© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnucis replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.