STRUCTURE OF THE MODIFIED HEME IN ALLYLBENZENE-INACTIVATED CHLOROPEROXIDASE DETERMINED BY Q-BAND CW AND PULSED ENDOR

During the epoxidation of allylbenzene, chloroperoxidase (CPO) is conv erted to an inactive green species in which the prosthetic heme has be en 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 (EN DOR) spectroscopy to study the CPO heme in situ following inactivation with allylbenzene, using samples prepared in natural isotopic abundan ce, with N-15-labeled enzyme, and with allylbenzene labeled with H-2 o r C-13 in specific vinylic positions. The electron paramagnetic resona nce (EPR) spectrum of the inactivated enzyme is dominated by a low-spi n ferric signal (g(1,2,3) = 2.32, 2.16, 1.95). N-14,N-15 ENDOR examina tion of allylbenzene-inactivated CPO reveals that three nitrogens of t he heme are similar, but the fourth nitrogen is markedly different, su ggesting that a single pyrrole ring has been covalently modified at th e unique nitrogen. These studies also reveal the orientation of the g tensor relative to the heme. C-13 ENDOR of allylbenzene-inactivated CP O with C-13-labeled allylbenzene shows that the C-1 and C-2 carbons of allylbenzene are covalently connected to the heme system. H-1,H-2 END OR plus mass analysis of CPO heme after inactivation with deuterated a llylbenzene show that all three vinylic protons are retained in the he me adduct. No strongly-coupled exchangeable protons are observed, indi cating that the axially bound water of frozen native CPO has been disp laced. The H-1 at the C-2 position of the alkene shows strong, mostly isotropic hyperfine coupling while the two hydrogens at the C-l positi on show weak, dipolar couplings. The hyperfine tensors of H-1,H-2 of t he C-1 position of allylbenzene have been determined, and give the pos ition of these atoms relative to the heme. These data, combined with m olecular modeling calculations, have been used to deduce that the ally lbenzene-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.