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1.
Molecules ; 27(5)2022 Mar 03.
Article in English | MEDLINE | ID: mdl-35268761

ABSTRACT

Soluble Mn(III)-L complexes appear to constitute a substantial portion of manganese (Mn) in many environments and serve as critical high-potential species for biogeochemical processes. However, the inherent reactivity and lability of these complexes-the same chemical characteristics that make them uniquely important in biogeochemistry-also make them incredibly difficult to measure. Here we present experimental results demonstrating the limits of common analytical methods used to quantify these complexes. The leucoberbelin-blue method is extremely useful for detecting many high-valent Mn species, but it is incompatible with the subset of Mn(III) complexes that rapidly decompose under low-pH conditions-a methodological requirement for the assay. The Cd-porphyrin method works well for measuring Mn(II) species, but it does not work for measuring Mn(III) species, because additional chemistry occurs that is inconsistent with the proposed reaction mechanism. In both cases, the behavior of Mn(III) species in these methods ultimately stems from inter- and intramolecular redox chemistry that curtails the use of these approaches as a reflection of ligand-binding strength. With growing appreciation for the importance of high-valent Mn species and their cycling in the environment, these results underscore the need for additional method development to enable quantifying such species rapidly and accurately in nature.

2.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Article in English | MEDLINE | ID: mdl-34161271

ABSTRACT

Desert varnish is a dark rock coating that forms in arid environments worldwide. It is highly and selectively enriched in manganese, the mechanism for which has been a long-standing geological mystery. We collected varnish samples from diverse sites across the western United States, examined them in petrographic thin section using microscale chemical imaging techniques, and investigated the associated microbial communities using 16S amplicon and shotgun metagenomic DNA sequencing. Our analyses described a material governed by sunlight, water, and manganese redox cycling that hosts an unusually aerobic microbial ecosystem characterized by a remarkable abundance of photosynthetic Cyanobacteria in the genus Chroococcidiopsis as the major autotrophic constituent. We then showed that diverse Cyanobacteria, including the relevant Chroococcidiopsis taxon, accumulate extraordinary amounts of intracellular manganese-over two orders of magnitude higher manganese content than other cells. The speciation of this manganese determined by advanced paramagnetic resonance techniques suggested that the Cyanobacteria use it as a catalytic antioxidant-a valuable adaptation for coping with the substantial oxidative stress present in this environment. Taken together, these results indicated that the manganese enrichment in varnish is related to its specific uptake and use by likely founding members of varnish microbial communities.


Subject(s)
Ecological and Environmental Phenomena , Geologic Sediments/chemistry , Manganese/analysis , Antioxidants/metabolism , Cyanobacteria/metabolism , Geologic Sediments/microbiology , Microbiota , Oxidation-Reduction , Sunlight , Water
3.
Front Neurosci ; 14: 619279, 2020.
Article in English | MEDLINE | ID: mdl-33679289

ABSTRACT

Wild-type human SOD1 forms a highly conserved intra-molecular disulfide bond between C57-C146, and in its native state is greatly stabilized by binding one copper and one zinc atom per monomer rendering the protein dimeric. Loss of copper extinguishes dismutase activity and destabilizes the protein, increasing accessibility of the disulfide with monomerization accompanying disulfide reduction. A further pair of free thiols exist at C6 and C111 distant from metal binding sites, raising the question of their function. Here we investigate their role in misfolding of SOD1 along a pathway that leads to formation of amyloid fibrils. We present the seeding reaction of a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) to exclude variables caused by these free cysteines. Completely reduced fibril seeds decreasing the kinetic barrier to cleave the highly conserved intramolecular disulfide bond, and accelerating SOD1 reduction and initiation of fibrillation. Presence or absence of the pair of free thiols affects kinetics of fibrillation. Previously, we showed full maturation with both Cu and Zn prevents this behavior while lack of Cu renders sensitivity to fibrillation, with presence of the native disulfide bond modulating this propensity much more strongly than presence of Zn or dimerization. Here we further investigate the role of reduction of the native C57-C146 disulfide bond in fibrillation of wild-type hSOD1, firstly through removal of free thiols by paired mutations C6A, C111S (AS-SOD1), and secondly in seeded fibrillation reactions modulated by reductant tris (2-carboxyethyl) phosphine (TCEP). Fibrillation of AS-SOD1 was dependent upon disulfide reduction and showed classic lag and exponential growth phases compared with wild-type hSOD1 whose fibrillation trajectories were typically somewhat perturbed. Electron microscopy showed that AS-SOD1 formed classic fibrils while wild-type fibrillation reactions showed the presence of smaller "sausage-like" oligomers in addition to fibrils, highlighting the potential for mixed disulfides involving C6/C111 to disrupt efficient fibrillation. Seeding by addition of sonicated fibrils lowered the TCEP concentration needed for fibrillation in both wild-type and AS-SOD1 providing evidence for template-driven structural disturbance that elevated susceptibility to reduction and thus propensity to fibrillate.

