NMR-STUDIES OF THE ACTIVE-SITE OF ISOPENICILLIN-N SYNTHASE, A NONHEME IRON(II) ENZYME

The active site structure of isopenicillin N synthase (IPNS) has been previously studied by the use of Mossbauer, EPR, electronic absorption, and NMR spectroscopies [Chen, V. J., Frolik, C. A., Orville, A. M., Harpel, M. R., Lipscomb, J. D., Surerus, K. K., & Munck, E. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1990) Inorg. Chem. 26, 1111-1112]. These studies have revealed three coordinated His residues along with three sites for substrate [delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine, ACV], NO, and water binding on the active Fe(II) of IPNS. We report here NMR studies of Fe(II)IPNS and its Co(II)-substituted derivative [Co(II)IPNS]. By the use of NOE techniques on the Co(II)IPNS-ACV complex, we have recognized a -CH2-CH < spin system at 14.6, 24.3, and 38.6 ppm that is assigned to the alpha and beta-protons of a coordinated Asp residue. Corresponding solvent nonexchangeable features are found near 40 ppm in Fe(II)IPNS and the Fe(II)IPNS-ACV complex, but the peaks are too broad for NOE effects to be observed. The binding of NO to the Fe(II) center results in a significant change in the configuration of the metal site: (a) The C(beta)H-2 resonances due to the coordinated Asp residue disappear. The loss of the signal may indicate a change of the carboxylate configuration from syn-like to anti-like or, less likely, its displacement by NO. (b) The imidazole NH resonance for one of the coordinated His residues in the Fe(II)IPNS-ACV complex also disappears, suggesting that this His residue is strongly perturbed and may be detached from the metal site. These results allow us to propose a scheme showing the effects of exogenous ligand binding on the active site of IPNS. To date, this is the first successful NMR study of the endogenous ligands of the Fe(II)-NO center in a non-heme Fe(II) protein.