The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Oxygen is a reactive chemical, and its gradual accumulation in the environment must have been a problem for early life forms. For kinetic reasons much of its toxicity is likely to be mediated by its partially reduced forms, superoxide (O_2-) and hydrogen peroxide (H₂O₂). Virtually all aerobic organisms contain enzymes devoted to eliminating these species from the cell: superoxide dismutase for O₂-, and catalases and peroxidases for H₂O₂. This implies that O_2- and H₂O₂ are formed somehow during aerobic metabolism and that, unless scavenged, they will harm the cell. Indeed, mutants that are unable to detoxify these oxygen species have difficulty growing in air.

Yet the most fundamental questions about O_2- and H₂O₂ toxicity remain unanswered: How do these species arise in the cell? What damage can they do? What do cells do to avoid or repair that damage? And when in nature does oxidative damage determine the fate of the cell?

Our laboratory pursues these questions primarily in E. coli, a model organism whose metabolism is well-understood and which is amenable to easy genetic manipulation. However, the mechanisms by which oxidants damage E. coli, and the strategies by which E. coli defends itself, are widely shared by other bacteria—and, indeed, by higher organisms.

How are superoxide and hydrogen peroxide formed inside cells? Mutant strains of E. coli that lack superoxide dismutase or catalases/peroxidases are very sick. Yet E. coli has no enzymes that produce O_2- or H₂O₂ as deliberate products. So what is the source of the O_2- and H₂O₂ that disable these mutants? We have found that some flavoenzymes inadvertently transfer electrons to dissolved oxygen when it bounces into their reduced cofactors. O₂- is formed if one electron is transferred, and H₂O₂ is formed if two electrons are transferred. Not surprisingly, this behavior is particularly marked if the flavin is solvent-exposed, has a high electron density, and has a low reduction potential. Several respiratory dehydrogenases have these traits, and they generate significant O_2- and H₂O₂ during respiration. However, genetic analyses show that the primary source(s) of oxidants inside E. coli lie outside the respiratory chain. The identification of these sources is currently an important goal of our lab.

What biomolecules does superoxide damage? Superoxide and hydrogen peroxide are used as weapons throughout the biological world: microbes and plants secrete antibiotics which, when ingested by competitors, redox-cycle to generate crippling doses of intracellular O_2- and H₂O₂, and mammals blast invasive bacteria with O_2- and H₂O₂ that is generated by specialized enzymes on the phagosomal membranes of neutrophils and macrophages. Yet we still are trying to identify the biological molecules these oxidants damage. Superoxide does not react directly with polypeptides, sugars, or nucleic acids, and it is unlikely to trigger lipid peroxidation because bacterial membranes lack polyunsaturated fatty acids. It has become clear that O_2- disables the TCA cycle and branched-chain biosynthetic pathways by destroying the iron-sulfur clusters of critical enzymes. This must not be its only toxic action, however, since SOD-deficient E. coli also exhibit other metabolic defects. We are analyzing these phenotypes in order to pinpoint additional biomolecules that O_2- damages.

What biomolecules does hydrogen peroxide damage? H₂O₂ oxidizes transition metals. This behavior has several consequences for cell health. First, like O_2-, H₂O₂ directly damages the iron-sulfur clusters of key enzymes. Second, H₂O₂ reacts with the intracellular pool of unincorporated ferrous iron and thereby generates hydroxyl radicals. The latter species react avidly with virtually all biomolecules—including DNA. This injury causes mutations, a fact that has led many scientists to postulate that in mammals chronic oxidative DNA damage is a root cause of carcinogenesis and, perhaps, aging. Finally, as a consequence of its reactions with iron, H₂O₂ generally disrupts iron homeostasis and metabolism. This makes it difficult for cells to deliver iron into new metalloproteins. We are working to illuminate details of these damage processes (see figure).

How does E. coli defend itself against oxidants? While superoxide dismutase, catalases, and peroxidases scavenge O_2- and H₂O₂, they can never diminish the intracellular levels to zero. Therefore many additional defensive measures are taken. E. coli repairs the iron-sulfur clusters of damaged enzymes by re-reducing their clusters and replacing their lost iron atoms. If the rate of enzyme damage exceeds the repair capacity, E. coli goes further, replacing some of the damaged iron-sulfur enzymes--aconitase and fumarase—with isozymes that are resistant to oxidation. During periods of oxidative stress E. coli also synthesizes Dps, an iron-sequestering protein that minimizes the amount of unincorporated iron that is available to react with H₂O₂. This action substantially suppresses DNA damage. Genetic analyses have identified many other genes that are deliberately induced in response to O_2- or H₂O₂. We are still working to identify their roles.

Why can't anaerobes grow aerobically? How do phagocytes kill bacteria? And other questions. . . . Oxidative stress determines the fate of bacteria in several situations. Obligate anaerobes cannot grow in the presence of oxygen, a feature that profoundly constrains their lifestyle but is not well understood. Most bacteria are overpowered by the oxidative burst of phagocytes—but a select few pathogens somehow tolerate it. We are hopeful that E. coli will provide insights into these problems, too. At this point we know that some of the enzymes that oxygen inactivates in obligate anaerobes are the same ones that superoxide damages in E. coli. We also know that some of the oxidative defenses that were identified in E. coli are essential to the success of invading pathogens. Thus the lessons from E. coli provide direction for investigations of oxidative stress in these other organisms as well.