Isopenicillin N synthase (IPNS) from Cephalosporium acremonium (Mr 38 400) is an ironcontaining enzyme that aerobically catalyzes the four-electron oxidative ring closure reactions of δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), forming the β-lactam and thiazolidine rings of isopenicillin N. Here, we report Fe K-edge X-ray absorption studies that provide insight into the iron coordination environment and the effect of substrate and nitric oxide binding. Our analysis reveals an iron(II) coordination environment consisting of two N/O-containing ligands at 2.01 ± 0.02 Å, three N/O ligands at 2.15 ± 0.02 Å, and one C/O scatterer at approximately 2.6–2.7 Å. Three His ligands are associated with the 2.15-Å shell, while an unsymmetrically chelated carboxylate is associated with a scatterer at 2.01 and at 2.6–2.7 Å, a combination which is consistent with the ligand environment deduced from 1H NMR studies [Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653–11659]. The remaining scatterer at 2.01 Å is assigned to a coordinated solvent molecule, most likely hydroxide, which can act as the proton acceptor for the incoming substrate. ACV binding to Fe(II)IPNS evinces an Fe-S interaction at 2.35 ± 0.02 Å, indicative of the coordination of substrate cysteine thiolate to the metal center. Analysis of the Fe(II)IPNS-ACV-NO data reveals one Fe-N at 1.71 ± 0.02 Å, three Fe-(N,O) at 2.04 ± 0.02 Å, one Fe-S at 2.32 ± 0.02 Å, and one Fe-(C,O) at 2.61 ± 0.02 Å, the short Fe-N bond being derived from the binding of NO. Our EXAFS conclusions, supported by corresponding analysis of relevant model complexes, corroborate and refine the working model for the Fe(II) coordination environment developed from previous spectroscopic studies.