Biochemical and spectroscopic characterization of Escherichia coli aconitases (AcnA and AcnB)

Escherichia coli contains two major aconitases (Acns), AcnA and AcnB. They are distantly related monomeric Fe-S proteins that contain different arrang ements of four structural domains. On the basis of the differential express ion of the acnA and acnB genes, AcnA has been designated as an aerobic-stat ionary-phase enzyme that is specifically induced by iron and oxidative stre ss, whereas AcnB functions as the major citric-acid-cycle enzyme during exp onential growth. The biochemical and kinetic properties of the purified enz ymes have now shown that AcnA is more stable than AcnB, has a higher affini ty for citrate, and operates optimally over a wider pH range, consistent wi th its role as a maintenance or survival enzyme during nutritional or oxida tive stress. In contrast, the better performance at high substrate concentr ations and greater instability of AcnB indicate that AcnB is specifically a dapted to function as the main catabolic enzyme and, by inactivation, to ra pidly modulate energy metabolism in response to oxidative or pH stress, eit her directly or indirectly by regulating post-transcriptional gene expressi on. EPR and magnetic-CD spectroscopy showed that the iron-sulphur clusters of the bacterial Acns land their binding sites) strongly resemble those of the mammalian enzymes. The EPR and MCD spectra of the oxidized inactive for m of AcnB confirmed the presence of a [3Fe-4S](1+) (S = 1/2) cluster. Compa risons showed that the EPR spectrum of AcnB more closely resembled that of mammalian mitochondrial Acn (m-Acn), whereas the spectrum of AcnA more clos ely resembled that of the cytoplasmic enzyme (c-Acn). The MCD spectra revea led spectroscopic signatures similar to that of m-Acn. Reconstitution of th e active [4Fe-4S](2+) forms followed by one-electron reduction gave rise to EPR spectra that are almost identical with those reported for the mammalia n enzymes.