The mechanism of type IB topoisomerase-mediated DNA relaxation was studied by modification of vaccinia topoisomerase I at the active site tyrosine (position 274) with several tyrosine analogues. These analogues had varied steric, electronic, and stereochemical features to permit assessment of those structural elements required to support topoisomerase function. Eleven tyrosine analogues were successfully incorporated into the active site of vaccinia topoisomerase I. It was found that only tyrosine analogues having the phenolic -OH group in the normal position relative to the protein backbone were active. Modifications that replaced the nucleophilic tyrosine OH (pKa approximately 10.0) group with NH2 (pKa 4.6), SH (pKa approximately 7.0), or I groups or that changed the orientation of the nucleophilic OH group essentially eliminated topoisomerase I function. For the active analogues, the electronic effects and H-bonding characteristics of substituents in the meta-position of the aromatic ring may be important in modulating topoisomerase I function. The pH profile for the functional analogues revealed a small shift toward lower pH when compared with wild-type topoisomerase I.