Isabel A. Abreu

Superoxide dismutases—a review of the metal-associated mechanistic variations

Superoxide dismutases are enzymes that function to catalytically convert superoxide radical to oxygen and hydrogen peroxide. These enzymes carry out catalysis at near diffusion controlled rate constants via a general mechanism that involves the sequential reduction and oxidation of the metal center, with the concomitant oxidation and reduction of superoxide radicals. That the catalytically active metal can be copper, iron, manganese or, recently, nickel is one of the fascinating features of this class of enzymes. In this review, we describe these enzymes in terms of the details of their catalytic properties, with an emphasis on the mechanistic differences between the enzymes. The focus here will be concentrated mainly on two of these enzymes, copper, zinc superoxide dismutase and manganese superoxide dismutase, and some relatively subtle variations in the mechanisms by which they function.

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.

Recent Updates on Salinity Stress in Rice: From Physiological to Molecular Responses

One-fifth of irrigated agriculture is negatively affected by high soil salinity. The expected population growth, over 9 billion by 2050, enhances the pressure for agricultural production in marginal saline lands. Rice (Oryza sativa L.), the staple food for more than half of the worlds population, is the most salt-sensitive cereal. The need for salt-tolerant rice varieties able to cope with several other stress conditions obviously puts a lot of pressure on breeders who must better comprehend the physiology and genetic control of salt tolerance. In spite of several good reviews recently published, an integrated vision of current information on rice tolerance to salt stress has been lacking. Here we present the most recent data on the salinity effect on rice physiology and stress adaptation, including implications on growth regulation and reproductive development. We have included an inventory of salt tolerance donors available for breeding programs and a comprehensive survey of current work on QTL detection and cloning as well as marker-assisted selection to introgress favorable alleles into elite rice lines. A schematic view of the rice chromosomes on which salt tolerance QTLs and candidate genes are positioned is also included. Finally, we focus on the most promising candidate genes involved in salt stress response. There, we discuss the available knowledge on salt stress signaling and ion homeostasis, LEAs and other stress-induced proteins, genes with unknown function and transcription regulators as well as the present knowledge on the role of post-translational modifications on the modulation of the response to salinity in rice. We conclude by highlighting still missing clues that could help to design better salt tolerant varieties, and we evaluate the significance of the data presented for the future of rice breeding and sustainability of the culture in marginal saline soils.

New allelic variants found in key rice salt-tolerance genes: an association study.

Salt stress is a complex physiological trait affecting plants by limiting growth and productivity. Rice, one of the most important food crops, is rated as salt-sensitive. High-throughput screening methods are required to exploit novel sources of genetic variation in rice and further improve salinity tolerance in breeding programmes. To search for genotypic differences related to salt stress, we genotyped 392 rice accessions by EcoTILLING. We targeted five key salt-related genes involved in mechanisms such as Na(+) /K(+) ratio equilibrium, signalling cascade and stress protection, and we found 40 new allelic variants in coding sequences. By performing association analyses using both general and mixed linear models, we identified 11 significant SNPs related to salinity. We further evaluated the putative consequences of these SNPs at the protein level using bioinformatic tools. Amongst the five nonsynonymous SNPs significantly associated with salt-stress traits, we found a T67K mutation that may cause the destabilization of one transmembrane domain in OsHKT1;5, and a P140A alteration that significantly increases the probability of OsHKT1;5 phosphorylation. The K24E mutation can putatively affect SalT interaction with other proteins thus impacting its function. Our results have uncovered allelic variants affecting salinity tolerance that may be important in breeding.

Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by Neelaredoxin

Archaeoglobus fulgidus is a hyperthermophilic sulphate‐reducing archaeon. It has an optimum growth temperature of 83°C and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O2·−) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O2·− scavenger in A. fulgidus, by studying both the wild‐type and recombinant proteins. Nlr is a 125‐amino‐acid blue‐coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium‐stained gel assays of cell‐soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O2·−. Furthermore, it is shown that Nlr is able to both reduce and dismutate O2·−, thus having a bifunctional reactivity towards O2·−. Kinetic and spectroscopic studies indicate that Nlrs superoxide reductase activity may allow the cell to eliminate O2·− quickly in a NAD(P)H‐dependent pathway. On the other hand, Nlrs superoxide dismutation activity will allow the cell to detoxify O2·− independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg−1 by the xanthine/xanthine oxidase assay at 25°C. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83°C, the rate constant is 5 × 106 M−1 s−1. Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.

