Differences between the manganese- and the iron-containing superoxide dismutases of Escherichia coli detected through sedimentation equilibrium, hydrodynamic, and spectroscopic studies.
The genome of Escherichia coli codes for two superoxide dismutases that may contain either iron (FeSOD) or manganese (MnSOD) at the active site. The crystal structures of MnSODs from two bacterial sources (but not E. coli) have been completed, and structural comparisons with the crystal structure of the FeSOD from either E. coli or Pseudomonas ovalis have been made. Despite the low degree (less than 50%) of sequence homology between the E. coli enzymes, the two proteins are suggested to be structurally homologous. Nonetheless, these enzymes exhibit absolute metal cofactor specificity in conferring enzymatic activity to the inactive apoenzyme. This observation is surprising considering the identity of the active site ligands and the similarities in their geometry and surrounding environment. Using analytical ultracentrifugation, we have determined that the solution properties of these two proteins are different. Thus dialysis of FeSOD but not of MnSOD against phosphate buffer in the presence or absence of EDTA caused dissociation of the homodimer. This dissociation appeared to be related to the loss of iron from native FeSOD. Thus, apoFeSOD but not apoMnSOD existed predominantly as a monomer at protein concentrations below 150 micrograms/mL. ApoMnSOD showed no evidence for dissociation under these conditions. Fluorescence data suggest that the tryptophan environments for the two enzymes are also different. The results of these physical measurements lead us to propose that subtle differences, perhaps at the subunit contact faces, exist in the structures of these crystallographically similar proteins.
Beyer
W F
WF
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Reynolds
J A
JA
Fridovich
I
I
D001051:Apoenzymes / Q000302:isolation & purification; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013482:Superoxide Dismutase / Q000378:metabolism*; D014364:Tryptophan; D014461:Ultracentrifugation
D001051:Apoenzymes; D014364:Tryptophan; D013482:Superoxide Dismutase