Iron enzyme ribulose-5-phosphate 3-epimerase in Escherichia coli is rapidly damaged by hydrogen peroxide but can be protected by manganese

Abstract

H 2 O 2 is commonly generated in biological habitats by environmental chemistry and by cellular immune responses. H 2 O 2 penetrates cells, disrupts metabolism, and blocks growth; it therefore is of interest to identify the major cellular molecules that H 2 O 2 damages and the strategies by which cells protect themselves from it. We used a strain of Escherichia coli that lacks catalases and peroxidases to impose protracted low-grade H 2 O 2 stress. Physiological analysis indicated that the pentose-phosphate pathway, in particular, was poisoned by submicromolar intracellular H 2 O 2 . Assays determined that ribulose-5-phosphate 3-epimerase (Rpe) was specifically inactivated. In vitro studies demonstrated that Rpe employs a ferrous iron atom as a solvent-exposed cofactor and that H 2 O 2 rapidly oxidizes this metal in a Fenton reaction. The oxidized iron is released immediately, causing a loss of activity. Most Rpe proteins could be reactivated by remetallation; however, a small fraction of proteins were irreversibly damaged by each oxidation cycle, and so repeated cycles of oxidation and remetallation progressively led to permanent inactivation of the entire Rpe pool. Manganese import and iron sequestration are key elements of the H 2 O 2 stress response, and we found that manganese can activate Rpe in vitro in place of iron, converting the enzyme to a form that is unaffected by H 2 O 2 . Indeed, the provision of manganese to H 2 O 2 -stressed cells protected Rpe and enabled the pentose-phosphate pathway to retain function. These data indicate that mononuclear iron enzymes can be primary targets of H 2 O 2 stress and that cells adapt by shifting from iron-to manganese-centered metabolism.hydroxyl radical | MntH | oxidative damage | OxyR

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

“…Furthermore, this dramatic drop in Mn content was accompanied by a 3-fold increase in Fe content, suggesting that E. faecalis compensates for lower levels of Mn by taking up additional Fe. While this appears counterintuitive, it is well known that Fe and Mn can act as interchangeable enzymatic cofactors (30,41). On the basis of the ability of reduced glutathione levels to rescue growth of the (p)ppGpp 0 strain under Mn-depleted conditions, we can conclude that its higher Mn requirement is directly linked to oxidative stress.…”

Regulation of Manganese Antioxidants by Nutrient Sensing Pathways in Saccharomyces cerevisiae

“…reactions at enzyme active sites (Anjem et al 2009;Sobota and Imlay 2011). Regardless of the mechanism, it was unclear whether the protective effect of Mn is part of the cell's tightly regulated battery of antioxidant responses or is a passive unregulated process.…”

Nramp1 and Other Transporters Involved in Metal Withholding during Infection

“…Iron is essential for ATP production and other metabolic pathways, but its concentration is limited to avoid production of ROS 2 catalyzed by excess levels of this redox-active metal (8). In some organisms, manganese can substitute for redox-active iron to protect against oxidative stress (9,10). Moreover, most pathogens require manganese to produce their own superoxide dismutase activity to thwart oxidative killing mechanisms exerted by the host (11)(12)(13).…”