Assay of metabolic superoxide production in Escherichia coli

Abstract

Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high SOD content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. SOD-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.