A novel A3 group aconitase tolerates oxidation and nitric oxide [Enzymology]

December 4th, 2014 by Doi, Y., Takaya, N.

Achromobacter denitrificans YD35 is an extremely nitrite (NO2−)-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, among which, acnA3 is essential for growth against 100 mM NO2−. Atmospheric oxygen inactivated AcnA3 at a rate of 1.6×10−3 min−1, which was 2.7- and 37-folds lower than those for AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S]2+ cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO2− levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8×10−3 min−1, which was 1.6- and 7.8-folds lower than those for AcnA4 and AcnB. When exposed to NO2−, the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate than the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO2− tolerance. AcnA3Tn accumulated less cellular NADH and ATP than YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO2− milieu. This novel aconitase is distributed in Alcaligenaceae bacteria including pathogens and denitrifiers and it appears to contribute to a novel NO2−-tolerance mechanism in this strain.