Barbara S. Berlett

Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals

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.

Methionine residues may protect proteins from critical oxidative damage.

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they form disulfide bonds which contribute to protein structure. In contrast, the key 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, providing for efficient scavenging of oxidants. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the sixteen methionine residues could be oxidized with little effect on activity. The oxidizable methionine residues were found to be relatively surface exposed while the intact residues were generally buried within the core of the protein. Further, the susceptible residues were physically arranged in an array which guarded the entrance to the active site. Methionine sulfoxide can be reduced back to methionine by the enzyme methionine sulfoxide reductase, providing a catalytic amplification of the antioxidant potential of each methionine residue. Given the importance of oxidative stress during aging, the potential function of methionine residues as antioxidants during aging should be investigated experimentally.

Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans

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.

A Low pKa Cysteine at the Active Site of Mouse Methionine Sulfoxide Reductase A

Background: The active site cysteine of methionine sulfoxide reductases has been reported to be 9.5. Results: The pKa of methionine sulfoxide reductase is 7.2. Conclusion: Methionine sulfoxide reductase has an active cysteine at its catalytic center. Significance: Methionine sulfoxide reductase is readily oxidized by low concentrations of hydrogen peroxide, supporting both antioxidant and redox signaling functions of the enzyme. Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pKa of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pKa of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pKa is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pKa renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.

Identification of enzymes and regulatory proteins in Escherichia coli that are oxidized under nitrogen, carbon, or phosphate starvation

Using proteomic technologies, we identified 62 proteins that are oxidized to carbonyl derivatives during growth of Escherichia coli under nitrogen starvation (NS), carbon starvation (CS), and phosphate starvation (PS) conditions. The carbonylated proteins were converted to 2,4-dinitrophenylhydrazone derivatives and these were identified using Western blotting and mass spectrometry by searching E. coli proteins in the Swiss-Prot and/or NCBI databases. Fourteen of the oxidized proteins were formed under both NS and CS conditions, and only three proteins were specifically oxidized under PS conditions. Interestingly, the carbonyl content of proteins in crude extracts of cells harvested after 48 h of stationary growth under NS and CS was significantly lower than that observed at mid-log and end-log phases of growth. In contrast, the carbonyl content of proteins in extracts of cells grown under PS conditions was fairly constant during comparable periods of growth.

Designing antioxidant peptides

Abstract Background Ionizing radiation causes the generation of damaging reactive oxygen species that lead to cellular damage and death. Organisms such as Deinococcus radiodurans have evolved mechanisms for extreme resistance to ionizing radiation, and resistance has been shown to be a consequence of protection of critical proteins from oxidative inactivation. Objectives D. radiodurans accumulates high levels of manganese and of small peptides that together are protective. Our aim was to rationally design antioxidant peptides. Methods Amino acid analysis was utilized to determine the rates of loss of the 20 amino acids exposed to varying doses of irradiation. The activity of glutamine synthetase and methionine sulfoxide reductase was assayed to follow their inactivation by irradiation. Results The ability of an amino acid to protect enzymes from inactivation by ionizing radiation paralleled its sensitivity to ionizing radiation. Based on this observation and the ability of histidine to confer water solubility, we synthesized the hexapeptide His-Met-His-Met-His-Met and found that it provided markedly increased protection against irradiation. Discussion Small peptides containing histidine and methionine were readily soluble and provided enzymes with remarkable protection from inactivation by ionizing radiation.