title: Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device.
abstract: To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). 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 approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s 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 approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
authors.forename: Motomasa
authors.initials: M
authors.lastname: Tanaka
authors.forename: Koji
authors.initials: K
authors.lastname: Matsuura
authors.forename: Shiro
authors.initials: S
authors.lastname: Yoshioka
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takahashi
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hori
authors.forename: Isao
authors.initials: I
authors.lastname: Morishima
mesh_terms: D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation* / Q000379:methods*; D004789:Enzyme Activation; D004867:Equipment Design; D005615:Freezing; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000295:instrumentation / Q000379:methods
chemical_list: D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase