NMR studies of the active site of isopenicillin N synthase, a non-heme iron(II) enzyme

• Published in: Biochemistry (Easton) Vol. 30; no. 50; pp. 11653 - 11659
• Main Authors: Ming, Li June, Que, Lawrence, Kriauciunas, Aidas, Frolik, Charles A, Chen, Victor J
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 17.12.1991
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Electron Spin Resonance Spectroscopy; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Heme - metabolism; Magnetic Resonance Spectroscopy; Miscellaneous; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Substrate Specificity; Fungi; Iron II Complexes; Ligand binding; Enzyme; Active site; Acremonium strictum; Overhauser effect; NMR spectrometry; Fungi Imperfecti; Cobalt II Complexes; Thallophyta; Two dimension spectrometry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00114a007

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 [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 less than 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 H2 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.