequence differences among the cytosol CuZn-SODS are greater than those among the chloroplast CuZn-SODS. These observations indicate that each type of isozyme had independently evolved after the acquisition of CuZn-SOD.     

蜡样芽胞杆菌M22铜锌超氧化物歧化酶基因的克隆及表达

采用PCR技术 ,从蜡样芽胞杆菌M2 2基因组DNA扩增到长 132 0bp的基因片段 .该片段含编码 179个氨基酸残基的开放阅读框 ,推定蛋白序列与报道的BacillusanthracisCu ,Zn SOD序列有96 %同源性 ,其中N端 16个氨基酸残基推定为信号肽序列 .将Cu ,Zn SOD编码区插入载体pET 2 2b(+ )构建表达载体pET 2 2b sodC ,转化E .coliBL2 1(DE3) ,IPTG诱导融合蛋白表达 .SDS PAGE显示 ,融合蛋白表观分子质量约 2 4kD ,占菌体裂解液中总蛋白的 2 1 3% .将此表达载体转入SOD缺陷型大肠杆菌PN132 ,赋予了该菌株对paraquat的抗性 .NBT光抑制法测定SOD活性显示 ,与PN132无SOD活性相比 ,重组子在IPTG诱导前SOD活性极低 (1 79U/mg) ,诱导后活性高达 6 9 76U/mg .非变性电泳结果显示 ,该酶活性不同程度地受到 5mmol LH2 O2 和 5mmol LKCN的抑制     

Effects of Cu,Zn-Superoxide Dismutase on Cell Proliferation and Neuroblast Differentiation in the Mouse Dentate Gyrus

Oxidative stress is one of the most important factors in reducing adult hippocampal neurogenesis in the adult brain. In this study, we observed the effects of Cu,Zn-superoxide dismutase (SOD1) on lipid peroxidation, cell proliferation, and neuroblast differentiation in the mouse dentate gyrus using malondialdehyde (MDA), Ki67, and doublecortin (DCX), respectively. We constructed an expression vector, PEP-1, fused PEP-1 with SOD1, and generated PEP-1-SOD1 fusion protein. We administered PEP-1 and 100 or 500 μg PEP-1-SOD1 intraperitoneally once a day for 3 weeks and sacrificed at 30 min after the last administrations. PEP-1 administration did not change the MDA levels compared to those in the vehicle-treated group, while PEP-1-SOD1 treatment significantly reduced MDA levels compared to the vehicle-treated group. In the PEP-1-treated group, the number of Ki67-positive nuclei was similar to that in the vehicle-treated group. In the 100 μg PEP-1-SOD1-treated group, the number of Ki67-positive nuclei was slightly decreased; however, in the 500 μg PEP-1-SOD1-treated group, Ki67-positive nuclei were decreased to 78.5% of the vehicle-treated group. The number of DCX-positive neuroblasts in the PEP-1-treated group was similar to that in the vehicle-treated group. However, the arborization of DCX-positive neuroblasts was significantly decreased in both the 100 and 500 μg PEP-1-SOD1-treated groups compared to that in the vehicle-treated group. The number of DCX-positive neuroblasts with tertiary dendrites was markedly decreased in the 500 μg PEP-1-SOD1-treated group. These results suggest that a SOD1 supplement to healthy mice may not be necessary to modulate cell proliferation and neuroblast differentiation in the dentate gyrus.     

乳酸克鲁维酵母Cu/Zn-SOD基因的克隆及在酿酒酵母中的表达研究

根据Genbank中乳酸克鲁维酵母(Kluyveromyces lactis)的Cu/Zn-SOD基因序列设计引物,通过PCR扩增得到Cu/Zn-SOD基因。在PGK1启动子驱动下,将该基因与荧光报告基因GFP融合,分别构建重组质粒YEplac195-PSGA和YCplac33-PSGA,并转化酿酒酵母(Saccharomyces cerevisiae)W303α菌株。通过菌落PCR和荧光显微观察证实乳酸克鲁维酵母(Kluyveromyces lactis)的Cu/Zn-SOD基因在W303α中成功表达。将获得的阳性转化子在添加20mmol/L百草枯的发酵培养基中进行发酵,SOD的比活力和总活力分别是不添加百草枯培养基中发酵菌体的6.7倍和4.7倍。通过热激胁迫处理进一步探讨Cu/Zn-SOD对宿主sod1Δ酿酒酵母菌株EG118耐受力的影响,结果显示抗热击能力的顺序为EG118(YEplac195-PSGA)EG118(YCplac33-PSGA)EG118。以上结果为发酵工业中防止菌体老化和增强菌体的发酵能力提供一定的理论指导,也为后续的Cu/Zn-SOD体外分子定向进化改造奠定必要的基础。     

