Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B.
Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.
D001386:Azides / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis; D001665:Binding Sites; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005459:Fluorides / Q000494:pharmacology; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013045:Species Specificity; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000302:isolation & purification / Q000378:metabolism
D001386:Azides; D001426:Bacterial Proteins; D007501:Iron; D013482:Superoxide Dismutase; D005459:Fluorides