4.
Free Radic Biol Med ; 140: 1-3, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31344436

ABSTRACT

Looking across our planet's four-and-a-half billion-year history, the rise of dioxygen-an interval sometimes called the Great Oxygenation Event (GOE)-is arguably the most important environmental change. This revolution occurred approximately 2.3 billion years ago, roughly at the mid-way point in Earth history, and it was ultimately driven by a biological innovation: the evolution of oxygenic photosynthesis. The evolution of oxygenic photosynthesis conferred the ability to use water as a photosynthetic substrate (earlier photosynthesis was anoxygenic and required reduced iron, sulfur, carbon, or hydrogen). Primary productivity-no longer limited by a source of electrons-greatly expanded across the Earth surface. In turn, dioxygen accumulated and became widely available for use in anabolic and catabolic metabolisms, forming a rich cascade of evolutionary potential and consequence. The modern biosphere figured out how to balance harmful oxidative stress with the beneficial ways dioxygen can be used. But how did life come to first tolerate and then thrive in an oxygenated world? It's this question that attracted the diverse perspectives reflected in this special issue.


Subject(s)
Biological Evolution , Oxygen/metabolism , Photosynthesis , Carbon/metabolism , Electrons , Humans , Hydrogen/metabolism , Iron/metabolism , Sulfur/metabolism
5.
Free Radic Biol Med ; 140: 113-125, 2019 08 20.
Article in English | MEDLINE | ID: mdl-30738765

ABSTRACT

Throughout the history of life on Earth, abiotic components of the environment have shaped the evolution of life, and in turn life has shaped the environment. The element manganese embodies a special aspect of this collaboration; its history is closely entwined with those of photosynthesis and O2-two reigning features that characterize the biosphere today. Manganese chemistry was central to the environmental context and evolutionary innovations that enabled the origin of oxygenic photosynthesis and the ensuing rise of O2. It was also manganese chemistry that provided an early, fortuitous antioxidant system that was instrumental in how life came to cope with oxidative stress and ultimately thrive in an aerobic world. Subsequently, the presence of O2 transformed the biogeochemical dynamics of the manganese cycle, enabling a rich suite of environmental and biological processes involving high-valent manganese and manganese redox cycling. Here, we describe insights from chemistry, biology, and geology, to examine manganese dynamics in the environment, and its unique role in the history of life.


Subject(s)
Biological Evolution , Earth, Planet , Manganese/metabolism , Oxygen/metabolism , Manganese/chemistry , Oxidation-Reduction , Oxygen/chemistry , Photosynthesis
6.
J Am Soc Mass Spectrom ; 30(2): 218-226, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30328005

ABSTRACT

Solvent-accessibility change plays a critical role in protein misfolding and aggregation, the culprit for several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mass spectrometry-based hydroxyl radical (·OH) protein footprinting has evolved as a powerful and fast tool in elucidating protein solvent accessibility. In this work, we used fast photochemical oxidation of protein (FPOP) hydroxyl radical (·OH) footprinting to investigate solvent accessibility in human copper-zinc superoxide dismutase (SOD1), misfolded or aggregated forms of which underlie a portion of ALS cases. ·OH-mediated modifications to 56 residues were detected with locations largely as predicted based on X-ray crystallography data, while the interior of SOD1 ß-barrel is hydrophobic and solvent-inaccessible and thus protected from modification. There were, however, two notable exceptions-two closely located residues inside the ß-barrel, predicted to have minimal or no solvent accessibility, that were found modified by FPOP (Phe20 and Ile112). Molecular dynamics (MD) simulations were consistent with differential access of peroxide versus quencher to SOD1's interior complicating surface accessibility considerations. Modification of these two residues could potentially be explained either by local motions of the ß-barrel that increased peroxide/solvent accessibility to the interior or by oxidative events within the interior that might include long-distance radical transfer to buried sites. Overall, comparison of modification patterns for the metal-free apoprotein versus zinc-bound forms demonstrated that binding of zinc protected the electrostatic loop and organized the copper-binding site. Our study highlights SOD1 hydrophobic groups that may contribute to early events in aggregation and discusses caveats to surface accessibility conclusions. Graphical Abstract.


