Contrasting Sensitivities of Escherichia coli Aconitases A and B to Oxidation and Iron Depletion

Abstract

Superoxide damages dehydratases that contain catalytic [4Fe-4S]2؉ clusters. Aconitases are members of that enzyme family, and previous work showed that most aconitase activity is lost when Escherichia coli is exposed to superoxide stress. More recently it was determined that E. coli synthesizes at least two isozymes of aconitase, AcnA and AcnB. Synthesis of AcnA, the less-abundant enzyme, is positively controlled by SoxS, a protein that is activated in the presence of superoxide-generating chemicals. We have determined that this arrangement exists because AcnA is resistant to superoxide in vivo. Surprisingly, purified AcnA is extremely sensitive to superoxide and other chemical oxidants unless it is combined with an uncharacterized factor that is present in cell extracts. In contrast, AcnB is highly sensitive to a variety of chemical oxidants in vivo, in extracts, and in its purified form. Thus, the induction of AcnA during oxidative stress provides a mechanism to circumvent a block in the tricarboxylic acid cycle. AcnA appears to be as catalytically competent as AcnB, so the retention of the latter as the primary housekeeping enzyme must provide some other advantage. We observed that the [4Fe-4S] cluster of AcnB is in dynamic equilibrium with the surrounding iron pool, so that AcnB is rapidly demetallated when intracellular iron pools drop. AcnA and other dehydratases do not show this trait. Demetallated AcnB is known to bind its cognate mRNA. The absence of AcnB activity also causes the accumulation and excretion of citrate, an iron chelator for which E. coli synthesizes a transport system. Thus, AcnB may be retained as the primary aconitase because the lability of its exposed cluster allows E. coli to sense and respond to iron depletion.

A minor catalase/peroxidase from Burkholderia cenocepacia is required for normal aconitase activity

“…The iron chelator a-a9-dipyridyl can enter the bacterial cytoplasm, and, under these conditions, some enzymes of the TCA cycle, like AcnB, can be rapidly demetallated (Varghese et al, 2003). We reasoned that a similar situation could occur in B. cenocepacia C5424, such that KatA could be required to counteract the effects of iron depletion on one or more TCA cycle enzymes.…”

“…Thus, it is possible that KatA acts on another form of aconitase, which may resemble the E. coli AcnA. In E. coli, AcnB is rapidly inactivated when cellular iron pools are low or under oxidative stress (Varghese et al, 2003). In contrast, AcnA synthesis is induced by oxidative stress and iron limitation, but it appears to require an unidentified cellular component for stability (Varghese et al, 2003).…”

“…In other bacteria, aconitase and fumarate reductase are known to possess ROS-sensitive iron-sulphur clusters (Oexle et al, 1999;Varghese et al, 2003). Thus, we determined the aconitase activity in MDL1 and MDL2 mutants.…”

“…In other bacteria, such as E. coli, the aconitase activity can be contributed by two enzymes: (i) AcnB, which is iron-and oxidative-stress-sensitive, and contributes the majority of the aconitase activity in growing cells, and (ii) AcnA, which is predominantly expressed and stable under conditions of oxidative stress, and in stationary phase (Varghese et al, 2003). To determine whether biochemically distinct forms of aconitase also exist in B. cenocepacia, cell-free lysates were pre-incubated with ferrous sulphate and DTT.…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…This conundrum remains unsolved. One idea that would explain the maintenance of aconitase B, however, is that its solvent exposure and general instability provide a mechanism through which the cell responds to iron starvation (75). A loss of activity may facilitate the accumulation and excretion of its substrate, citrate; citrate is an excellent iron chelator and serves as a siderophore with its own dedicated import system.…”