The mismetallation of enzymes during oxidative stress

Abstract

Mononuclear iron enzymes can tightly bind non-activating metals. How do cells avoid mismetallation? The model bacterium Escherichia coli may control its metal pools so that thermodynamics favor the correct metallation of each enzyme. This system is disrupted, however, by superoxide and hydrogen peroxide. These species oxidize ferrous iron and thereby displace it from many iron-dependent mononuclear enzymes. Ultimately, zinc binds in its place, confers little activity, and imposes metabolic bottlenecks. Data suggest that E. coli compensates by using thiols to extract the zinc and by importing manganese to replace the catalytic iron atom. Manganese resists oxidants and provides substantial activity.

Keywords: Hydrogen Peroxide; Iron Metabolism; Manganese; Superoxide Ion; Zinc.

FIGURE 1.

The role of Fe²⁺ in catalysis by ribulose-5-phosphate 3-epimerase. Proton abstraction from the chiral carbon is possible only because the divalent metal stabilizes the resonance structure of the oxyanion. Reprotonation follows, with inversion of configuration.

FIGURE 2.

Immediate and delayed phenotypes of superoxide stress in E. coli. A SOD⁻ mutant growing in anoxic medium was shifted at time 0 into oxic conditions. When Ile and Val were absent, growth stopped immediately due to the inactivation of dihydroxyacid dehydratase, a [4Fe-4S] enzyme. In contrast, growth continued for several hours in the aromatic-deficient medium. Enzyme analysis showed that mononuclear iron enzymes lost activity only slowly. At time 0 these enzymes were populated by ferrous iron; at 6 h they were populated by zinc. all AAs, all amino acids.

FIGURE 3.

Model for metallation of mononuclear iron enzymes during oxidative stress. Superoxide oxidizes bound ferrous cofactor to Fe³⁺, which dissociates, leaving behind an inactive apoprotein. Remetallation by cellular Fe²⁺ is rapid, and steady-state activity initially remains high. However, with each cycle of demetallation a subfraction of enzyme is inappropriately metallated by Zn²⁺. Because zinc-cofactored enzyme has minimal activity, the pool of enzyme activity progressively declines, and metabolic bottlenecks ensue. Supplementation with Mn²⁺ restores enzyme activity and pathway functions, presumably due to the alternative metallation of these enzymes by manganese.

FIGURE 4.

Primary controls upon synthesis of the E. coli manganese importer. The mntH gene is repressed in unstressed, iron-sufficient cells. When iron levels fall, the Fur repressor is inactivated by demetallation, and transcription is induced. Transcription is also induced when H₂O₂ activates the OxyR transcription factor. Under both conditions, intracellular manganese levels rise and mononuclear enzymes retain function.