MOSSBAUER AND ELECTRON-PARAMAGNETIC-RESONANCE STUDIES OF CHLOROPEROXIDASE FOLLOWING MECHANISM-BASED INACTIVATION WITH ALLYLBENZENE

We have used Mossbauer and electron paramagnetic resonance (EPR) spect roscopy to study a heme-N-alkylated derivative of chloroperoxidase (CP O) prepared by mechanism-based inactivation with allylbenzene and hydr ogen peroxide. The freshly prepared inactivated enzyme (''green CPO'') displayed a nearly pure low-spin ferric EPR signal with g = 1.94, 2.1 5, 2.31. The Mossbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in term s of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was si mulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which t he three T-2(2) levels of the ((3)d)(5) configuration in green CPO wer e lowest in energy, followed by a quartet. In native CPO, the (6)A(1) states follow the T-2(2) ground state doublet. The alkene-mediated ina ctivation of CPO is spontaneously reversible, Warming of a sample of g reen CPO to 22 degrees C for increasing times before freezing revealed slow conversion of the novel EPR species to two further spin S = 1/2 ferric species. One of these species displayed g = 1.82, 2.25, 2.60 in distinguishable from native CPO, By subtracting spectral components du e to native and green CPO, a third species with g = 1.86, 2.24, 2.50 c ould be generated. The EPR spectrum of this ''quasi-native CPO,'' whic h appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.