Coping with abiotic stress: Proteome changes for crop improvement☆

Plant breeders need new and more precise tools to accelerate breeding programs that address the increasing needs for food, feed, energy and raw materials, while facing a changing environment in which high salinity and drought have major impacts on crop losses worldwide. This review covers the achievements and bottlenecks in the identification and validation of proteins with relevance in abiotic stress tolerance, also mentioning the unexpected consequences of the stress in allergen expression. While addressing the key pathways regulating abiotic stress plant adaptation, comprehensive data is presented on the proteins confirmed as relevant to confer tolerance. Promising candidates still to be confirmed are also highlighted, as well as the specific protein families and protein modifications for which detection and characterization is still a challenge. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Kinetics of ultra-fast excited state proton transfer from 7-hydroxy-4-methylflavylium chloride to water

Abstract Excited state proton transfer from 7-hydroxy-4-methylflavylium chloride to water is reported. From a modified analysis of picosecond time-resolved fluorescence data (not using the lifetime of a parent compound), all rate constants were determined: the deprotonation rate constant of the flavylium cation, k d =1.4×10 11 s −1 , the protonation rate constant of the base form, k p =2.3×10 10 l mol −1 s −1 and the reciprocal fluorescence lifetimes of these species, k AH + =7.8×10 9 s −1 ( τ AH + =128 ps) and k A =7.6×10 9 s −1 ( τ A =132 ps), in water, at 20°C. The value of k d is the largest measured value for an intermolecular proton transfer (to water).

The Mechanism of Superoxide Scavenging by Archaeoglobus fulgidus Neelaredoxin

Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens. It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein reduces superoxide very efficiently (k = 1.5 × 109 m −1s−1), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted ∼50 nm from the absorption of the resting ferric state. The role of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.

Superoxide reduction by Archaeoglobus fulgidus desulfoferrodoxin: comparison with neelaredoxin

Superoxide reductases (SORs) are non-heme iron-containing enzymes that remove superoxide by reducing it to hydrogen peroxide. The active center of SORs consists of a ferrous ion coordinated by four histidines and one cysteine in a square-pyramidal geometry. In the 2Fe-SOR, a distinct family of SORs, there is an additional desulforedoxin-like site that does not appear to be involved in SOR activity. Our previous studies on recombinant Archaeoglobus fulgidus neelaredoxin (1Fe-SOR) have shown that the reaction with superoxide involves the formation of a transient ferric form that, upon protonation, decays to yield an Fe3+–OH species, followed by binding of glutamate to the ferric ion via replacement of hydroxide (Rodrigues et al. in Biochemistry 45:9266–9278, 2006). Here, we report the characterization of recombinant desulfoferrodoxin from the same organism, which is a member of the 2Fe-SOR family, and show that the steps involved in the superoxide reduction are similar in both families of SOR. The electron donation to the SOR from its redox partner, rubredoxin, is also presented here.

Trinucleotide Repeats: A Structural Perspective

Trinucleotide repeat (TNR) expansions are present in a wide range of genes involved in several neurological disorders, being directly involved in the molecular mechanisms underlying pathogenesis through modulation of gene expression and/or the function of the RNA or protein it encodes. Structural and functional information on the role of TNR sequences in RNA and protein is crucial to understand the effect of TNR expansions in neurodegeneration. Therefore, this review intends to provide to the reader a structural and functional view of TNR and encoded homopeptide expansions, with a particular emphasis on polyQ expansions and its role at inducing the self-assembly, aggregation and functional alterations of the carrier protein, which culminates in neuronal toxicity and cell death. Detail will be given to the Machado-Joseph Disease-causative and polyQ-containing protein, ataxin-3, providing clues for the impact of polyQ expansion and its flanking regions in the modulation of ataxin-3 molecular interactions, function, and aggregation.