Joan Selverstone Valentine

Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis

A subset of individuals with familial amyotrophic lateral sclerosis (FALS) possesses dominantly inherited mutations in the gene that encodes copper-zinc superoxide dismutase (CuZnSOD). A4V and G93A, two of the mutant enzymes associated with FALS, were shown to catalyze the oxidation of a model substrate (spin trap 5,5′-dimethyl-1-pyrroline N-oxide) by hydrogen peroxide at a higher rate than that seen with the wild-type enzyme. Catalysis of this reaction by A4V and G93A was more sensitive to inhibition by the copper chelators diethyldithiocarbamate and penicillamine than was catalysis by wild-type CuZnSOD. The same two chelators reversed the apoptosis-inducing effect of mutant enzymes expressed in a neural cell line. These results suggest that oxidative reactions catalyzed by mutant CuZnSOD enzymes initiate the neuropathologic changes in FALS.

Misfolded CuZnSOD and amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of motor neurons. The inherited form of the disease, familial ALS, represents 5–10% of the total cases, and the best documented of these are due to lesions in SOD1, the gene encoding copper–zinc superoxide dismutase (CuZnSOD). The mechanism by which mutations in SOD1 cause familial ALS is currently unknown. Two hypotheses have dominated recent discussion of the toxicity of ALS mutant CuZnSOD proteins: the oligomerization hypothesis and the oxidative damage hypothesis. The oligomerization hypothesis maintains that mutant CuZnSOD proteins are, or become, misfolded and consequently oligomerize into increasingly high-molecular-weight species that ultimately lead to the death of motor neurons. The oxidative damage hypothesis maintains that ALS mutant CuZnSOD proteins catalyze oxidative reactions that damage substrates critical for viability of the affected cells. This perspective reviews some of the properties of both wild-type and mutant CuZnSOD proteins, suggests how these properties may be relevant to these two hypotheses, and proposes that these two hypotheses are not necessarily mutually exclusive.

Superoxide Dismutase Activity Is Essential for Stationary Phase Survival in Saccharomyces cerevisiae MITOCHONDRIAL PRODUCTION OF TOXIC OXYGEN SPECIES IN VIVO

Yeast lacking copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (SOD), catalase T, or metallothionein were studied using long term stationary phase (10-45 days) as a simple model system to study the roles of antioxidant enzymes in aging. In well aerated cultures, the lack of either SOD resulted in dramatic loss of viability over the first few weeks of culture, with the CuZnSOD mutant showing the more severe defect. The double SOD mutant died within a few days. The severity reversed in low aeration; the CuZnSOD mutant remained viable longer than the manganese SOD mutant. To test whether reactive oxygen species generated during respiration play an important role in the observed cellular death, growth in nonfermentable carbon sources was measured. All strains grew under low aeration, indicating respiratory competence. High aeration caused much reduced growth in single SOD mutants, and the double mutant failed to grow. However, removal of respiration via another mutation dramatically increased short term survival and reversed the known air-dependent methionine and lysine auxotrophies. Our results suggest strongly that mitochondrial respiration is a major source of reactive oxygen species in vivo, as has been shown in vitro, and that these species are produced even under low aeration.

Molecular confinement influences protein structure and enhances thermal protein stability.

The sol‐gel method of encapsulating proteins in a silica matrix was investigated as a potential experimental system for testing the effects of molecular confinement on the structure and stability of proteins. We demonstrate that silica entrapment (1) is fully compatible with structure analysis by circular dichroism, (2) allows conformational studies in contact with solvents that would otherwise promote aggregation in solution, and (3) generally enhances thermal protein stability. Lysozyme, α‐lactalbumin, and metmyoglobin retained native‐like solution structures following sol‐gel encapsulation, but apomyoglobin was found to be largely unfolded within the silica matrix under control buffer conditions. The secondary structure of encapsulated apomyoglobin was unaltered by changes in pH and ionic strength of KCl. Intriguingly, the addition of other neutral salts resulted in an increase in the α‐helical content of encapsulated apomyoglobin in accordance with the Hofmeister ion series. We hypothesize that protein conformation is influenced directly by the properties of confined water in the pores of the silica. Further work is needed to differentiate the steric effects of the silica matrix from the solvent effects of confined water on protein structure and to determine the extent to which this experimental system mimics the effects of crowding and confinement on the function of macromolecules in vivo.

Amyloid-Like Filaments and Water-Filled Nanotubes Formed by Sod1 Mutant Proteins Linked to Familial Als

Mutations in the SOD1 gene cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human FALS patients and in transgenic mice expressing these mutant proteins, aggregates containing FALS SOD1 are observed. Accumulation of SOD1 aggregates is believed to interfere with axonal transport, protein degradation and anti-apoptotic functions of the neuronal cellular machinery. Here we show that metal-deficient, pathogenic SOD1 mutant proteins crystallize in three different crystal forms, all of which reveal higher-order assemblies of aligned β-sheets. Amyloid-like filaments and water-filled nanotubes arise through extensive interactions between loop and β-barrel elements of neighboring mutant SOD1 molecules. In all cases, non-native conformational changes permit a gain of interaction between dimers that leads to higher-order arrays. Normal β-sheet–containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions. Loss of this protection through conformational rearrangement in the metal-deficient enzyme could be a toxic property common to mutants of SOD1 linked to FALS.

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die.

Superoxide dismutases and superoxide reductases.

