Abstract:Bacteroides thetaiotaomicron was examined to determine whether its obligate anaerobiosis is imposed by endogenous reactive oxygen species or by molecular oxygen itself. Previous analyses established that aerated B. thetaiotaomicron loses some enzyme activities due to a high rate of endogenous superoxide formation. However, the present study establishes that another key step in central metabolism is poisoned by molecular oxygen itself. Pyruvate dissimilation was shown to depend upon two enzymes, pyruvate:formate lyase (PFL) and pyruvate:ferredoxin oxidoreductase (PFOR), that lose activity upon aeration. PFL is a glycyl-radical enzyme whose vulnerability to oxygen is already understood. The rate of PFOR damage was unaffected by the level of superoxide or peroxide, showing that molecular oxygen itself is the culprit. The cell cannot repair PFOR, which amplifies the impact of damage. The rates of PFOR and fumarase inactivation are similar, suggesting that superoxide dismutase is calibrated so the oxygen- and superoxide-sensitive enzymes are equally sensitive to aeration. The physiological purpose of PFL and PFOR is to degrade pyruvate without disrupting the redox balance, and they do so using catalytic mechanisms that are intrinsically vulnerable to oxygen. In this way the anaerobic excellence and oxygen sensitivity of B. thetaiotaomicron are two sides of the same coin.