Article
Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films
Journal of Inorganic Biochemistry
(2013)
Abstract
Electrochemical methods continue to present an attractive means for achieving in vitro biocatalysis with cytochromes P450; however understanding fully the nature of electrode-bound P450 remains elusive. Herein we report thermodynamic parameters using electrochemical analysis of full-length mammalian microsomal cytochrome P450 2B4 (CYP 2B4) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of CYP 2B4–DDAB films on silica slides reveal an absorption maximum at 418 nm, characteristic of low-spin, six-coordinate, water-ligated FeIII heme in P450. The FeIII/II and FeII/I redox couples (E1/2) of substrate-free CYP 2B4 measured by cyclic voltammetry are − 0.23 V and − 1.02 V (vs. SCE, or 14 mV and − 776 mV vs. NHE) at 21 °C. The standard heterogeneous rate constant for electron transfer from the electrode to the heme for the FeIII/II couple was estimated at 170 s− 1. Experiments indicate that the system is capable of catalytic reduction of dioxygen, however substrate oxidation was not observed. From the variation of E1/2 with temperature (18–40 °C), we have measured entropy and enthalpy changes that accompany heme reduction, − 151 J mol− 1 K− 1 and − 46 kJ mol− 1, respectfully. The corresponding entropy and enthalpy values are less for the six-coordinate low-spin, imidazole-ligated enzyme (− 59 J mol− 1 K− 1 and − 18 kJ mol− 1), consistent with limited conformational changes upon reduction. These thermodynamic parameters are comparable to those measured for bacterial P450 from Bacillus megaterium (CYP BM3), confirming our prior reports that the surfactant environment exerts a strong influence on the redox properties of the heme.
Keywords
- Cytochrome P450 2B4,
- Thin film electrochemistry,
- Biocatalysis
Disciplines
Publication Date
August 14, 2013
DOI
http://doi.org/10.1016/j.jinorgbio.2013.07.039
Citation Information
Katharine D. Hagen, James M. Gillan, Sang-Choul Im, Sally Landefeld, et al.. "Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films" Journal of Inorganic Biochemistry Vol. 129 (2013) p. 30 - 34 Available at: http://works.bepress.com/udit/34/