We report analyses of electrochemical and spectroscopic measurements on cytochrome P450 BM3 ( BM3) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of BM3-DDAB films on silica slides reveal the characteristic low-spin Fe-III heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 Fe-II-CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at similar to 420 nm is present. Infrared absorption spectra of the BM3 Fe-II-CO complex in DDAB dispersions feature a time-dependent shift of the carbonyl stretching frequency from 1950 to 2080 cm(-1). Voltammetry of BM3-DDAB films on graphite electrodes gave the following results: Fe-III/(II) E-1/2 at -260 mV (vs SCE), similar to 300 mV positive of the value measured in solution; Delta S degrees(rc), Delta S degrees, and Delta H degrees values for water-ligated BM3 in DDAB are -98 J mol(-1) K-1, -163 J mol(-1) K-1, and -47 kJ mol(-1,) respectively; values for the imidazole-ligated enzyme are -8 J mol(-1) K-1, -73 J mol(-1) K-1, and -21 kJ mol(-1). Taken together, the data suggest that BM3 adopts a compact conformation within DDAB that in turn strengthens hydrogen bonding interactions with the heme axial cysteine, producing a P420-like species with decreased electron density around the metal center.