Subject(s)
Hydroxyl Radical/chemistry , Protein Footprinting/methods , Solvents/chemistry , Superoxide Dismutase-1/chemistry , Glutamine/chemistry , Hydrogen Peroxide/chemistry , Molecular Dynamics Simulation , Oxidation-Reduction , Protein Conformation , Spectrometry, Mass, Electrospray Ionization , Static Electricity , Superoxide Dismutase-1/analysis , Superoxide Dismutase-1/metabolism , Tandem Mass Spectrometry , Zinc/metabolism
7.
J Am Chem Soc ; 139(32): 10960-10963, 2017 08 16.
Article in English | MEDLINE | ID: mdl-28758392

ABSTRACT

A mononuclear side-on peroxocobalt(III) complex with a tetradentate macrocyclic ligand, [CoIII(TBDAP)(O2)]+ (1), shows a novel and facile mode of dioxygenase-like reactivity with nitriles (R-C≡N; R = Me, Et, and Ph) to produce the corresponding mononuclear hydroximatocobalt(III) complexes, [CoIII(TBDAP)(R-C(═NO)O)]+, in which the nitrile moiety is oxidized by two oxygen atoms of the peroxo group. The overall reaction proceeds in one-pot under ambient conditions (ca. 1 h, 40 °C). 18O-Labeling experiments confirm that both oxygen atoms are derived from the peroxo ligand. The structures of all products, hydroximatocobalt(III) complexes, were confirmed by X-ray crystallography and various spectroscopic techniques. Kinetic studies including the Hammett analysis and isotope labeling experiments suggest that the mechanistic mode of 1 for activation of nitriles occurs via a concerted mechanism. This novel reaction would be significantly valuable for expanding the chemistry for nitrile activation and utilization.

8.
J Neurochem ; 140(1): 140-150, 2017 01.
Article in English | MEDLINE | ID: mdl-27727458

ABSTRACT

A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In this study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time stamp that distinguishes pre-existing and newly made proteins. In cells that transiently over-express the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu co-factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly made protein, are prone to misfolding and aggregation.


Subject(s)
Inclusion Bodies/enzymology , Inclusion Bodies/genetics , Mutation/physiology , Superoxide Dismutase-1/biosynthesis , Superoxide Dismutase-1/genetics , Animals , CHO Cells , Cricetinae , Cricetulus , HEK293 Cells , Humans , Protein Aggregates/physiology , Protein Folding
9.
Curr Opin Chem Biol ; 31: 166-78, 2016 04.
Article in English | MEDLINE | ID: mdl-27043270

ABSTRACT

Life on Earth originated and evolved in anoxic environments. Around 2.4 billion-years-ago, ancestors of Cyanobacteria invented oxygenic photosynthesis, producing substantial amounts of O2 as a byproduct of phototrophic water oxidation. The sudden appearance of O2 would have led to significant oxidative stress due to incompatibilities with core cellular biochemical processes. Here we examine this problem through the lens of Cyanobacteria-the first taxa to observe significant fluxes of intracellular dioxygen. These early oxygenic organisms likely adapted to the oxidative stress by co-opting preexisting systems (exaptation) with fortuitous antioxidant properties. Over time more advanced antioxidant systems evolved, allowing Cyanobacteria to adapt to an aerobic lifestyle and become the most important environmental engineers in Earth history.


Subject(s)
Earth, Planet , Oxygen/chemistry , Antioxidants/chemistry , Cyanobacteria/metabolism , Geology , Reactive Oxygen Species/chemistry , Reactive Oxygen Species/metabolism
10.
J Biol Chem ; 290(51): 30624-36, 2015 Dec 18.
Article in English | MEDLINE | ID: mdl-26511321

ABSTRACT

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of post-translational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57-Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.


Subject(s)
Amyloid/chemistry , Amyotrophic Lateral Sclerosis/enzymology , Disulfides/chemistry , Protein Multimerization , Superoxide Dismutase/chemistry , Zinc/chemistry , Amyloid/genetics , Amyloid/metabolism , Amyotrophic Lateral Sclerosis/genetics , Disulfides/metabolism , Enzyme Stability/genetics , Humans , Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Zinc/metabolism
11.
J Biol Chem ; 290(4): 2405-18, 2015 Jan 23.
Article in English | MEDLINE | ID: mdl-25433341

ABSTRACT

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.


