title: Absence of a detectable intermediate in the compound I formation of horseradish peroxidase at ambient temperature.
abstract: A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H(2)O(2) by HRP is significantly faster than 1 x 10(4) s(-1).
authors.affiliation: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo, Kyoto 615-8510, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Shintaku
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: Isao
authors.initials: I
authors.lastname: Morishima
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods; D013696:Temperature
chemical_list: D005290:Ferric Compounds; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase