We report thermodynamic properties of the drug-metabolizing human cytochrome P450 2B4 within surfactant films based on electrochemical methods. The protein was embedded in didodecyldimethylammonium bromide (DDAB) bilayer film on a basal plane graphite electrode. Cyclic voltammetry at 25 mV/s was conducted in 50 mM KPi 50 mM KCl pH 7.0 buffer anaerobically between 18.4-40.4 ?C. Analysis of the half-wave potential dependence on temperature revealed decreases in entropy and enthalpy upon heme reduction. These thermodynamic changes are coupled to the expulsion of the heme distal axial water ligand; this is indicative of hydrophobic collapse around the catalytic center, resulting in a more rigid and stable structure. Spectroscopic analysis of the protein in the film revealed the Fe<sup>III</sup> heme Soret band at 418 nm, identical to its value in solution. This confirms protein integrity within the film, thereby affirming the physiological relevance of our thermodynamic data.