Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase

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Item Type:	Article
Title:	Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase
Creators Name:	Schuenemann, V., Jung, C., Terner, J., Trautwein, A.X. and Weiss, R.
Abstract:	It is generally assumed that the putative compound I (cpd I) in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases and catalases and many synthetic cpd I analogues. In these systems one oxidation equivalent resides on the Fe(IV)=O unit (d4, S=1) and one is located on the porphyrin (S′=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin π-cation radical system. However, this laboratory has recently reported detection of a ferryl iron (S=1) and a tyrosyl radical (S′=1/2), via Moessbauer and EPR studies of 8 ms-reaction intermediates of substrate-free P450cam from Pseudomonas putida, prepared by a freeze-quench method using peroxyacetic acid as the oxidizing agent [Schuenemann et al., FEBS Lett. 479 (2000) 149]. In the present study we show that under the same reaction conditions, but in the presence of the substrate camphor, only trace amounts of the tyrosine radical are formed and no Fe(IV) is detectable. We conclude that camphor restricts the access of the heme pocket by peroxyacetic acid. This conclusion is supported by the additional finding that binding of camphor and metyrapone inhibit heme bleaching at room temperature and longer reaction times, forming only trace amounts of 5-hydroxy-camphor, the hydroxylation product of camphor, during peroxyacetic acid oxidation. As a control we performed freeze-quench experiments with chloroperoxidase from Caldariomyces fumago using peroxyacetic acid under the identical conditions used for the substrate-free P450cam oxidations. We were able to confirm earlier findings [Rutter et al., Biochemistry 23 (1984) 6809], that an antiferromagnetically coupled Fe(IV)=O porphyrin {pi}-cation radical system is formed. We conclude that CPO and P450 behave differently when reacting with peracids during an 8-ms reaction time. In P450cam the formation of Fe(IV) is accompanied by the formation of a tyrosine radical, whereas in CPO Fe(IV) formation is accompanied by the formation of a porphyrin radical.
Keywords:	Binding Sites, Camphor 5-Monooxygenase, Chloride Peroxidase, Electron Spin Resonance Spectroscopy, Escherichia coli, Freezing, Kinetics, Molecular Cloning, Molecular Models, Peracetic Acid, Protein Conformation, Pseudomonas putida, Recombinant Proteins
Source:	Journal of Inorganic Biochemistry
ISSN:	0162-0134
Publisher:	Elsevier
Volume:	91
Number:	4
Page Range:	586-596
Date:	20 September 2002
Official Publication:	https://doi.org/10.1016/S0162-0134(02)00476-2
PubMed:	View item in PubMed

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