Chapter 1 Oxidation of Proteins in Plants—Mechanisms and Consequences

Lee J. Sweetlove*1 (email: Lee.sweetlove@plants.ox.ac.uk) and Ian M. Møller†

*Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom †Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Forsøgsvej 1, DK-4200 Slagelse, Denmark 1Corresponding author: Abstract The production of reactive oxygen and reactive nitrogen species in plant cells can lead to a variety of modifications of proteins through oxidation of amino acid side groups. The widespread occurrence of such modifications is becoming appreciated as new proteomic approaches allow their systematic identification. Oxidized amino acid residues can be identified directly by mass spectrometry if the modification is stable, but it is more common to covalently tag the oxidized group by reaction with a marker molecule. The marker molecule generally allows visualization through immuno-detection and isolation of modified proteins by affinity purification. Although there are several technical caveats with such approaches, they have been useful in documenting the extent of oxidative modification of proteins and have highlighted a number of proteins where oxidative modification is critical for protein function. A view that such modifications could have signalling ramifications is emerging. However, in many cases there is a lack of information as to the effect of oxidation on protein activity or function. Severe protein oxidation is costly to the cell since oxidatively damaged proteins need to be degraded by specific proteases or damaged cellular components recycled via the autophagy pathway. Avoiding this cost is clearly advantageous, and it has been proposed that proteins may have an over-representation of easily oxidizable amino acids on their surface to act as decoy or sacrificial residues, thus preventing or postponing oxidation of residues more important for the function of the protein.

I. Introduction

Oxidative stress occurs when the rate of production of reactive oxygen (ROS) and/or reactive nitrogen species (RNS) is greater than the capacity of the cell's antioxidant defences to detoxify them. As a consequence, the extent to which ROS and RNS oxidize key cellular macromolecules is dramatically increased. Some of these oxidation events may prevent the normal functioning of the target macromolecules, and the resulting change in cellular homeostasis is referred to as oxidative stress. Along with lipids, proteins are the key class of macromolecules in the cell that can be oxidized in a way that contributes to cellular oxidative stress. The aim of this chapter is to review recent advances in our understanding of the process of protein oxidation. We will briefly introduce the production of ROS and RNS, summarize the main mechanisms by which proteins can be oxidized, before reviewing recent work providing an overview of the proteins that are targets of oxidation, and the importance of specific oxidation events in the regulation of cellular processes. We will also consider the cost of protein oxidation and describe our current understanding of the mechanisms by which oxidized proteins are processed and removed.

