Inactivation-reactivation of aconitase in Escherichia coli. A sensitive measure of superoxide radical.
The rapid inactivation of aconitase by O2-, previously seen to occur in vitro, was explored in vivo. A fraction of the aconitase in growing, aerobic, Escherichia coli is inactive at any instant but can be activated by imposition of anaerobic conditions. This reactivation occurred in the absence of protein synthesis and was inhibited by the ferrous chelator alpha,alpha'-dipyridyl. This fraction of inactive, but activatable, aconitase was increased by augmenting O2- production with paraquat, decreased by elevation of superoxide dismutase, and increased by inhibiting reactivation with alpha,alpha'-dipyridyl. The balance between inactive and active aconitase thus represented a pseudoequilibrium between inactivation by O2- and reactivation by restoration of Fe(II), and it provided, for the first time, a measure of the steady-state concentration of O2- within E. coli. On this basis, [O2-] was estimated to be approximately 20-40 pM in aerobic log phase E. coli containing wild type levels of superoxide dismutase and approximately 300 pM in a mutant strain lacking superoxide dismutase.
Gardner
P R
PR
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Fridovich
I
I
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002374:Catalase / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals; D007700:Kinetics; D010269:Paraquat / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D005609:Free Radicals; D013481:Superoxides; D015082:2,2'-Dipyridyl; D002374:Catalase; D013482:Superoxide Dismutase; D000154:Aconitate Hydratase; D010269:Paraquat