Isopenicillin N synthase (IPNS) from Streptomyces jumonjinensis (M(r) 37,902) is a non-heme ferrous iron-containing enzyme that catalyzes the oxidative cyclization of the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies [reviewed in Cooper, R. D. (1993) Biomed. Chem. 1, 1-17] have led to a model for the coordination environment of the iron atom possessing three histidine and one aspartic acid endogenous ligands and a solvent molecule. A refinement of that model proposes that formation of the Fe(II) IPNS-ACV complex occurs with displacement of the H2O from the metal center and that one of the histidines is subsequently replaced by a solvent molecule on binding of dioxygen. Here we report genetic studies to determine the nature and location of the endogenous ligands in the S. jumonjinensis IPNS primary amino acid sequence that constitute the ferrous active site. Site-directed mutagenesis was used to exchange each of the seven histidines and the five aspartic acids that are conserved in bacterial and fungal IPNS proteins. Biochemical analysis of the alanine-substituted mutant proteins shows that two histidines, His212 and His268, and one aspartic acid, Asp214, are essential for enzyme activity. The other mutant enzymes have specific activities 5-68% that of wild type. Sequence analysis of 10 IPNS and 42 other non-heme ferrous iron-dependent dioxygenases reveal the presence of a common motif, HisXAsp(53-57)XHis, which in IPNS contains the identical two histidines and one aspartic acid essential for function. Accordingly, we have assigned residues His212, His268, and Asp214 as three of the four endogenous ligands postulated to form the IPNS ferrous active site. Compelling support for these conclusions comes from the recent crystal structure determination of the manganese form of a fungal IPNS [Roach et al. (1995) Nature 375, 700-704].