II. The Formation of ROS and RNS

The biochemistry of formation of ROS and RNS has been extensively reviewed and we do not wish to reproduce this exhaustive information here. Our aim in this section is to briefly outline the main points and provide the context for the rest of the chapter. For a more detailed account of the chemistry and biochemistry of ROS and RNS formation, the reader should consult Halliwell and Gutteridge's excellent textbook (Halliwell and Gutteridge, 2007). In addition, there are numerous relevant plant-specific reviews that we wholeheartedly recommend (Apel and Hirt, 2004, del Rio et al., 2006, Delledonne, 2005, Halliwell, 2006, Moller, 2001, Møller et al., 2007, Noctor and Foyer, 1998, Noctor et al., 2007 and Rinalducci et al., 2008). ROS are produced either by partial, single-electron reduction of oxygen to generate superoxide, hydrogen peroxide and hydroxyl radicals or by alteration of oxygen electron spin states by photoactivation to generate singlet oxygen (Fig. 1). The latter happens exclusively in the chloroplast via a photosensitization reaction involving photosystem II. In the light, the chloroplast is also the main source of superoxide (Foyer and Noctor, 2003) as a result of ‘electron leakage’ from the photosynthetic electron transport chain. However, significant quantities of superoxide are also generated as a by-product of the mitochondrial electron transport chain (Foyer and Noctor, 2003 and Moller, 2001) and in the apoplast as a consequence of NADPH oxidase activity in response to biotic (Sagi and Fluhr, 2001) and abiotic (Achard et al., 2008) stresses. Superoxide will rapidly chemically dismutate to form hydrogen peroxide, a reaction that is accelerated manyfold by the presence of superoxide dismutases in most subcellular compartments. Hydrogen peroxide is also generated directly in very large quantities in peroxisomes as a by-product of photorespiratory metabolism and the ?-oxidation of fatty acids. In the presence of reduced transition metals, Fenton chemistry reduces hydrogen peroxide to the extremely reactive hydroxyl radical. Fig. 1 Formation of the most important reactive oxygen species (A) and reactive nitrogen species (B).
Different ROS vary significantly in terms of their properties and reactivity; the order of reactivity being hydroxyl radical > superoxide > hydrogen peroxide. Their reactivity sets limits on how far different ROS can propagate from their site of production. The hydroxyl radical is so reactive that it will react more or less indiscriminately with the first molecule it encounters. In contrast, the relatively low reactivity of hydrogen peroxide means that it can accumulate to significant concentrations and can diffuse as far as 1 ?m from its site of production (Møller et al., 2007). Superoxide will travel a shorter distance (up to 30 nm) and moreover, as a charged species at cellular pH (pKa 4.8—Halliwell and Gutteridge, 2007), it is confined to the subcellular compartment in which it is produced. The superoxide formed by the NADPH oxidase outside the plasma membrane, where the pH is normally significantly lower than inside the cell, could be substantially protonated and may find it easier to enter the cell by crossing the plasma membrane as a neutral molecule. Plants also produce RNS, particularly nitric oxide (NO•) and peroxynitrite (ONOO?). It remains unclear what is the most significant source of NO in plant cells. Efforts to uncover a canonical nitric oxide synthase, analogous to that found in mammals, have been beset with controversy (Zemojtel et al., 2006). A putative nitric oxide synthase in Arabidopsis (Guo and Crawford, 2005) was ultimately revealed to be incapable of NO synthesis from arginine and instead was established as a plastid-localized GTPase (Gas et al., 2009). This protein is still linked with NO• production (knockouts reduce NO• levels) but the mechanism is unclear. There are two other potential sources of NO• in plants: nitrate reductase and the mitochondrial electron transport chain under anoxia. Nitrate reductase can reduce nitrite to NO•. This could occur in vivo when the nitrite accumulates (the preferred substrate of nitrate reductase is, of course, nitrate) such as during anoxia when nitrite reductase is inhibited (Meyer et al., 2005). Anoxia is also a prerequisite for NO• production by mitochondria, allowing nitrite to serve as an alternative terminal electron acceptor in the electron transport chain. Because NO• inhibits complex IV of the respiratory chain, but not the alternative oxidase, NO• production has been proposed as a mechanism for controlling respiratory balance under low oxygen (Benamar et al., 2008 and Borisjuk et al., 2007). Whatever the mechanisms of NO• synthesis, there are reliable measurements demonstrating its presence in a range of plant tissues. Although NO• is a radical, its reactivity with proteins is limited. However, NO• does react extremely readily with superoxide to form peroxynitrite and this anion is more significant in terms of protein oxidation.

III. Mechanisms of Oxidation of Proteins

The following protein amino acids contain side groups that can be oxidized by different ROS and RNS leading to stable covalent modifications: Cys, Met, His, Arg, Lys, Pro, Tyr and Trp. The reactions are summarized in Fig. 2. Most of these reactions are essentially irreversible, although in the specific case of oxidation of thiols, enzyme-catalyzed re-reduction is possible (Bechtold et al., 2004 and Rouhier et al., 2006). Considering only the oxidation by ROS, most of the oxidation reactions shown in Fig. 2 are found to be only triggered by the highly reactive hydroxyl radical or singlet oxygen. This means that such oxidation events are only likely to occur in proteins that are localized extremely close to the site of production of these radicals. ...