Electron Spin Echo Envelope Modulation Studies of the Cu(II)-Substituted Derivative of Isopenicillin N Synthase: A Structural and Spectroscopic Model

Electron spin echo evelope modulation spectroscopy (ESEEM) was used to study the active site structure of isopenicillin N synthase (IPNS) from Cephalosporium acremonium with Cu(II) as a spectroscopic probe. Fourier transform of the stimulated electron spin-echo envelope for the Cu(II)-substituted enzyme, Cu(II)IPNS, revealed two nearly magnetically equivalent, equatorially coordinated His imidazoles. The superhyperfine coupling constant, A_iso, for the remote ¹⁴N of each imidazole was 1.65 MHz. The binding of substrate to the enzyme altered the magnetic coupling so that A_iso is 1.30 MHz for one nitrogen and 2.16 MHz for the other. From a comparison of the ESEEM of Cu(II)IPNS in D₂O and H₂O, it is suggested that water is a ligand of Cu(II) and this is displaced upon the addition of substrate.