Activation of hydrogen peroxide in horseradish peroxidase occurs within ∼200 μs observed by a new freeze-quench device

Motomasa Tanaka, Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Hiroshi Hori, Isao Morishima

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45 Citations (Scopus)

Abstract

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freezequench device with ∼200 μs of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H2O2). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at ∼1 ms after the mixing of HRP and H2O2 suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at ∼200 μs show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within ∼200 μs after mixing HRP and H2O2. We conclude that the activation of H2O2 in HRP at ambient temperature completes within ∼200 μs. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.

Original languageEnglish
Pages (from-to)1998-2004
Number of pages7
JournalBiophysical Journal
Volume84
Issue number3
DOIs
Publication statusPublished - 2003 Mar 1
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics

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