Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films

详细信息    查看全文

• 作者：Katharine D. Hagen ; James M. Gillan ; Sang-Choul Im ; Sally L ; efeld ; Griffin Mead ; Megan Hiley ; Lucy A. Waskell ; Michael G. Hill ; Andrew K. Udit
• 关键词：Cytochrome P450 2B4 ; Thin film electrochemistry ; Biocatalysis
• 刊名：Journal of Inorganic Biochemistry
• 出版年：December, 2013
• 年：2013
• 卷：129
• 期：Complete
• 页码：30-34
• 全文大小：350 K

文摘

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 Fe^III heme in P450. The Fe^III/II and Fe^II/I redox couples (E_1/2) of substrate-free CYP 2B4 measured by cyclic voltammetry are 鈭?#xA0;0.23 V and 鈭?#xA0;1.02 V (vs. SCE, or 14 mV and 鈭?#xA0;776 mV vs. NHE) at 21 掳C. The standard heterogeneous rate constant for electron transfer from the electrode to the heme for the Fe^III/II couple was estimated at 170 s^鈭?#xA0;1. Experiments indicate that the system is capable of catalytic reduction of dioxygen, however substrate oxidation was not observed. From the variation of E_1/2 with temperature (18-40 掳C), we have measured entropy and enthalpy changes that accompany heme reduction, 鈭?#xA0;151 J mol^鈭?#xA0;1 K^鈭?#xA0;1 and 鈭?#xA0;46 kJ mol^鈭?#xA0;1, respectfully. The corresponding entropy and enthalpy values are less for the six-coordinate low-spin, imidazole-ligated enzyme (鈭?#xA0;59 J mol^鈭?#xA0;1 K^鈭?#xA0;1 and 鈭?#xA0;18 kJ mol^鈭?#xA0;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.