NMR Studies of the Active Site of Isopenicillin N Synthase, a Non-Heme Iron(II) Enzyme

Li June Ming, Lawrence Que, Aidas Kriauciunas, Charles A. Frolik, Victor J. Chen

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The active site structure of isopenicillin N synthase (IPNS) has been previously studied by the use of Mössbauer, EPR, electronic absorption, and NMR spectroscopies [Chen, V. J., Frolik, C. A., Orville, A. M., Harpel, M. R., Lipscomb, J. D., Surerus, K. K., & Münck, 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 [δ-(L-α-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 α and β 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 CβH2 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.

Original languageEnglish (US)
Pages (from-to)11653-11659
Number of pages7
Issue number50
StatePublished - Dec 1 1991


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