Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase.
The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain. Growth of these strains on a gluconate minimal medium was also impaired under these conditions. The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation. The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E. coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1. The dehydratase was much less sensitive to O2 or H2O2 than to O2-. The virtual substrate, alpha-glycerophosphate, provided partial protection. Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme. Fluoride ions stabilized the dehydratase and blocked the effect of oxidants. The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.
Gardner
P R
PR
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Fridovich
I
I
D001426:Bacterial Proteins / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005459:Fluorides / Q000737:chemistry; D005609:Free Radicals; D006836:Hydro-Lyases / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D010269:Paraquat / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013481:Superoxides / Q000737:chemistry*
D001426:Bacterial Proteins; D002614:Chelating Agents; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D013438:Sulfhydryl Compounds; D013481:Superoxides; D006861:Hydrogen Peroxide; D006836:Hydro-Lyases; C022362:phosphogluconate dehydratase; D010269:Paraquat; D005459:Fluorides