Mössbauer and electron paramagnetic resonance studies of chloroperoxidase following mechanism-based inactivation with allylbenzene.
We have used Mössbauer and electron paramagnetic resonance (EPR) spectroscopy to study a heme-N-alkylated derivative of chloroperoxidase (CPO) prepared by mechanism-based inactivation with allylbenzene and hydrogen peroxide. The freshly prepared inactivated enzyme ("green CPO") displayed a nearly pure low-spin ferric EPR signal with g = 1.94, 2.15, 2.31. The Mössbauer spectrum of the same species recorded at 4.2 K showed magnetic hyperfine splittings, which could be simulated in terms of a spin Hamiltonian with a complete set of hyperfine parameters in the slow spin fluctuation limit. The EPR spectrum of green CPO was simulated using a three-term crystal field model including g-strain. The best-fit parameters implied a very strong octahedral field in which the three 2T2 levels of the (3d)5 configuration in green CPO were lowest in energy, followed by a quartet. In native CPO, the 6A1 states follow the 2T2 ground state doublet. The alkene-mediated inactivation of CPO is spontaneously reversible. Warming of a sample of green 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 indistinguishable from native CPO. By subtracting spectral components due to native and green CPO, a third species with g = 1.86, 2.24, 2.50 could be generated. The EPR spectrum of this "quasi-native CPO," which appears at intermediate times during the reactivation, was simulated using best-fit parameters similar to those used for native CPO.
Debrunner
P G
PG
Department of Physics, University of Illinois, Urbana 61801, USA.
Dexter
A F
AF
Schulz
C E
CE
Xia
Y M
YM
Hager
L P
LP
D000498:Allyl Compounds / Q000494:pharmacology*; D001555:Benzene Derivatives / Q000494:pharmacology*; D002711:Chloride Peroxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D003904:Mitosporic Fungi / Q000201:enzymology; D011166:Porphyrins; D015204:Spectroscopy, Mossbauer; D000081248:Allylbenzene Derivatives
D000498:Allyl Compounds; D001555:Benzene Derivatives; D002711:Chloride Peroxidase; D004791:Enzyme Inhibitors; D006861:Hydrogen Peroxide; D011166:Porphyrins; C102347:allylbenzene; D000081248:Allylbenzene Derivatives