Superoxide, O2•–, is formed in all living organisms that come in contact with air, and, depending upon its biological context, it may act as a signaling agent, a toxic species, or a harmless intermediate that decomposes spontaneously. Its levels are limited in vivo by two different types of enzymes, superoxide reductase (SOR) and superoxide dismutase (SOD). Although superoxide has long been an important factor in evolution, it was not so when life first emerged on Earth at least 3.5 billion years ago. At that time, the early biosphere was highly reducing and lacking in any significant concentrations of dioxygen (O2), very different from what it is today. Consequently, there was little or no O2•– and therefore no reason for SOR or SOD enzymes to evolve. Instead, the history of biological O2•– probably commences somewhere around 2.4 billion years ago, when the biosphere started to experience what has been termed the “Great Oxidation Event”, a transformation driven by the increase in O2 levels, formed by cyanobacteria as a product of oxygenic photosynthesis.1 The rise of O2 on Earth caused a reshaping of existing metabolic pathways, and it triggered the development of new ones.2 Its appearance led to the formation of the so-called “reactive oxygen species” (ROS), for example, superoxide, hydrogen peroxide, and hydroxyl radical, and to a need for antioxidant enzymes and other antioxidant systems to protect against the growing levels of oxidative damage to living systems. Dioxygen is a powerful four-electron oxidizing agent, and the product of this reduction is water. 1 When O2 is reduced in four sequential one-electron steps, the intermediates formed are the three major ROS, that is, O2•–, H2O2, and HO•. 2 3 4 5 Each of these intermediates is a potent oxidizing agent. The consequences of their presence to early life must have been an enormous evolutionary challenge. In the case of superoxide, we find the SOD and SOR enzymes to be widely distributed throughout current living organisms, both aerobic and anaerobic, suggesting that, from the start of the rise of O2 on Earth, the chemistry of superoxide has been an important factor during evolution. The SORs and three very different types of SOD enzymes are redox-active metalloenzymes that have evolved entirely independently from one another for the purpose of lowering superoxide concentrations. SORs catalyze the one-electron reduction of O2•– to give H2O2, a reaction requiring two protons per superoxide reacted as well as an external reductant to provide the electron (eq 6). SODs catalyze the disproportionation of superoxide to give O2 and H2O2, a reaction requiring one proton per superoxide reacted, but no external reductant (eq 7). 6 7 All of the SOR enzymes contain only iron, while the three types of SODs are the nickel-containing SODs (NiSOD), the iron- or manganese-containing SODs (FeSOD and MnSOD), and the copper- and zinc-containing SODs (CuZnSOD). Although the structures and other properties of these four types of metalloenzymes are quite different, they all share several characteristics, including the ability to react rapidly and selectively with the small anionic substrate O2•–. Consequently, there are some striking similarities between these otherwise dissimilar enzymes, many of which can be explained by considering the nature of the chemical reactivity of O2•– (see below). Numerous valuable reviews describing the SOD and SOR enzymes have appeared over the years, but few have covered and compared all four classes of these enzymes, as we attempt to do here. Thus, the purpose of this Review is to describe, compare, and contrast the properties of the SOR and the four SOD enzymes; to summarize what is known about their evolutionary pathways; and to analyze the properties of these enzymes in light of what is known of the inherent chemical reactivity of superoxide.

Structure and reactivity of a mononuclear non-haem iron( III )–peroxo complex

Oxygen-containing mononuclear iron species—iron(iii)–peroxo, iron(iii)–hydroperoxo and iron(iv)–oxo—are key intermediates in the catalytic activation of dioxygen by iron-containing metalloenzymes. It has been difficult to generate synthetic analogues of these three active iron–oxygen species in identical host complexes, which is necessary to elucidate changes to the structure of the iron centre during catalysis and the factors that control their chemical reactivities with substrates. Here we report the high-resolution crystal structure of a mononuclear non-haem side-on iron(iii)–peroxo complex, [Fe(iii)(TMC)(OO)]+. We also report a series of chemical reactions in which this iron(iii)–peroxo complex is cleanly converted to the iron(iii)–hydroperoxo complex, [Fe(iii)(TMC)(OOH)]2+, via a short-lived intermediate on protonation. This iron(iii)–hydroperoxo complex then cleanly converts to the ferryl complex, [Fe(iv)(TMC)(O)]2+, via homolytic O–O bond cleavage of the iron(iii)–hydroperoxo species. All three of these iron species—the three most biologically relevant iron–oxygen intermediates—have been spectroscopically characterized; we note that they have been obtained using a simple macrocyclic ligand. We have performed relative reactivity studies on these three iron species which reveal that the iron(iii)–hydroperoxo complex is the most reactive of the three in the deformylation of aldehydes and that it has a similar reactivity to the iron(iv)–oxo complex in C–H bond activation of alkylaromatics. These reactivity results demonstrate that iron(iii)–hydroperoxo species are viable oxidants in both nucleophilic and electrophilic reactions by iron-containing enzymes.

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper–zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of ≈650 kDa under physiological conditions, i.e., 37°C, pH 7.0, and 100 μM protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.

The dark side of dioxygen biochemistry

The cellular biochemistry of dioxygen is Janus-faced. The good side includes numerous enzyme-catalyzed reactions of dioxygen that occur in respiration and normal metabolism, while the dark side encompasses deleterious reactions of species derived from dioxygen that lead to damage of cellular components. These reactive oxygen species have historically been perceived almost exclusively as agents of the dark side, but it has recently become clear that they play beneficial roles as well.