Escherichia coli DNA topoisomerase I contains three Zn(II) in each enzyme molecule required for relaxation of negatively supercoiled DNA. Apoenzymes were prepared from both the intact topoisomerase (M(r) 97,000) and the truncated active form top85 (M(r) 85,000) that lacks the carboxyl terminal domain but still contains the three Zn(II). Fluorescence and circular dichroism spectroscopy were used to compare the apoenzymes with topoisomerase and top85 reconstituted with Zn2+. The results indicated structural changes affecting the environment of the tryptophan residues and increasing the alpha-helical and beta-sheets content of the protein occurred upon zinc removal. These structural changes probably account for the loss of enzyme activity.