ISSN:
1075-4261
Keywords:
Chemistry
;
Analytical Chemistry and Spectroscopy
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Physics
Notes:
Simultaneous measurements of resonance Raman and absorption spectra were performed for intermediates generated upon addition of hydrogen peroxide to ferric Arthromyces ramosus peroxidase (ARP) using the microcirculating system constructed in this laboratory, which enables generation of desirable intermediates under steady-state conditions. Compound I of ARP generated at neutral pH was stable over tens of minutes in the absence of laser illumination with this circulation system, but was gradually degraded under laser illumination, giving rise to a new irreversible species with an iron-oxo heme. Such photosensitivity was not observed for compound II in the steady state at alkaline pH. Surprisingly, the Raman spectrum of compound I of ARP in the high-frequency region, where characteristic frequency shifts are expected upon oxidation of the macrocycle, was quite close to that of compound II, despite the fact that the reduced Soret absorption indicated the formation of a π-cation radical. The Fe=O stretching (νFe=O) frequency of compound I was observed at 781 cm-1 for the 16O derivative but appeared as a doublet at 744 and 731 cm-1 for the 18O derivative. The isotope sensitivity of the νFe=O mode of compound I was seen upon H216O/H218O solvent substitution but not upon H216O2/H218O2 peroxide substitution in H216O at neutral pH. This directly indicates the occurrence of an oxygen atom exchange between the oxo-heme and bulk water, providing the first example of such exchange in compound I of peroxidases. The oxygen exchange was abolished for compound II at alkaline pH, for which the νFe=o mode was seen at 787/749 cm-1 only upon H216O2/H218O2 peroxide substitution. The oxygen exchangeability seems to depend on protonation of a nearby residue with pKα ∼ 9 and to correlate with stability of compound I. © 1996 John Wiley & Sons, Inc.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
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