Structure of the modified heme in allylbenzene-inactivated chloroperoxidase determined by Q-band CW and pulsed ENDOR

Hong In Lee, Annette F. Dexter, Yang Cheng Fann, Frederick J. Lakner, Lowell P. Hager, Brian M Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

During the epoxidation of allylbenzene, chloroperoxidase (CPO) is converted to an inactive green species in which the prosthetic heme has been modified by addition of the alkene plus an oxygen atom (Dexter, A. F.; Hager, L. P. J. Am. Chem. Soc. 1995, 117, 817-818). We have used Q-band continuous wave and pulsed electron-nuclear double resonance (ENDOR) spectroscopy to study the CPO heme in situ following inactivation with allylbenzene, using samples prepared in natural isotopic abundance, with 15 N-labeled enzyme, and with allylbenzene labeled with 2 H or 13 C in specific vinylic positions. The electron paramagnetic resonance (EPR) spectrum of the inactivated enzyme is dominated by a low-spin ferric signal (g 1,2,3 = 2.32, 2.16, 1.95). 14,15 N ENDOR examination of allylbenzene-inactivated CPO reveals that three nitrogens of the heme are similar, but the fourth nitrogen is markedly different, suggesting that a single pyrrole ring has been covalently modified at the unique nitrogen. These studies also reveal the orientation of the g tensor relative to the heme. 13 C ENDOR of allylbenzene-inactivated CPO with 13 C-labeled allylbenzene shows that the C-1 and C-2 carbons of allylbenzene are covalently connected to the heme system. 1,2 H ENDOR plus mass analysis of CPO heme after inactivation with deuterated allylbenzene show that all three vinylic protons are retained in the heme adduct. No strongly-coupled exchangeable protons are observed, indicating that the axially bound water of frozen native CPO has been displaced. The 1 H at the C-2 position of the alkene shows strong, mostly isotropic hyperfine coupling while the two hydrogens at the C-1 position show weak, dipolar couplings. The hyperfine tensors of 1,2 H of the C-1 position of allylbenzene have been determined, and give the position of these atoms relative to the heme. These data, combined with molecular modeling calculations, have been used to deduce that the allylbenzene-bound heme of inactivated CPO is an N-alkylhemin metallocycle with C-1 of allylbenzene bonded to the pyrrole nitrogen and to obtain metrical details of its structure.

Original languageEnglish (US)
Pages (from-to)4059-4069
Number of pages11
JournalJournal of the American Chemical Society
Volume119
Issue number17
DOIs
StatePublished - Apr 30 1997

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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