The demonstration that oxidatively modified forms of proteins accumulate during aging, oxidative stress, and in some pathological conditions has focused attention on physiological and nonphysiological mechanisms for the generation of reactive oxygen species (ROS) 1 and on the modification of biological molecules by various kinds of ROS. Basic principles that govern the oxidation of proteins by ROS were established in the pioneering studies of Swallow (1), Garrison (2, 3), and Scheussler and Schilling (4) who characterized reaction products formed when proteins were exposed to ionizing radiation under conditions where only ⅐ OH, O 2. , or a mixture of both was made available. Results of these studies demonstrated that the modification of proteins is initiated mainly by reactions with ⅐ OH; however, the course of the oxidation process is determined by the availability of O 2 and O 2 . or its protonated form (HO ⅐ 2 ). Collectively, these ROS can lead to oxidation of amino acid residue side chains, formation of protein-protein cross-linkages, and oxidation of the protein backbone resulting in protein fragmentation. In the meantime, it has been shown that other forms of ROS may yield similar products and that transition metal ions can substitute for ⅐ OH and O 2 . in some of the reactions.

Cysteine and methionine are the two sulfurcontaining residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they can form disulfide bonds that contribute to protein structure. In contrast, the specific functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, available as an efficient oxidant scavenger. Reduction back to methionine by methionine sulfoxide reductases would allow the antioxidant system to function catalytically. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the 16 methionine residues could be oxidized with little effect on catalytic activity of the enzyme. The oxidizable methionine residues were found to be relatively surface exposed, whereas the intact residues were generally buried within the core of the protein. Furthermore, the susceptible residues were physically arranged in an array that guarded the entrance to the active site.

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders. P rotein-bound methionine residues are among the most susceptible to oxidation by reactive oxygen species (ROS), resulting in formation of methionine sulfoxide [Met(O)] residues. However, this modification can be repaired by methionine sulfoxide reductase (MsrA), which catalyzes the thioredoxindependent reduction of free and protein-bound Met(O) to methionine, both in vitro (1) and in vivo (2). Bacteria and yeast cells lacking the msrA gene show increased sensitivity to oxidative stress and lower survival rates (3, 4), with yeast showing accumulation of high levels of both free and protein-bound Met(O) (2, 4). In addition, overexpression of the MsrA enzyme in human T cells prolongs their life under conditions of oxidative stress (4). Because methionine residues are particularly susceptible to oxidation by ROS, MsrA could have at least three important functions in cellular metabolism: (i) as an antioxidant enzyme that scavenges ROS by facilitating the cyclic interconversion of methionine͞protein-methionine residues between oxidized and reduced forms (2); (ii) as a repair enzyme by keeping critical methionine residues in their reduced form; and (iii) as a regulator of critical enzyme activity through cyclic interconversion of specific methionine residues between oxidized and reduced forms (5, 6). Escherichia coli and Saccharomyces cerevisiae both contain at least two Msrs. One (MsrA) is able to reduce both free and protein-bound Met(O), and the other can reduce only free Met(O). The MsrA protein is highly expressed in liver, kidney, pigment epithelial cells of the retina, macrophages, cerebellum, and brain neurons (7). These tissues͞ cells are sensitive to oxidative stress damages. Therefore, abolishing the MsrA enzyme could lead to loss of antioxidant defense, resulting in enhanced oxidative damage and a decreased lifespan. To investigate the possible role of MsrA as an antioxidant in mammals and its possible influence on lifespan, we created a strain of mouse lacking the MsrA protein. Materials and ...

Highly reactive oxygen species that are formed during normal metabolism and under conditions of oxidative stress are able to oxidize proteins or convert lipid and carbohydrate derivatives to compounds that react with functional groups on proteins. Among other changes, these ROS-mediated reactions lead to the formation of protein carbonyl derivatives, which serves as a marker of ROS-mediated protein damage. On the basis of this marker, it is established that oxidatively damaged protein is associated with aging and some diseases. The accumulation of oxidatively damaged protein reflects the balance among a myriad of factors that govern the rates of ROS generation and the rate at which damaged protein is degraded. Peroxynitrite, which is formed under normal physiological conditions, is able to oxidize methionine residues in proteins and to nitrate tyrosine residues; however, its ability to do so is dependent on the availability of CO2, which stimulates the nitration of tyrosine residues but inhibits the oxidation of methionine residues. Nitration of tyrosine residues may contribute to peroxynitrite toxicity, as nitration precludes the phosphorylation or nucleotidylation of tyrosine residues and thereby seriously compromises one of the most important mechanisms of cellular regulation and signal transduction.

For Deinococcus radiodurans and other bacteria which are extremely resistant to ionizing radiation, ultraviolet radiation, and desiccation, a mechanistic link exists between resistance, manganese accumulation, and protein protection. We show that ultrafiltered, protein-free preparations of D. radiodurans cell extracts prevent protein oxidation at massive doses of ionizing radiation. In contrast, ultrafiltrates from ionizing radiation-sensitive bacteria were not protective. The D. radiodurans ultrafiltrate was enriched in Mn, phosphate, nucleosides and bases, and peptides. When reconstituted in vitro at concentrations approximating those in the D. radiodurans cytosol, peptides interacted synergistically with Mn2+ and orthophosphate, and preserved the activity of large, multimeric enzymes exposed to 50,000 Gy, conditions which obliterated DNA. When applied ex vivo, the D. radiodurans ultrafiltrate protected Escherichia coli cells and human Jurkat T cells from extreme cellular insults caused by ionizing radiation. By establishing that Mn2+-metabolite complexes of D. radiodurans specifically protect proteins against indirect damage caused by gamma-rays delivered in vast doses, our findings provide the basis for a new approach to radioprotection and insight into how surplus Mn budgets in cells combat reactive oxygen species.

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The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in a Saccharomyces cerevisiae null mutant of msrA by using a high-copy plasmid harboring the msrA gene and its promoter. The resulting strain had about 25-fold higher MsrA activity than its parent strain. When exposed to either hydrogen peroxide, paraquat, or 2,2-azobis-(2-amidinopropane) dihydrochloride treatment, the MsrA overexpressed strain grew better, had lower free and protein-bound methionine sulfoxide and had a better survival rate under these conditions than did the msrA mutant and its parent strain. Substitution of methionine with methionine sulfoxide in a medium lacking hydrogen peroxide had little effect on the growth pattern, which suggests that the oxidation of free methionine in the growth medium was not the main cause of growth inhibition of the msrA mutant. Ultraviolet A radiation did not result in obvious differences in survival rates among the three strains. An enhanced resistance to hydrogen peroxide treatment was shown in human T lymphocyte cells (Molt-4) that were stably transfected with the bovine msrA and exposed to hydrogen peroxide. The survival rate of the transfected strain was much better than its parent strain when grown in the presence of hydrogen peroxide. These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress.