Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron
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Date
2017-12-29
Publication Type
Journal Article
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yes
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Abstract
Cytochrome P450cam (CYP101Fe³⁺) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe³⁺ by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO₂]²⁺. Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH]²⁺ (compound 0) accumulates within 5 μs and decays rapidly to CYP101Fe³⁺, with a k440 nm of 9.6 × 104 s⁻¹. All processes are complete within 40 μs at 4 °C. Importantly, no transient absorbance bands could be assigned to CYP101[FeO²⁺por•⁺] (compound 1) or CYP101[FeO²⁺] (compound 2). However, indirect evidence for the involvement of compound 1 was obtained from the kinetics of formation and decay of a tyrosyl radical. 5-Hydroxycamphor was formed quantitatively, and the catalytic activity of the enzyme was not impaired by exposure to radiation during the pulse radiolysis experiment. The rapid decay of compound 0 enabled calculation of the limits for the Gibbs activation energies for the conversions of compound 0 → compound 1 → compound 2 → CYP101Fe³⁺, yielding a ΔG‡ of 45, 39, and 39 kJ/mol, respectively. At 37 °C, the steps from compound 0 to the iron(III) state would take only 4 μs. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time.
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published
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Journal / series
Volume
292 (52)
Pages / Article No.
21481 - 21489
Publisher
American Society for Biochemistry and Molecular Biology
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Subject
Cytochrome P450; electron transfer; isotope effect; kinetics; oxidation-reduction (redox)
Organisational unit
03512 - Günther, Detlef / Günther, Detlef
02513 - Laboratorium für Anorganische Chemie / Laboratory of Inorganic Chemistry