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Spectroscopic and Kinetic Evidence for the Crucial Role of Compound 0 in the P450 cam ‐Catalyzed Hydroxylation of Camphor by Hydrogen Peroxide
Author(s) -
Franke Alicja,
van Eldik Rudi
Publication year - 2015
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201501886
Subject(s) - chemistry , catalytic cycle , catalysis , deprotonation , hydrogen peroxide , hydroxylation , heterolysis , reactivity (psychology) , peroxide , medicinal chemistry , homolysis , bond cleavage , camphor , stereochemistry , substrate (aquarium) , photochemistry , ion , organic chemistry , enzyme , radical , medicine , oceanography , alternative medicine , pathology , geology
The hydroperoxo iron(III) intermediate P450 cam Fe III –OOH, being the true Compound 0 (Cpd 0) involved in the natural catalytic cycle of P450 cam , 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 Cpd 0 enabled us to kinetically examine the formation and reactivity of P450 cam Fe III –OOH species as a function of varying reaction conditions, such as pH, and concentration of H 2 O 2 , camphor, and potassium ions. The mechanism of hydrogen peroxide binding to the substrate‐free form of P450 cam 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 H 2 O 2 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 Cpd I or Cpd II as products of heterolytic or homolytic OO bond cleavage, respectively, in Cpd 0 could be observed under the selected reaction conditions. The kinetic data obtained from the reactivity studies involving (1 R )‐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 P450 cam .