Subject(s)
Mutant Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Superoxide Dismutase/chemistry , Amyotrophic Lateral Sclerosis/genetics , Apoproteins/chemistry , Calorimetry, Differential Scanning , Disulfides/chemistry , Electron Spin Resonance Spectroscopy , Humans , Mass Spectrometry , Metals/chemistry , Mutation , Oxidative Stress , Protein Binding , Protein Conformation , Saccharomyces cerevisiae/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry , Superoxides/chemistry , Zinc/chemistry
13.
J Biol Inorg Chem ; 19(4-5): 647-57, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24692094

ABSTRACT

The dimeric Cu-Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu2Co2SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co(2+), Ni(2+) is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu2Ni2SOD1 and acting as a very good mimic of the native Cu ion in Ni2Zn2SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini's paramagnetic NMR pursuits of metalloproteins. We report Ni(2+) binding to apo wild-type SOD1 and a time-dependent Ni(2+) migration from the Zn site to the Cu site, and the preparation and characterization of Ni2Ni2SOD1, which shows coordination properties similar to those of Cu2Cu2SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , Copper/chemistry , Copper/metabolism , Magnetic Resonance Spectroscopy , Protein Folding , Superoxides/metabolism , Zinc/chemistry , Zinc/metabolism
14.
J Biol Inorg Chem ; 18(8): 985-92, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24061560

ABSTRACT

Copper-zinc superoxide dismutase (Sod1) is an abundant intracellular enzyme that catalyzes the disproportionation of superoxide to give hydrogen peroxide and dioxygen. In most organisms, Sod1 acquires copper by a combination of two pathways, one dependent on the copper chaperone for Sod1 (CCS), and the other independent of CCS. Examples have been reported of two exceptions: Saccharomyces cerevisiae, in which Sod1 appeared to be fully dependent on CCS, and Caenorhabditis elegans, in which Sod1 was completely independent of CCS. Here, however, using overexpressed Sod1, we show there is also a significant amount of CCS-independent activation of S. cerevisiae Sod1, even in low-copper medium. In addition, we show CCS-independent oxidation of the disulfide bond in S. cerevisiae Sod1. There appears to be a continuum between CCS-dependent and CCS-independent activation of Sod1, with yeast falling near but not at the CCS-dependent end.


Subject(s)
Copper/metabolism , Enzyme Activation , Saccharomyces cerevisiae/enzymology , Superoxide Dismutase/metabolism , Oxidation-Reduction , Saccharomyces cerevisiae/metabolism , Superoxide Dismutase-1 , Zinc/metabolism
15.
Proc Natl Acad Sci U S A ; 110(27): 10934-9, 2013 Jul 02.
Article in English | MEDLINE | ID: mdl-23781106

ABSTRACT

Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALS-mutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.


Subject(s)
Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/genetics , Mutant Proteins/chemistry , Mutant Proteins/genetics , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Amino Acid Sequence , Amino Acid Substitution/genetics , Amyloid/chemistry , Amyloid/genetics , Amyloid/ultrastructure , Humans , Microscopy, Atomic Force , Models, Molecular , Molecular Sequence Data , Mutant Proteins/ultrastructure , Proteolysis , Superoxide Dismutase/ultrastructure , Superoxide Dismutase-1
16.
PLoS One ; 8(5): e62446, 2013.
Article in English | MEDLINE | ID: mdl-23667478

ABSTRACT

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.


Subject(s)
Protein Multimerization , Superoxide Dismutase/chemistry , Amino Acid Sequence , Candida albicans/cytology , Cytosol/enzymology , Enzyme Activation , Enzyme Stability , Hot Temperature , Mitochondria/enzymology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Denaturation , Protein Structure, Quaternary , Saccharomyces cerevisiae/cytology , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism
17.
Proc Natl Acad Sci U S A ; 109(36): 14314-9, 2012 Sep 04.
Article in English | MEDLINE | ID: mdl-22908245

ABSTRACT

Reduction of superoxide (O2-) by manganese-containing superoxide dismutase occurs through either a "prompt protonation" pathway, or an "inner-sphere" pathway, with the latter leading to formation of an observable Mn-peroxo complex. We recently reported that wild-type (WT) manganese superoxide dismutases (MnSODs) from Saccharomyces cerevisiae and Candida albicans are more gated toward the "prompt protonation" pathway than human and bacterial MnSODs and suggested that this could result from small structural changes in the second coordination sphere of manganese. We report here that substitution of a second-sphere residue, Tyr34, by phenylalanine (Y34F) causes the MnSOD from S. cerevisiae to react exclusively through the "inner-sphere" pathway. At neutral pH, we have a surprising observation that protonation of the Mn-peroxo complex in the mutant yeast enzyme occurs through a fast pathway, leading to a putative six-coordinate Mn(3+) species, which actively oxidizes O2- in the catalytic cycle. Upon increasing pH, the fast pathway is gradually replaced by a slow proton-transfer pathway, leading to the well-characterized five-coordinate Mn(3+). We here propose and compare two hypothetical mechanisms for the mutant yeast enzyme, differing in the structure of the Mn-peroxo complex yet both involving formation of the active six-coordinate Mn(3+) and proton transfer from a second-sphere water molecule, which has substituted for the -OH of Tyr34, to the Mn-peroxo complex. Because WT and the mutant yeast MnSOD both rest in the 2+ state and become six-coordinate when oxidized up from Mn(2+), six-coordinate Mn(3+) species could also actively function in the mechanism of WT yeast MnSODs.


