Thiolate Ligation of the Active Site Fe²⁺of Isopenicillin N Synthase Derives from Substrate Rather Than Endogenous Cysteine: Spectroscopic Studies of Site-Specific Cys Â-Ser Mutated Enzymes

Isopenicillin N synthase (IPNS) catalyzes double ring closure of the tripeptide (L-π-amino-δ-adipoyl)-L-cysteinyl-D-valine (ACV) to form the (8-lactam and thiazolidine rings of penicillin-type antibiotics. Our previous spectroscopic study using IPNS from Cephalosporium acremonium expressed in Escherichia coli [Chen, V.J., Orville, A.M., Harpel, M.R., Frolik, C.A., Surerus, K.K., Miinck, E., & Lipscomb, J.D. (1989) J. Biol. Chem. 264, 21677-21681] indicated that a thiolate enters the coordination of the essential active site Fe²⁺when ACV binds to IPNS. The presence of an Fe-S bond in the IPNS-ACV complex is confirmed by EXAFS data presented in the preceding paper [Scott, R.A., Wang, S., Eidsness, M.K., Kriauciunas, A., Frolik, C.A. & Chen, V.J. (1992) Biochemistry (preceding paper in this issue)]. However, these studies leave unclear whether the coordinating thiolate derives from ACV or an endogenous cysteine. Here, we examine the spectroscopic properties of three genetically engineered variants of IPNS in which the only two endogenous cysteines are individually and collectively replaced by serine. The EPR, Mossbauer, and optical spectra of the mutant enzymes and their complexes with ACV, NO, or both ACV and NO are found to be essentially the same as those of wild-type IPNS, showing that the endogenous cysteines are not Fe²⁺ligands in any of these complexes. Spectral quantitations show that the double Cys → Ser mutation decreases the affinity of the enzyme for ACV by about 6-fold, suggesting that the endogenous cysteines influence the structure of the substrate binding pocket remote from the iron. Thiolate complexation of the Fe²⁺is also examined using ACV analogues. All ACV analogues examined in which the cysteinyl thiol moiety is unaltered are found to bind to the IPNS-NO complex to give optical and EPR spectra very similar to those of the ACV complex. In contrast, analogues in which the cysteinyl moiety of ACV is replaced with serine or cysteic acid fail to elicit the characteristic EPR and optical features despite the fact that they are bound with reasonable affinity to the enzyme. These results demonstrate that the thiolate of ACV coordinates the Fe²⁺. The EPR spectra of both the IPNS-NO and IPNS-ACV-NO complexes are broadened for samples prepared in ¹⁷O-enriched water, showing that water (or hydroxide) is also an iron ligand in each case. Thus, the Fe²⁺coordination of the IPNS-ACV-NO complex accommodates at least three exogenous ligands. Photodissociation of NO from this complex results in a species exhibiting nearly the same Mossbauer spectrum as the IPNS-ACV complex, suggesting that NO addition does not result in a major change in other aspects of the Fe2+ coordination.