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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 and Isao Morishima^*

^* Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan; and Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka Osaka 560-8531, Japan

Correspondence: Address reprint requests to Dr. Satoshi Takahashi or Prof. Isao Morishima, Dept. of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan. Tel.: 81-75-753-5921; Fax: 81-75-751-7611; E-mail: st{at}mds.moleng.kyoto-u.ac.jp or morisima{at}mds.moleng.kyoto-u.ac.jp.

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench 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.

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