[Show abstract][Hide abstract] ABSTRACT: We have studied the characterization of thermophilic cytochrome P450 (P450st)-didodecyldimethylammonium bromide (DDAB) films by using UV-vis absorption, resonance Raman spectroscopy, and electrochemical methods. The observed Raman spectrum indicated near-native conformation of the heme iron in DDAB film on the surface of a glass slide, while on the surface of a plastic-formed carbon (PFC) electrode, the conformation of P450st-DDAB was very similar to that of heme-DDAB film, suggesting the release of heme from P450st in DDAB films on PFC electrodes. When NaBr was added as salt to the casting solution, the result of Raman spectrum indicated near-native conformation of P450st in DDAB film even on the PFC electrode, but no redox potential of P450st which has near native structure was observed. This study suggests the essential experimental conditions when working with heme protein-DDAB films as, in some cases, heme iron from proteins is released on the surface of the electrode.
[Show abstract][Hide abstract] ABSTRACT: Myoglobin (Mb) was chemically modified with activated poly(ethylene oxide) (PEO) (average molecular weight of 2000) to solubilize it in various organic solvents. UV-vis, circular dichroism, and Raman spectroscopy were used to characterize the structure correlated with the electron-transfer reactions of PEO-modified Mb (PEO-Mb). Spectroscopic data indicated changes in heme coordination geometry for PEO-Mb in various organic solvents that are different from that in water. The Raman spectrum showed the characteristics of PEO-Mb in PEO oligomer (average MW of 200) in the five-coordinate high-spin state, which facilitates fast electron-transfer reactions between protein and the glassy carbon electrode. These results suggest heme environment effects on the properties of proteins in organic solvents.
No preview · Article · Oct 2006 · Biotechnology Progress
[Show abstract][Hide abstract] ABSTRACT: Direct electron transfer of cytochrome P450 from the thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7, (P450st) entrapped onto the surface of a plastic formed carbon electrode in poly(ethylene oxide) (PEO) containing 0.5 M KCl was achieved. The formal potential for wild-type P450st is −103 mV (vs. Ag wire). Thermophilic P450st was modified with PEO having an average molecular weight of 2000 (PEO2000) to increase its stability in PEO solvent. The P450st enzyme was dissolved in several organic media and PEO oligomers after the modification. Resonance Raman spectra revealed no effects of chemical modification with PEO2000 and PEO solvent on the heme structure of P450st. The electrochemical responses of PEO2000-modified P450st in PEO solvent were well-defined and stable compared with those of wild-type P450st. Voltammetry of the modified protein on the surface of a plastic formed carbon electrode in PEO solvent showed a FeIII/FeII potential at −114 mV vs. Ag wire, which is similar to that of wild-type P450st.
No preview · Article · Aug 2006 · Electrochemistry Communications
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st) was chemically modified with activated poly(ethylene oxide) (PEO) to make it more soluble in ionic liquid and in which Raman spectra results show the structural stability in the five coordinate high-spin state even at a temperature of 120 degrees C.
No preview · Article · Jul 2006 · Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: Ionic liquids exhibit good physical characteristics including the ability to dissolve various kinds of compounds. The use of ionic liquids in biocatalytic reactions has gained much attention recently. In the case of heme proteins, however, all native proteins are not dissolved in ionic liquids. In the present study, cytochrome P450 (P450) which catalyzes selective oxidation of inert hydrocarbon molecules was dissolved in 1-ethyl-3-methyl-imidazolium bis(trifluoromethansulfonyl)imide ([emim][TFSI]) by polyethylene oxide) (PEO) modification. The absorption and resonance Raman spectra of PEO-P450 in [emim][TFSI] were similar to those of PEO-P450 in water. These results prove that the dissolution in [emim][TFSI] does not cause large structural change of PEO-P450. The clear redox response of PEO-P450 was also observed in [emim][TFSI].