Revisiting the reactions of superoxide with glutathione and other thiols

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Abstract

The reaction between GSH and superoxide has long been of interest in the free radical biology. Early studies were confusing, as some reports suggested that the reaction could be a major pathway for superoxide removal whereas others questioned whether it happened at all. Further research by several investigators, including Helmut Sies, was required to clarify this complex reaction. We now know that superoxide does react with GSH, but the reaction is relatively slow and occurs mostly by a chain reaction that consumes oxygen and regenerates superoxide. Most of the GSH is converted to GSSG, with a small amount of sulfonic acid. As shown by Sies and colleagues, singlet oxygen is a by-product. Although removal of superoxide by GSH may be a minor pathway, GSH and superoxide have a strong physiological connection. GSH is an efficient free radical scavenger, and when it does so, thiyl radicals are generated. These further react to generate superoxide. Therefore, radical scavenging by GSH and other thiols is a source of superoxide and hydrogen peroxide, and to be an antioxidant pathway, there must be efficient removal of these species.

Introduction

I first met Helmut Sies at a 1983 EMBO workshop on Oxidative Damage and Related Enzymes, held in Frascati, just outside Rome. I have fond memories of this meeting, partly for the excellent food and ambience of the surrounding countryside, but more particularly as my first introduction to people like Helmut who were pioneering the free radical field. As illustrated in this special issue of ABB, Helmut Sies has made an enormous contribution to redox biology, in fields as diverse as the biological chemistry of carotenoids, glutathione and singlet oxygen, to formulating concepts of oxidative stress and defining what we mean by antioxidant. This article is based on one aspect of Helmut's work that had particular impact on my research, namely the reaction of superoxide with glutathione. But I would first like to mention some of his earliest work, carried out with Britton Chance and Nozomu Oshino in the early 1970s [1], [2], [3], in which they used the spectral characteristics of the redox states of catalase to show that hydrogen peroxide is produced during normal metabolism. Using this technology, they were able to estimate H2O2 production rates in liver tissue, and steady state concentrations in the nanomolar range. These data remain some of the best quantitative information available and are still widely quoted. However, I believe that the methodology used by Sies and colleagues has been underutilized and may have a place in today's efforts to clarify cellular functions of H2O2. The development of fluorescent probes and their use in live cell imaging has led to considerable advances in our understanding of redox metabolism. But powerful as these techniques are, quantification remains challenging. Measuring the redox state of catalase in cells or tissues may well provide complementary information.

The focus of this article relates to a 1983 paper by Sies and Heribert Wefers on the reaction of superoxide with GSH in which they showed that one of the products is singlet oxygen [4]. At that time there was considerable interest in identifying the biological molecules that react with superoxide radicals and establishing which of these reactions contribute to or protect against superoxide toxicity. We were interested in GSH as a free radical scavenger, and the fate of the glutathionyl radicals (GSradical dot) that are produced in the scavenging reaction. As discussed below, these go on to generate superoxide, so it was important to understand the fate of superoxide in such systems. This article describes what we now know about superoxide thiol interactions and how this relates to Sies's earlier findings.

Section snippets

GSH oxidation by superoxide

When we started our investigation [5], several investigators including Wefers and Sies had already shown that superoxide reacts with GSH. However, there was considerable disagreement about whether the reaction was fast enough for GSH to be a major physiological target or was much slower and would therefore be insignificant in cells containing superoxide dismutase (SOD). Reported rate constants ranged from >105 M−1s−1 (which supported the former conclusion) to 15 M−1s−1 (which supported the

Mechanistic considerations

Although the main reactions involved in thiol oxidation by superoxide are shown in Scheme 1, there have been no detailed mechanistic studies on how the contributions of the different pathways vary under different conditions. There is some uncertainty about the origin of the thiyl radical. The most obvious mechanism is via direct electron (or hydrogen) transfer from GSH to superoxide (reaction 4). However, this reaction produces H2O2 and experimental studies with GSH, dithiothreitol and

Superoxide production during radical scavenging by GSH

Superoxide is also an important participant in GSH biochemistry when GSH functions as a free radical scavenger. GSH scavenges a wide range of biologically relevant radicals. GSradical dot is produced (reaction 5) and this is followed by the reactions described above that result in superoxide production. There numerous reported studies showing that radical scavenging by GSH produces superoxide and (via dismutation) hydrogen peroxide (for example [22], [23], [24], [25], [26]). Furthermore, when the initial

Conclusions

The reaction of superoxide with thiols has been investigated in a number of laboratories, including Helmut Sies's study on product characterization. We now know that GSH and other thiols are oxidized by superoxide, but the reaction is relatively slow and may be significant physiologically only where little SOD is present. Therefore, it may be less relevant intracellularly than in compartments such as phagosomes or endosomes (redoxosomes) [28], [29] where NADPH oxidase activity generates large

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