Titration of Escherichia coli DNA topoisomerase I with PMPS and 65Zn(II) binding showed independent release and binding of the three Zn(II) in each enzyme molecule. Removal of Zn(II) from topoisomerase I or top85 (truncated topoisomerase I with the Zn(II) binding domain at the carboxyl terminal) affected their sensitivity to Glu-C and Asp-N endoproteases but there was no significant effect on their rate of proteolysis by trypsin or Lys-C endoprotease. This suggested that Zn(II) removal did not result in complete unfolding of topoisomerase enzyme structure but only affected folding of small local regions. Digestion with carboxypeptidase Y further demonstrated that the folding of the zinc binding region itself was altered upon Zn(II) removal.