Spectroscopy and Electrochemistry of Cytochrome P450 BM3-Surfactant Film Assemblies

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 FeIII heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 FeII−CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at ∼420 nm is present. Infrared absorption spectra of the BM3 FeII−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: FeIII/II E 1/2 at −260 mV (vs SCE), ∼300 mV positive of the value measured in solution; ΔS°rc, ΔS°, and ΔH° 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.