Spectroscopic and Kinetic Evidence for the Crucial Role of Compoundâ
0 in the P450cam -Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide.
Chemistry
; 21(43): 15201-10, 2015 Oct 19.
Article
em En
| MEDLINE
| ID: mdl-26353996
ABSTRACT
The hydroperoxo iron(III) intermediate P450cam Fe(III) -OOH, being the true Compoundâ
0 (Cpdâ
0) involved in the natural catalytic cycle of P450cam , could be transiently observed in the peroxo-shunt oxidation of the substrate-free enzyme by hydrogen peroxide under mild basic conditions and low temperature. The prolonged lifetime of Cpdâ
0 enabled us to kinetically examine the formation and reactivity of P450cam Fe(III) -OOH species as a function of varying reaction conditions, such as pH, and concentration of H2 O2 , camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate-free form of P450cam differs completely from that observed for other heme proteins possessing the distal histidine as a general acid-base catalyst and is mainly governed by the ability of H2 O2 to undergo deprotonation at the hydroxo ligand coordinated to the iron(III) center under conditions of pH≥p${K{{{\rm P450}\hfill \atop {\rm a}\hfill}}}$. Notably, no spectroscopic evidence for the formation of either Cpdâ
I or Cpdâ
II as products of heterolytic or homolytic OO bond cleavage, respectively, in Cpdâ
0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1R)-camphor, provide, for the first time, experimental evidence for the catalytic activity of the P450Fe(III) -OOH intermediate in the oxidation of the natural substrate of P450cam .
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MEDLINE
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Ano de publicação:
2015
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Article