Cu,Zn-Superoxide Dismutase Increases the Therapeutic Potential of Adipose-derived Mesenchymal Stem Cells by Maintaining Antioxidant Enzyme Levels

In the present study, we investigated the ability of Cu, Zn-superoxide dismutase (SOD1) to improve the therapeutic potential of adipose tissue-derived mesenchymal stem cells (Ad-MSCs) against ischemic damage in the spinal cord. Animals were divided into four groups: the control group, vehicle (PEP-1 peptide and artificial cerebrospinal fluid)-treated group, Ad-MSC alone group, and Ad-MSC-treated group with PEP-1-SOD1. The abdominal aorta of the rabbit was occluded for 30 min in the subrenal region to induce ischemic damage, and immediately after reperfusion, artificial cerebrospinal fluid or Ad-MSCs (2?×?10⁵) were administered intrathecally. In addition, PEP-1 or 0.5 mg/kg PEP-1-SOD1 was administered intraperitoneally to the Ad-MSC-treated rabbits. Motor behaviors and NeuN-immunoreactive neurons were significantly decreased in the vehicle-treated group after ischemia/reperfusion. Administration of Ad-MSCs significantly ameliorated the changes in motor behavior and NeuN-immunoreactive neuronal survival. In addition, the combination of PEP-1-SOD1 and Ad-MSCs further increased the ameliorative effects of Ad-MSCs in the spinal cord after ischemia. Furthermore, the administration of Ad-MSCs with PEP-1-SOD1 decreased lipid peroxidation and maintained levels of antioxidants such as SOD1 and glutathione peroxidase compared to the Ad-MSC alone group. These results suggest that combination therapy using Ad-MSCs and PEP-1-SOD1 strongly protects neurons from ischemic damage by modulating the balance of lipid peroxidation and antioxidants.     

Interaction of Cu(II)with His-Val-Gly-Asp and of Zn(II) with His-Val-His,Two Peptides at the Active Site of Cu,Zn-Superoxide Dismutase

His-Val-His and His-Val-Gly-Asp are two naturally occurring peptide sequences, present at the active site of Cu,Zn-superoxide dismutase (Cu,Zn-SOD). We have already studied the interaction of His-Val-His=A (copper binding site) with Cu(II) and of His-Val-Gly-Asp=B (zinc binding site) with Zn(II). As a continuation of this work and for comparison purposes we have also studied the interaction of Zn(II) with His-Val-His and Cu(II) with His-Val-Gly-Asp using both potentiometric and spectroscopic methods (visible, EPR, NMR). The stoichiometry, stability constants and solution structure of the complexes formed have been determined. Histamine type of coordination is observed for/ZnAH/²⁺, /ZnA/⁺, /ZnA²H/⁺ and/ZnA²/ in acidic pH while deprotonation of coordinated water molecules is observed at higher pH. /CUB/ species is characterized by the formation of a macrochelate and histamine type coordination. Its stability results in the suppression of amide deprotonation which occurs at high pH resulting in the formation of the highly distorted from square planar geometry 4N complex/CuBH_-3/³.     

Purification of Cu, Zn-Superoxide Dismutase Isoenzymes from Fish Liver: Appearance of New Isoforms as a Consequence of Pollution

Liver cell-free extracts of fish (Mugil sp.) from polluted environments show new Cu, Zn-SOD isoenzymes when analyzed by polyacrylamide gel electrophoresis or isoelectrofocusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were present both in control and problem samples while the isoenzymes of intermediate pI value showed significant differences. Fish from control areas showed three intermediate isoenzymes with pI 5.7, 5.5 and 5.4 (the last one quite faint) while polluted animals showed three bands of pI 5.9, 5.45 and 5.35, this last very intense. To further characterize their utility as biomarkers, Cu, Zn-SOD isoenzymes from polluted fish livers were purified to homogeneity. Five superoxide dismutase peaks were purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Isoenzymes I and V displayed the highest specific activity. Upon incubation with moderate H₂O₂ concentrations, pure isoenzyme I yielded more acidic bands with pI 5.5, 5.45 and 5.35, this last being predominant. The pure isoenzyme V generated only a new band of pI 5.0. Concomitant with oxidation, the activity of peaks I and V was lost in a H₂O₂ concentration-dependent manner. The pattern of the new acidic bands generated upon the oxidixing treatment of isoenzyme I closely resembles that observed in crude extracts from polluted animals.     

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.     

Probing the Structural Basis for Enzyme-Substrate Recognition In Cu,Zn Superoxide Dismutase

A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu. Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stcreochemistry. supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu132, Glu133, Lys136, Thr137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzyme's rapid catalytic rate.     

Direct Magnetic Resonance Evidence for Peroxymonocarbonate Involvement in the Cu,Zn-Superoxide Dismutase Peroxidase Catalytic Cycle

Cu,Zn-superoxide dismutase (SOD1) is a copper- and zinc-dependent enzyme. The main function of SOD1 is believed to be the scavenging and detoxification of superoxide radicals. Nevertheless, the last 30 years have seen a rapid accumulation of evidence indicating that SOD1 may also act as a peroxidase, an alternative function that was implicated in the onset and progression of familial amyotrophic lateral sclerosis. Although SOD1 peroxidase activity and its dependence on carbon dioxide have been well described, the molecular basis of the SOD1 peroxidase cycle remains obscure, because none of the proposed catalytic intermediates have so far been identified. In view of recent observations, we hypothesized that the SOD1 peroxidase cycle relies on two steps: 1) reduction of SOD-Cu(II) by hydrogen peroxide followed by 2) oxidation of SOD-Cu(I) by peroxymonocarbonate, the product of the spontaneous reaction of bicarbonate with hydrogen peroxide, to produce SOD-Cu(II) and carbonate radical anion. This hypothesis has been investigated through electron paramagnetic resonance and nuclear magnetic resonance to provide direct evidence for a peroxycarbonate-driven, SOD1-catalyzed carbonate radical production. The results gathered herein indicate that peroxymonocarbonate () is a key intermediate in the SOD1 peroxidase cycle and identify this species as the precursor of carbonate radical anions.Cytosolic Cu,Zn-superoxide dismutase (SOD1)² is a metal-dependent enzyme capable of accelerating the rate of spontaneous superoxide dismutation into O₂ and H₂O₂ through the redox cycling of its copper ion (1, 2). SOD1 is widely distributed in mammalian cells and tissues and has been demonstrated to be located in the cytosol and in the intermembrane space of the mitochondria (see Ref. 3 and references therein). Because of that, SOD1 is believed to be a major player in the first line defense against reactive oxygen species, in particular superoxide anion.In addition to its dismutase activity, SOD1 possesses a well described but incompletely understood peroxidase activity which is dependent on hydrogen peroxide and markedly stimulated by small oxidizable anions such as nitrite and the ubiquitous carbon dioxide (3-12). The peroxidase activity of SOD1 has been proposed to impact the onset and progression of familial amyotrophic lateral sclerosis, a severely debilitating fatal disease characterized by the selective death of motor neurons (13-18). Although several reports exist in the literature indicating the formation of SOD1 aggregates and accumulation as a potential cause in the pathology progression, conflicting hypotheses are still under debate concerning the mechanisms that lead to the formation of SOD1-protein aggregates (19-21). Although some support the suggestion that free radical-induced covalent cross-links among SOD1 amino acids play a fundamental role in aggregate formation (22, 23), others support the view that metal loss from the enzyme structure leads to an unstable apo-form of SOD1 with increased capacity to form aggregates (24, 25). A detailed understanding of the SOD1 peroxidase cycle is essential to unraveling the mechanisms through which SOD1 aggregates are produced.The SOD1 peroxidase cycle is initiated when SOD1-Cu(II) is reduced by H₂O₂ or its deprotonated form (12), the peroxide anion (HOO^-), to SOD1-Cu(I). This latter species is subsequently oxidized to a hypervalent intermediate (proposed to be either SOD1-Cu(III), SOD1-Cu(II)-·_OH, or SOD1-Cu=O) (8, 9) that remains to be characterized. The reduction of this hypervalent intermediate by small anions is supposed to close the cycle, leading to the native enzyme and diffusible highly reactive radicals derived from the anionic substrates (6, 10).During its peroxidase cycle, a considerable fraction of SOD1 is inactivated due to the oxidation of the copper-binding histidines to oxohistidine, presumably by the hypervalent intermediate, in a process that can be prevented by the presence of reducing substrates and, in their absence, unavoidably leads to copper loss (26). Although this process is well described for the heme-dependent peroxidase cycle, current literature data (9, 27-30) and the fact that the proposed SOD1-bound hypervalent copper states (Cu(III), Cu(II)=O, and Cu(II)/^._OH) have never been characterized suggested to us that an alternative mechanism may take place, leading to production from and H₂O₂ by the enzyme, a process that does not involve copper oxidation beyond the thermodynamically stable Cu(II) form. In the presence of , a significant fraction of H₂O₂ is promptly converted to through the perhydration of CO₂ (27, 28, 31, 32). The peroxo bond in peroxymonocarbonate can be cleaved by reduced metals to produce and H₂O (30 33) (Reactions 1-5),where Reactions 1 and 2 represent SOD1 reduction, Reactions 3 and 4 represent SOD1 oxidation, and Reaction 5 represents peroxycarbonate formation.Interestingly, studies employing molecular modeling of the SOD1 active site indicate that and H₂O₂ gain access to the SOD1 active site, where they react to produce in close proximity to the copper ion (29). This interaction of with Cu(I) may result in production.On the basis of these new data, we hypothesized that SOD1-Cu(I), which is the predominant form of SOD1 exposed to excess hydrogen peroxide (8, 9), is oxidized back to the native form of the enzyme by more efficiently than by H₂O₂ itself or HOO^-. The latter two oxidations would slowly produce ^.OH radicals (or the equivalent SOD1-Cu(III), SOD1-Cu(II)=O, or Cu(II)/·_OH) in the enzyme active site, leading to the observed inactivation of SOD1 (see Scheme 1). Here we present data that strongly support this hypothesis; they indicate that is a key substrate for reduced SOD1, which mediates SOD1-Cu(I) reoxidation back to the resting SOD1-Cu(II) severalfold faster than H₂O₂ itself and, in doing so, serves as the carbonate radical anion precursor.Open in a separate windowSCHEME 1.Schematic representation of the peroxidase catalytic cycle of Cu,Zn-SOD in the presence of /CO₂. Native Cu,Zn-SOD is reduced by the peroxide anion, which gains access to the copper through the enzyme''s anion channel. Reduced Cu,Zn-SOD is reoxidized by the peroxycarbonate anion (), which is in equilibrium with H₂O₂//CO₂, leading to carbonate radical anion production. Superoxide anion (), the product of HOO^--induced SOD1 reduction, can oxidize SOD1-Cu(I) back to its resting SOD-Cu(II) state at diffusion-limited rates; however, it can alternatively reduce another molecule of SOD1-Cu(II) to SOD1-Cu(I), considerably accelerating the rate of SOD1-Cu(II) reduction by H₂O₂. Whether will act as a reductant of SOD1-Cu(II) or an oxidant of SOD1-Cu(I) will depend on the ratio of SOD1-Cu(II)/SOD1-Cu(I) at a given time, because the rate constants for the reaction of with both SOD1 states are close to the diffusion limits.     

Chemiluminescence Assays of Cu2Zn2 Superoxide Dismutase Mimicking Cu-Complexes

The aqueous decay of K₃CrO₈ was used to compare the reactivity of Cu₂Zn₂ superoxide dismutase and two active centre analogues where the first shell atoms around the copper are four unsaturated nitrogens. Unlike the acetate or biuret type Cu(II) chelates these di-Schiff-base complexes had an identical reactivity compared to that of the intact enzyme. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K₃CrO₈ induced chemiluminescence by 50%.

Furthermore, a lucigenin amplified chemiluminescence assay based on isolated polymorph nuclear leucocytes in the absence and presence of whole, unseparated blood was developed and successfully employed. CuPu(Im), and CuPu(Py), equivalent to 0.5 and 0.8 SOD units. only, were required to inhibit the photon emission by 50% in the absence of bovine serum albumin. Even in the presence of 600 μM albumin mimicking the competitive copper chelation in biological fluids Cu-Pu(Py)₂ and CuPu(lm)₂ remained active, whereas the carboxylate-and biuret type chelates Cu(Sal)₂ and Cu(Ser)₂ reacted like CuSO₄ The same reactivity of these low M, SOD mimics was seen in human blood.     

Purification and Characterization of Cu,Zn Superoxide Dismutase from Ark Shell Scapharca broughtonii

A superoxide dismutase has been purified to apparent homogeneity from the muscular tissue of the ark shell, Scapharca broughtonii, by ammonium sulfate fractionation, and consecutive column chromatographies using DEAE-Sephadex and Sephadex G-100. This enzyme has a molecular weight of 71,700 and is composed of two identical subunits of M _r 35,800, which are joined by noncovalent interactions. The purified enzyme was stable over the range of pH 5.0-10.0 at 4°C for 24 h and at temperatures below 45°C. Cyanide at 0.1 and 1 mM inhibited the activity of the superoxide dismutase 56 and 100%, but 5 mM azide caused 8% inhibition. The optical spectrum of this enzyme had a maximum at 265 nm, and the amino acid composition of the enzyme was similar to that of the other Cu, Zn superoxide dismutases except for the contents of threonine, serine, proline, and leucine. Atomic absorption spectroscopy showed that this enzyme has approximately 2 atoms of Cu²⁺ and Zn²⁺ per mole of enzyme. These results indicate that the purified enzyme from ark shell, Scapharca broughtonii, is a Cu, Zn superoxide dismutase.