Subject(s)
Candida albicans/enzymology , Manganese/metabolism , Models, Molecular , Saccharomyces cerevisiae/enzymology , Superoxide Dismutase/metabolism , Amino Acid Substitution/genetics , Catalysis , Catalytic Domain , Crystallography , Oxidation-Reduction , Oxygen/metabolism , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics
18.
Proc Natl Acad Sci U S A ; 109(18): 6892-7, 2012 May 01.
Article in English | MEDLINE | ID: mdl-22505740

ABSTRACT

Nonenzymatic manganese was first shown to provide protection against superoxide toxicity in vivo in 1981, but the chemical mechanism responsible for this protection subsequently became controversial due to conflicting reports concerning the ability of Mn to catalyze superoxide disproportionation in vitro. In a recent communication, we reported that low concentrations of a simple Mn phosphate salt under physiologically relevant conditions will indeed catalyze superoxide disproportionation in vitro. We report now that two of the four Mn complexes that are expected to be most abundant in vivo, Mn phosphate and Mn carbonate, can catalyze superoxide disproportionation at physiologically relevant concentrations and pH, whereas Mn pyrophosphate and citrate complexes cannot. Additionally, the chemical mechanisms of these reactions have been studied in detail, and the rates of reactions of the catalytic removal of superoxide by Mn phosphate and carbonate have been modeled. Physiologically relevant concentrations of these compounds were found to be sufficient to mimic an effective concentration of enzymatic superoxide dismutase found in vivo. This mechanism provides a likely explanation as to how Mn combats superoxide stress in cellular systems.


Subject(s)
Manganese/pharmacology , Superoxides/antagonists & inhibitors , Antioxidants/metabolism , Antioxidants/pharmacology , Carbonates/metabolism , Carbonates/pharmacology , Catalysis , In Vitro Techniques , Kinetics , Ligands , Manganese/metabolism , Models, Biological , Organometallic Compounds/metabolism , Organometallic Compounds/pharmacology , Oxidative Stress/drug effects , Superoxide Dismutase/metabolism , Superoxides/metabolism
19.
Curr Top Med Chem ; 12(22): 2560-72, 2012.
Article in English | MEDLINE | ID: mdl-23339308

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the death of motor neurons. About 10% of ALS cases are inherited (familial), and a large subset of them are caused by mutations in the gene encoding the copper-zinc superoxide dismutase (SOD1). The detection of SOD1-positive inclusions in familial ALS patients suggests the role of SOD1 aggregation underlying the pathology of familial ALS. Although SOD1 mutant proteins are different in structure, stability and activity, they all exhibit a higher aggregation propensity than wild-type SOD1. We here review the recent studies on the role of metallation states and disulfide status in the unfolding, misfolding, and aggregation of SOD1. Investigations of the mechanism of SOD1 aggregation enhance our understanding of onset and progression of ALS and have implications for therapeutic approaches for treating ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Disulfides/metabolism , Superoxide Dismutase/metabolism , Amyotrophic Lateral Sclerosis/genetics , Animals , Humans , Mutation , Protein Folding , Protein Processing, Post-Translational , Superoxide Dismutase/chemistry , Superoxide Dismutase/genetics , Superoxide Dismutase-1
20.
J Am Chem Soc ; 133(51): 20878-89, 2011 Dec 28.
Article in English | MEDLINE | ID: mdl-22077216

ABSTRACT

Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O(2)(-)). This behavior limits the amount of H(2)O(2) produced at high [O(2)(-)]; its desirability can be explained by the multiple roles of H(2)O(2) in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O(2)(-)], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn(3+) species in yeast Mn(3+)SODs, including the well-characterized 5-coordinate Mn(3+) species and a 6-coordinate L-Mn(3+) species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O(2)(-)].


Subject(s)
Candida albicans/enzymology , Saccharomyces cerevisiae/enzymology , Superoxide Dismutase/metabolism , Candida albicans/chemistry , Candida albicans/metabolism , Catalytic Domain , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy , Kinetics , Models, Molecular , Oxidation-Reduction , Protein Multimerization , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Superoxide Dismutase/chemistry
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